UC Davis

Currently in the agricultural industry, the application of pesticides to benefit crop health and performance is unavoidable, yet major reform is needed for safer and more sustainable practices. Plant-beneficial bacteria are of interest because they are able to address many of the issues traditional pesticides target. These biopesticides are isolated from the environment thus completely natural, and are not harmful to humans. Unfortunately, the types of viable bacteria for biopesticide products are mostly limited to spore-formers due to some challenges in commercialization. Microencapsulation, a process where a wall material surrounds and protects a cargo component to form a microcapsule, is perhaps the best method for non-spore-forming plant-beneficial bacteria to be implemented in the agricultural industry. Encapsulation of non-spore-formers appears to be most promising in dry alginate beads. Alginate is a highly desirable naturally-derived wall material for its safety and ability to form a gel in the presence of multivalent cations. However, forming dry alginate beads is a tedious process requiring a gelling step, curing step, collection step, and drying step, making it difficult to efficiently scale up. A recently developed UC Davis spray-drying process forms in situ cross-linked alginate microcapsules (CLAMs) in a single step. In this work, I hypothesized that formulation and process development of the single step encapsulation process by spray-drying or fluidized bed spray-coating, will stabilize non-spore-forming bacteria such that high viability is maintained throughout the encapsulation process and in long term storage. To identify key formulation and process parameters during spray-drying which influenced survival of bacteria, the non-spore-forming plant-protective antifungal bacterium Collimonas arenae Cal35 was encapsulated in CLAMs. Response surface methodology was used in this study with the optimization objectives of maximizing survival of Cal35, and maximizing yield of spray-dried powder. For the control parameters, only inlet temperature significantly impacted survival, while inlet temperature and spray rate influenced yield. Alginate concentration of the CLAMs formulation only impacted yield. By lowering inlet temperature to 95°C, the greatest survival was achieved, whereas greater inlet temperatures, and low spray rates and alginate concentrations produced the highest yield. When Cal35 was spray-dried with common excipients, maltodextrin and modified starch, there was no survival. Combining the CLAMs formulation with modified starch yielded the lowest loss of 3 log units while extending the shelf survival to over three weeks in a low oxygen and low humidity storage environment. Most importantly, Cal35 was able to retain its antifungal activity after spray-drying and shelf storage supporting its potential as a biofungicide. Since the physical encapsulation of bacteria in spray-dried CLAMs is driven by atomization, fluidized bed spray-coating was investigated as another potential encapsulation method for seed coating applications. Here, enzyme was used as a substitute for bacteria to facilitate the process development of forming in situ cross-linked alginate matrix shell (CLAMshell) particles by fluidized bed spray-coating. The CLAMshell process maximized process efficiency while simultaneously improving the coating quality. Incorporation of the CLAMshell formulation allowed a 40% faster spray rate to be achieved over coatings with cargo-only, with coating efficiencies above 90%, and aggregates and fines below 2% of the product. Process interruptions were also eliminated. Increasing alginate concentration and using polyvinyl alcohol as an excipient in the CLAMshell formulation produced coatings with the greatest mechanical strength with minor particle damage and low attrition. CLAMshell particles also delayed dissolution of the salt core in solution, indicating potential for controlled release. Finally, the non-spore-forming plant-beneficial bacteria Collimonas arenae Cal35 was encapsulated using the same formulation in CLAMs by spray-drying, and in CLAMshells by fluidized bed spray-coating, as both encapsulation processes are feasible in practice depending on how a bacterial biopesticide might be applied. Cal35 encapsulation in CLAMs had greater survival with a 3 log reduction in viability. Encapsulation in CLAMshells resulted in a 4 log reduction. Testing the sensitivity of Cal35 to heat and desiccation individually revealed the bacteria is somewhat tolerant to high temperatures, but not tolerant to desiccation. High temperatures and fast drying time are the conditions seen during spray-drying, which had better survival. Overall, CLAMs were between 5-20 µm allowing them to be applied to crops by spraying, while CLAMshells ranging between 250-300 µm would be more suitable for applying biopesticides in seed coatings. Overall, this research demonstrates that through formulation and process development of the highly scalable CLAMs and CLAMshell encapsulation methods, there is potential for industrially application of non-spore-forming bacterial biopesticides. Low in-process drying temperatures but fast drying times were found to be most beneficial to the survival of Cal35. Addition of excipients in the formulation such as modified starch which is known for its oxygen barrier properties improved the shelf stability of Cal35, however, controlling the direct storage environment greatly affected its stability. Future work is highly recommended for scale up of both processes.