Progress 09/01/17 to 08/31/20
Outputs
Target Audience:During the Phase II period of performance, 3Bar provided internships to six (6) undergraduate students with majors in Bioengineering, Biology, and Horticulture who supported work performed on the project. 3Bar hired one of the interns as a full time employee; four of the interns went on to pursuegraduate degrees in agriculturebiosciences or engineering and one went into industry. The greenhouse and field trial workconducted by The Ohio State University was supported by a PhD student who graduated in 2020. All students benefited from the experiental learning contributing toworkforce development. Changes/Problems:During 2019, due to the late release of the second half of funds by the Government, work supported by a sub-contract with The Ohio State University was postponed. The delays resulted in a no-cost extension being requested and approved to extend the project through August 31, 2020. Efforts directly impacted by the delays included 1) completion of field trials and associated sample analysis to support SCN-control efficacy and yield improvement under field conditions, and 2) completion of stability testing of new formulations under a range of storage conditions and extended timeframes to support basis for label and use instructions needed for commercialization. The original goal of the Phase II project was to commercialize a bionematicide product utilizing 3Bar's delivery system; however, varied and inconsistent efficacy resultsfrom the greenhouse and field trials did not support a clear candidate for SCN control to move forward with commercialization. Nonetheless, several important research findings led to improvements in the packaged delivery system and discovery of a new bacteria strain (Pantoea agglomerans MBSA-3B1) with important characteristics for commercialization of a plant health product. Commercial potential of MBSA-3B1 was demonstrated by improved shelf-stability and growth in the delivery system, along with feasibility to apply MBSA-3B1 as a seed treatment using a commercial seed treater. Subsequently, a pivot of commercialization plans led to a new plant health product being developed with plans to commercialize as a seed treatment for row crops in 2021. What opportunities for training and professional development has the project provided?On a regular basis, 3Bar Biologics employs 2-3 undergraduate students as student interns, who work part-time while going to school and full-time during the summer. Students learn basic microbiology skills, including aseptic technique, plating and enumeration, creating media, etc, and lead a project during the summer that supports 3Bar's technology development goals. 3Bar Biologics works with the Tech Ohio Diversity and Inclusion internship program to hire student interns. The program provides college students with hands-on experience in some of Ohio's most innovative companies, while providing startup tech companies with great talent to help them grow.3Bar highly values the student intern program as we gain exceptional support from the students, while contributing to bioeconomy workforce development. 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?
3Bar Biologics, in collaboration with Dr. Christopher Taylor at The Ohio State University, evaluated the commercial potential for applying plant growth promoting bacteria (PGPB) for plant-parasitic nematode control. Specifically, soybean cyst nematode (SCN; Heterodera glycines) is the major pathogen of soybean in North America, causing $1.5 billion in crop losses annually.Phase II efforts built upon the previous work, with the goal toincrease the scale of production and testing to develop a more stable and robust product. Changes in product formulation and packaging were driven by the need for a scaled-up (>10 L) product configuration to accommodate large farm (>1,000 acres) and Ag retail seed treatment applications. Commercial potential of eight bacteria (from the genera Pseudomonas, Bacillus, and Pantoea) to act as nematode control agents and plant growth promoters was further investigated in greenhouse and microplot studies over two seasons in 2017 and 2018. This final report for Award No. 2017-33610-27301 (System for Biologic Control of Soybean Cyst Nematode) summarizes several key advancements made with respect to the delivery system and general understanding of the SCN-active Pseudomonas strains and their modes of action that led to changes in direction regarding product formulation, packaging, and commercialization strategy. The R&D efforts were partially funded under Award No. 2019-33610-27301, with the remainder funded through 3Bar Biologics-generated revenue. Identification of new strain MBSA-3B1: Based on genome sequence verification during commercial transfer of the strains from The Ohio State University to 3Bar Biologics, one of the leading SCN-active strains (48C10) was reclassified from Pseudomonas spp. to Pantoea agglomerans. With the reclassification, the strain has been renamed MBSA-3B1 and the sequence was submitted to GenBank. MBSA-3B1 was found to have improved shelf-life and significantly improves germination rates and plant biomass. Biochemical analysis has shown that MBSA-3B1 produces high levels of auxin, a plant growth promoting hormone. Stability of dry formulation: Previous efforts have shown encapsulation of gram-negative bacteria can provide a shelf stable dry formulation >6 months. While efforts to scale-up the encapsulation process were initiated, variability in control of thedrying