Microencapsulation of Bacillus Strains for Improving Wheat (Triticum turgidum Subsp. durum) Growth and Development.

Bio-formulation technologies have a limited impact on agricultural productivity in developing countries, especially those based on plant growth-promoting rhizobacteria. Thus, calcium alginate microbeads were synthesized and used for the protection and delivery of three beneficial Bacillus strains for agricultural applications. The process of encapsulation had a high yield per gram for all bacteria and the microbeads protected the Bacillus strains, allowing their survival, after 12 months of storage at room temperature. Microbead analysis was carried out by observing the rate of swelling and biodegradation of the beads and the released-establishment of bacteria in the soil. These results showed that there is an increase of around 75% in bead swelling on average, which allows for larger pores, and the effective release and subsequent establishment of the bacteria in the soil. Biodegradation of microbeads in the soil was gradual: in the first week, they increased their weight (75%), which consistently results in the swelling ratio. The co-inoculation of the encapsulated strain TRQ8 with the other two encapsulated strains showed plant growth promotion. TRQ8 + TRQ65 and TRQ8 + TE3T bacteria showed increases in different biometric parameters of wheat plants, such as stem height, root length, dry weight, and chlorophyll content. Thus, here we demonstrated that the application of alginate microbeads containing the studied strains showed a positive effect on wheat plants.

Polycationic Surfaces Promote Whole-Cell Immobilization and Induce Microgranulation of Clostridium saccharoperbutylacetonicum N1-4 for Enhanced Biobutanol Production.

Immobilization is a common strategy used to protect microbial cells to improve the performance of bioprocesses. However, the interaction mechanism between the cells and the immobilization material is generally poorly understood. In this study, we employed natural polysaccharide-based materials as immobilization carriers for clostridial fermentation in an attempt to enhance the production of butanol (a valuable biofuel/biochemical but highly toxic to the host cells) and meanwhile elucidate the interaction mechanisms related to immobilization. The utilization of chitosan powder as the immobilization carrier enhanced butanol productivity by 97% in the fermentation with Clostridium saccharoperbutylacetonicum N1-4 and improved butanol titer by 21% in the fermentation with Clostridium beijerinckii NCIMB 8052. Additionally, analogue derivatives using microcrystalline cellulose (MCC) and cotton cationized on the surface with 3-chloro-2-hydroxypropyltrymethylammonium (CHPTA) and 2-chloro-N,N-diethylaminoethyl chloride (DEAEC) were prepared and used as immobilization carriers for similar fermentation conditions. The CHPTA derivatives showed slightly increased production of butanol and total solvent with C. saccharoperbutylacetonicum. Overall, our results indicated that the interaction between the cell and the carrier material occurs through a double mechanism involving adsorption immobilization and induced aggregation. This work provides insights concerning the effects of the chemical properties of the carrier material (such as the cation density and surface area) on fermentation performance, enabling a better understanding of the interaction between bacterial cells and the cationic materials. The derivatization strategies employed in this study can be applied to most cellulosic materials to modulate the properties and enhance the interaction between the cell and the carrier material for immobilization, thus improving the bioprocess performance.

Identification and Investigation of Autolysin Genes in Clostridium saccharoperbutylacetonicum Strain N1-4 for Enhanced Biobutanol Production.

Biobutanol is a valuable biochemical and one of the most promising biofuels. Clostridium saccharoperbutylacetonicum N1-4 is a hyperbutanol-producing strain. However, its strong autolytic behavior leads to poor cell stability, especially during continuous fermentation, thus limiting the applicability of the strain for long-term and industrial-scale processes. In this study, we aimed to evaluate the role of autolysin genes within the C. saccharoperbutylacetonicum genome related to cell autolysis and further develop more stable strains for enhanced butanol production. First, putative autolysin-encoding genes were identified in the strain based on comparison of amino acid sequence with homologous genes in other strains. Then, by overexpressing all these putative autolysin genes individually and characterizing the corresponding recombinant strains, four key genes were pinpointed to be responsible for significant cell autolysis activities. Further, these key genes were deleted using CRISPR-Cas9. Fermentation characterization demonstrated enhanced performance of the resultant mutants. Results from this study reveal valuable insights concerning the role of autolysins for cell stability and solvent production, and they provide an essential reference for developing robust strains for enhanced biofuel and biochemical production.IMPORTANCE Severe autolytic behavior is a common issue in Clostridium and many other microorganisms. This study revealed the key genes responsible for the cell autolysis within Clostridium saccharoperbutylacetonicum, a prominent platform for biosolvent production from lignocellulosic materials. The knowledge generated in this study provides insights concerning cell autolysis in relevant microbial systems and gives essential references for enhancing strain stability through rational genome engineering.

Enhancing the tolerance of Clostridium saccharoperbutylacetonicum to lignocellulosic-biomass-derived inhibitors for efficient biobutanol production by overexpressing efflux pumps genes from Pseudomonas putida.

Furan aldehydes and phenolic compounds generated during biomass pretreatment can inhibit fermentation for biofuel production. Efflux pumps actively transport small molecules out of cells, thus sustaining normal microbial metabolism. Pseudomonas putida has outstanding tolerance to butanol and other small molecules, and we hypothesize that its efflux pump could play essential roles for such robustness. Here, we overexpressed efflux pump genes from P. putida to enhance tolerance of hyper-butanol producing Clostridium saccharoperbutylacetonicum to fermentation inhibitors. Interestingly, overexpression of the whole unit resulted in decreased tolerance, while overexpression of the subunit (srpB) alone exerted significant enhanced robustness of the strain. Compared to the control, the engineered strain had enhanced capability to grow in media containing 17% more furfural or 50% more ferulic acid, and produced ~14 g/L butanol (comparable to fermentation under regular conditions without inhibitors). This study provided valuable reference for boosting microbial robustness towards efficient biofuel production from lignocellulosic materials.