processes remain technical hurdles for scale-up from bench to pilot scale. Due to these technical challenges for scale-up, existing commercially scaled methods were pursued. In particular, lyophilization (freeze drying) was explored as a commercially available option. Freeze-dried MBSA-3B1 showed improved stability at room temperature (22oF) at levels of 1010 CFU/g for at least 40 weeks and can survive at elevated temperature (35oC) for approximately 20 weeks. Scalable packaging: To scale the package configuration,flexible plastic bags were selected based on the ease in sterilizing bags using gamma irradiation, opportunity for gas exchange with high-permeability bags, and ability to scale the package size (from 1 L to >100 L totes). A second generation delivery system (named LiveMicrobeTM) was developed involving a plastic bag-in-box configuration. To mass-produce the LiveMicrobeTMdelivery system, a novel biomanufacturing footprint is being proposed called small batch fermentation (SFB). By not fermenting in large vessels as currently used in conventional industrial fermentation, 3Bar's SBF "factory of the future" can improve microbial product viability, quality, and costs for production. Liquid nutrient source: With larger package sizes, the ability to store the nutrients for fermentation in the cap (separate from liquid) becomes problematic for reasonable operation of the two-chamber system. Subsequently, the nutrients for fermentation were moved to the liquid chamber. Optimization of nutrient source and amount was investigated; based on results a low concentration (10%) of nutrient rich media (tryptic soy broth, TSB) performed as good or better than alternatives. For scale up, next steps involve replacement of nutrient components with lower cost alternatives. Recommended product storage and use: Temperature during fermentation is a major factor impacting the bacterial growth and is strain dependent. At cooler temperatures, the bacteria grow at a slower rate, but reach a maximum population within 2 to 3 days post activation (DPA). Testing of the plastic bag package configuration has consistently shown 108CFU/mL growth and stability at this population for greater than 28 DPA. For label use instructions, it is recommended that the product be stored and used at temperatures ranging between 15-26oC (60-80oF) and used within 28 days. SCN efficacy in field trials: Microplot trials were performed in 2017 and 2018 to determine the effect bacterial inoculants have on SCN control and soybean yield increase. There were no treatments that significantly reduced SCN populations or increased yield in all experiments, and the results varied from year to year; of note, treatments containing MSBA-3B1 consistently resulted in higher yields than other bacteria treatments. A commercial product Poncho/VOTiVO® was also tested, and neither SCN eggs nor yield were significantly different from the controls. Mode of action and priming of soil: The work found thePseudomonasstrains produced substantial amounts of two classes of volatile organic compounds (VOCs): cyanides and organosulfurs. Hydrogen cyanide (HCN) is a known contributor to Pseudomonas spp. bioactivity both against nematode and fungal pathogens. One organosulfur compound of interest is dimethyl disulfide (DMDS). Synthetically produced DMDS is sold commercially as a soil fumigant alternative to methyl bromide. As a priming agent, addition of L-methionine to a minimal media increased the production and bioactivity of organosulfurs by multiple log-fold seen both on agar- and soil-based systems. Thus, these results broadly demonstrate the potential to selectively produce specific volatile compounds through addition of certain amino acids to the soil system. Although the original goal of the Phase II project was to commercialize a bionematicide product utilizing 3Bar's delivery system, results from the greenhouse and microplot trials were varied and efficacy of individual treatments were inconsistent from trial to trial. Subsequently, based on the research findings, a pivot in the commercialization strategy is planned. Specifically, MBSA-3B1 was found to be considerably more stable than 3Bar's current commercialized Pseudomonas spp.as a dry formulation, particularly at higher storage temperatures. MBSA-3B1 was confirmed to produce a high amount of indole-3-acetic acid (IAA, auxin) to provide growth promotion benefits such as improved seed germination and elongation of roots for improved nutrient and water uptake. Consistent growth of MBSA-3B1 in the packaged bioreactor was confirmed and feasibility to apply MBSA-3B1 as a seed treatment was validated using a commercial seed treater. Subsequently, a new plant health product Bio-YIELD ST is being developed with plans to commercialize in 2021. In summary, our vision for the short term involves continuing to prove out the LiveMicrobeTMdelivery system with farmers by launching a new plant health product Bio-YIELD ST as a seed treatment for corn and soybeans. Bio-YIELD ST widens 3Bar's market reach with seed treatment in row crops, and opens new markets including specialty crops. This Phase II project made possible the discovery of a new microbe strain with considerable plant health benefits and improvement of the packaged bioreactor for scaling the packaging size and mass-production. Born out of this work is 3Bar'sproposed SBF "factory of the future" which revisions the biomanufacturing process to improve the quality, cost, time to commercialization, and flexibility for producing microbial products.
Publications
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2020
Citation:
Kimmelfield RB. 2020. Establishing the use of Pseudomonas spp. as biocontrol agents of fungal and nematode pathogens. Dissertation, The Ohio State University. 294 pp.
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:During 2019, due to the late release of the second half of funds by the Government, work supported by a sub-contract with The Ohio State University was postponed. The delays resulted in a no-cost extension being requested and approved to extend the project through August 31, 2020. Efforts directly impacted by the delays included 1) completion of field trials and associated sample analysis to support SCN-control efficacy and yield improvement under field conditions, and 2) completion of stability testing of new formulations under a range of storage conditions and extended timeframes to support basis for label and use instructions needed for commercialization. What opportunities for training and professional development has the project provided?On a regular basis, 3Bar Biologics employs 2-3 undergraduate students as student interns. Students learn basic microbiology skills, including aseptic technique, plating and enumeration, creating media, etc. 3Bar Biologics works with the Tech Ohio Diversity and Inclusion internship program to hire student interns. The program provides college students with hands-on experience in some of Ohio's most innovative companies, while providing startup tech companies with great talent to help them grow. 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?
3Bar Biologics is dedicated to improving yield, profitability, and sustainability of farms using beneficial microbial products to increase crop yield while reducing dependence on chemical fertilizers and pesticides. 3Bar's novel microbial inoculant delivery system is activated on-farm to deliver the freshest, most viable biological product to the field. 3Bar Biologics, in collaboration with Dr. Christopher Taylor at The Ohio State University (OSU), is developing a biopesticide product for soybean cyst nematode (SCN) control. SCN is the major pathogen of soybean in North America, causing $1.5 billion in crop losses annually. As part of Phase I efforts, preliminary evaluation of lead candidate strains under laboratory, greenhouse, and field conditions demonstrated feasibility to continue commercial product development. Phase II efforts build upon the previous work, with the goal to scale up the research and development to commercialization. Progress during second year of SBIR project: During 2019, due to the late release of the second half of funds by the Government, work supported by a sub-contract with The Ohio State University was postponed. The delays resulted in a no-cost extension being requested and approved to extend the project through August 31, 2020. Efforts directly impacted by the delays included 1) completion of field trialsample analysis to support SCN-control efficacy and yield improvement under field conditions, and 2) completion of stability testing of new formulations under a range of storage conditions and extended timeframes to support basis for label and use instructions needed for commercialization. Included in this interim report is a summary of progress, which highlights several key areas of research and development (R&D) that led to changes in direction regarding product formulation and packaging. The R&D efforts were partially funded under Award No. 2019-33610-27301, with the remainder funded through 3Bar Biologics-generated revenue. The changes in product formulation and packaging were driven by the need for a scaled-up (>10 L) product configuration to accommodate large farm (>1,000 acres) and Ag retail seed treatment applications. The following provides a summary of each of the key efforts and findings during the reporting period: Strain identification: Based on gene sequence verification during commercial transfer of the strains from The Ohio State University to 3Bar Biologics, one of the leading SCN-active strains (48C10) was reclassified from Pseudomonas spp. to Pantoea agglomerans. With the reclassification, the strain has been renamed 3BB1. Stability dry formulation: Previous efforts have shown encapsulation of gram-negative bacteria can provide a shelf-stable dry formulation >6 months. While efforts to scale-up the encapsulation process were initiated, variability in control of the formulation mixing and drying processes remain technical hurdles for scale-up from bench to pilot scale. Due to these technical challenges for scale-up, existing commercially scaled methods were pursued. In particular, lyophilization (freeze drying) was explored as a commercially available option. Freeze dry powder of Pseudomonas spp. strain Wood3 was formulated by a commercial partner, and shelf stability at room temperature (22oC) was found improvedcompared to encapsulation. A freeze dry formulation of P. agglomerans strain 3BB1 (formerly strain 48C10) was recently produced and storage stability measurements have been initiated at room temperature (22oF) and elevated temperature (37oC). Stability in packaged product: The minimally viable prototype (MVP) package (3 L) involves a hard-sided plastic bottle with the dry formulated microbes and nutrients for fermentation contained in the cap portion of the package. Stability of Pseudomonas spp. Wood3 in the packaged product was evaluated over the course of one year at 22oC (laboratory conditions). Stability of the encapsulated cells declined to approximately 1E5 CFU/g over the course of a year. The corresponding microbial recovery with longer storage times (and subsequently lower initial inoculum) maintained 1E8 CFU/mL growth in the package; however, the number of days to reach the maximum population increased from 2 to 3 days post activation (DPA) to 14 DPA. Improvements in the dry formulation and packaging are ongoing to improve consistency in product viability for greater than one year. Scalable packaging: To scale the package configuration, either larger hard-sided containers or flexible plastic bags were considered. Flexible plastic bags were selected, based on the ease in sterilizing bags (through irradiation), opportunity for gas exchange with low-permeability bags, and ability to scale the package size (from 1 L to >100 L totes). A utility patent application for the package configuration is in the process of being submitted. Liquid nutrient source: With larger package sizes, the ability to store the nutrients for fermentation in the cap (separate from liquid) becomes problematic for reasonable operation of the two-chamber system. Subsequently, the nutrients for fermentation were moved to the liquid chamber. Optimization of nutrient source and amount was investigated; based on preliminary results a low concentration (10%) of nutrient rich media (tryptic soy broth, TSB) performed as good or better than alternatives. For scale up, next steps involve replacement of nutrient components with lower cost alternatives. Stability testing of plastic bag package configuration: Different liquid volumes and gaseous headspace were evaluated, to determine growth kinetics and stability of the bacteria culture in the plastic bag package. Preliminary results indicate that as the surface area: volume (SA:V) ratio increases, the cell density (CFU/mL) increases, supporting the importance of an adequate gaseous head space for oxygen exchange during fermentation. Similar total CFU (1E12 CFU) were obtained for both 10 L and 5 L liquid in 20 L bags, suggesting that similar total cells can be achieved in the system while lowering the amount of media to save costs. Additional testing to further evaluate the SA:V ratio and impacts of diffuse oxygen transfer with different plastic bag gas transmissibility is recommended. Based on these results, 10 L of liquid in 20 L bag is recommended to achieve the target 1E8 CFU/mL. Recommended product storage: Temperature during fermentation is a major factor impacting the bacterial growth and is strain dependent. Growth kinetics of P. agglomerans strain 3BB1 at 22oC (laboratory conditions) and cooler conditions representative of a warehouse or barn (12oC to 15oC) was evaluated.At cooler temperatures, the bacteria grow at a slower rate, but reach a maximum population within 2 to 3 days post activation (DPA). For label use instructions, it is recommended that the product be stored and used at temperatures ranging between 15oC - 26oC (60oF - 80oF) to ensure growth of the bacteria to 1E8 CFU/mL within 48 hours. Recommended product usage: Preliminary testing of the plastic bag package configuration (strain 3BB1 dry formulation, 10% TSB liquid nutrient, 10 L liquid in 20 L bag) has consistently shown 1E8 CFU/mL growth and stability at this population for minimally 28 days post activation. For label use instructions, it is recommended that once the product is activated, it be used within a 28 day window. Microplot trials: Replicated field trials in microplots were conducted during the 2017 and 2018 seasons. For the 2018 season, nematode populations are in the process of being counted with an expected completion date by the end of 2019. Data collected in this study include soybean yield per microplot and (soybean cyst nematode (SCN) eggs in 100 cubic centimeters of soil. ?
Publications
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?On a regular basis, 3Bar Biologics employs 2-3 undergraduate students as student interns. Students learn basic microbiology skills, including aseptic technique, plating and enumeration, creating media, etc. 3Bar Biologics works with the Tech Ohio Diversity and Inclusion internship program to hire student interns. The program providescollege students with hands-on experience in some of Ohio's most innovative companies, while providing startup tech companies with great talent to help them grow. 3Bar Biologics also partnered with Worthington Linworth HS to host two high school students for an 8-week internship as part of the schools "walk about" program. The program provides high school students the opportunity to shadow fields they may have interest. 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?
3Bar Biologics is dedicated to improving yield, profitability, and sustainability of farms using beneficial microbial products to increase crop yield while reducing dependence on chemical fertilizers and pesticides. 3Bar's novel microbial inoculant delivery system is activated on-farm to deliver the freshest, most viable biological product to the field. 3Bar Biologics, in collaboration with Dr. Christopher Taylor at The Ohio State University (OSU), is developing a biopesticide product for soybean cyst nematode (SCN) control. SCN is the major pathogen of soybean in North America, causing $1.5 billion in crop losses annually. As part of Phase I efforts, preliminary evaluation of lead candidate strains under laboratory, greenhouse, and field conditions demonstrated feasibility to continue commercial product development. Phase II efforts build upon the previous work, with the goal to scale up the research and development to commercialization. Key findings to date: Objective 1: Confirm yields and stability in longer term/broader range of conditions SCN-active Pseudomonas strain 48C10 has demonstrated stability and robustness at different storage temperatures and compatibility with all of the starter fertilizers, insecticides, fungicides, and herbicides tested to date. Additional SCN-active strains will be evaluated based on microplot trial results. Consistent recovery of bacteria in the bioreactor was demonstrated after storage at room temperature for 40 weeks. Even as the inoculum decreases to levels <109CFU/g, sufficient viable encapsulated cells remain to grow in the bioreactor system. However, at longer storage durations, the exponential growth phase is slowed so that stationary phase takes longer to reach. Efforts to improve storage stability of inoculum will help consistency in reaching stationary phase within 48 hr at longer storage times. Replacing soybean with animproved nutrient source (e.g., tryptic soybean broth, TSB) increased cell concentrations by 0.5 log. Optimization of nutrient source and amount can further increase cellular yields. Objective 2: Optimizate and scale-up alginate bead formulation process Encapsulation of Pseudomonas strains was improved by concentrating the bacteria culture 10X, leading to a ten-fold increase in bacterial loading and improved storage stability. Overall, stability of encapsulated bacteria is maintained at approximately 106 CFU/mL (109 CFU/g) for at least 6 months. Several formulation parameters were evaluated, including amount of alginate (1%, 2%, 3% w/v), skim milk (1%, 2%, 5%, 10% w/v), and cross-linking duration (30 min, minimal). Based on results to date, the candidate formulation includes 2% alginate, 5 to 10% skim milk, and 30 min of cross-linking duration. Desiccation loss during drying was reduced on average to 0.6 log loss (range: 0.2 log to 1.2 log), largely driven by addition of skim milk providing a protective effect. Other strategies were evaluated (resuspending concentrate in spent media, adding trehalose, preconditioning cells), but in general did not providefurther benefit in reducing desiccation loss or improving storage stability of encapsulated cells. Production of exopolysaccharides is a known bacterial defense mechanism against environmental stressors including drying. By resuspending the concentrated bacterial culture in supernatant, the cells were less susceptible to drying, resulting in a drying loss of approximately 0.3 log compared to the ≥ 1 log drying loss previously observed. Based on initial scale-up efforts, thealginate bead production rate was increased by roughly 4X. By adding multiple needle nozzles and a multi-channel peristaltic pump, scale up can be further increased 10X and greater. Objective 3: Develop basis for label and pamphlet and use instructions As a benchmark, growth kinetics of Wood3 in a bioreactor atroom temperature (RT; 20-22oC) demonstrated thepopulation reached 107CFU/mL between 36-48 hours, and steadily increased and maintained a population of approximately 108CFU/mLfor a month and longer.Growth kinetics studies are planned with candidate strainsand optimized solid substrate (TSB),at temperatures of 4oC (refrigerated), 12-15oC (workshop or barn), and 22oC (RT; office). Objective 4: Demonstrate SCN-control efficiacy and yield improvement in field microplot and open field replicated trials. Replicated field trials in microplots were conducted during the 2017 and 2018 seasons. The microplot systemallows direct application of microbes and pests under controlled field conditions. Each microplot consists of a large PVC tube (10 inch wide by 12 inch deep) buried 11 inch in the soil.Single and multiple strains of SCN-activePseudomonasstrains were applied as soil drench and seed treatment under microplot conditions.Bacteria drench treatments were applied 3 weeks after planting; SCNeggs were inoculated 3 days after soil drench inoculation. Soil cores were taken for SCN egg counts.Soil samples from 2017 and 2018 seasons are currently being processed to determine efficacy. Microplot trials evaluated soybean yield (grams of shelled soybean). For 2017 microplot trial, on average each microplot produced approximately 160 grams of shelled soybean. There were no significant differences in yield between any of the treatments (Tukey Test, p<0.05). It should also be noted that no differences were observed between the drench treated or seed treated samples. Strain and group-specific primers were developed to track the presence and longevity of specific SCN-active Pseudomonas strains in collected soil samples. Soil samples from the 2018 season are being processed to evaluate presence ofPseudomonasspecies.
Publications
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