Characterization of lignin and hemicellulose degrading bacteria isolated from cow rumen and forest soil: Unveiling a novel enzymatic model for rice straw deconstruction.

Application of greener pretreatment technology using robust ligninolytic bacteria for short duration to deconstruct rice straw and enhance bioethanol production is currently lacking. The objective of this study is to characterize three bacterial strains isolated from the milieux of cow rumen and forest soil and explore their capabilities of breaking down lignocellulose - an essential process in bioethanol production. Using biochemical and genomic analyses these strains were identified as Bacillus sp. HSTU-bmb18, Bacillus sp. HSTU-bmb19, and Citrobacter sp. HSTU-bmb20. Genomic analysis of the strains unveiled validated model hemicellulases, multicopper oxidases, and pectate lyases. These enzymes exhibited interactions with distinct lignocellulose substrates, further affirmed by their stability in molecular dynamic simulations. A comprehensive expression of ligninolytic pathways, including ß-ketoadipate, phenyl acetate, and benzoate, was observed within the HSTU-bmb20 genome. The strains secreted approximately 75-82 U/mL of cellulase, xylase, pectinase, and lignin peroxidase. FT-IR analysis of the bacterial treated rice straw fibers revealed that the intensity of lignin-related peaks decreased, while cellulose-related peaks sharpened. The values of crystallinity index for the untreated control and the treated rice straw with either HSTU-bmb18, or HSTU-bmb19, or HSTU-bmb20 were recorded to be 34.48, 28.49, 29.36, 31.75, respectively, which are much higher than that of 13.53 noted for those treated with the bacterial consortium. The ratio of fermentable cellulose in rice straw increased by 1.25-, 1.79-, 1.93- and 2.17-fold following treatments with HSTU-bmb18, HSTU-bmb20, HSTU-bmb19, and a mixed consortium of these three strains, respectively. These aggregative results suggested a novel model for rice straw deconstruction utilizing hydrolytic enzymes of the consortium, revealing superior efficacy compared to individual strains, and advancing cost-effective, affordable, and sustainable green technology.

Characterization of Growth-Promoting Activities of Consortia of Chlorpyrifos Mineralizing Endophytic Bacteria Naturally Harboring in Rice Plants-A Potential Bio-Stimulant to Develop a Safe and Sustainable Agriculture.

Eighteen pesticide-degrading endophytic bacteria were isolated from the roots, stems, and leaves of healthy rice plants and identified through 16S rRNA gene sequencing. Furthermore, biochemical properties, including enzyme production, dye degradation, anti-bacterial activities, plant-growth-promoting traits, including N-fixation, P-solubilization, auxin production, and ACC-deaminase activities of these naturally occurring endophytic bacteria along with their four consortia, were characterized. Enterobacter cloacae HSTU-ABk39 and Enterobacter sp. HSTU-ABk36 displayed inhibition zones of 41.5 ± 1.5 mm, and 29 ± 09 mm against multidrug-resistant human pathogenic bacteria Staphylococcus aureus and Staphylococcus epidermidis, respectively. FT-IR analysis revealed that all eighteen isolates were able to degrade chlorpyrifos pesticide. Our study confirms that pesticide-degrading endophytic bacteria from rice plants play a key role in enhancing plant growth. Notably, rice plants grown in pots containing reduced urea (30%) mixed with either endophytic bacterial consortium-1, consortium-2, consortium-3, or consortia-4 demonstrated an increase of 17.3%, 38.6%, 18.2%, and 39.1% yields, respectively, compared to the control plants grown in pots containing 100% fertilizer. GC-MS/MS analysis confirmed that consortia treatment caused the degradation of chlorpyrifos into different non-toxic metabolites, including 2-Hydroxy-3,5,6 trichloropyridine, Diethyl methane phosphonate, Phorate sulfoxide, and Carbonochloridic. Thus, these isolates could be deployed as bio-stimulants to improve crop production by creating a sustainable biological system.

Enhanced rice plant (BRRI-28) growth at lower doses of urea caused by diazinon mineralizing endophytic bacterial consortia and explorations of relevant regulatory genes in a Klebsiella sp. strain HSTU-F2D4R.

Endophytic biostimulant with pesticide bioremediation activities may reduce agrochemicals application in rice cultivation. The present study evaluates diazinon-degrading endophytic bacteria, isolated from rice plants grown in the fields with pesticide amalgamation, leading to increased productivity in high-yielding rice plants. These endophytes showed capabilities of decomposing diazinon, confirmed by FT-IR spectra analysis. Growth promoting activities of these endophytes can be attributed to their abilities to produce an increased level of IAA content and to demonstrate high level ACC-deaminase activities. Furthermore, these endophytes demonstrated enhanced level of extracellular cellulase, xylanase, amylase, protease and lignin degrading activities. Five genera including Enterobacter, Pantoea, Shigella, Acinetobacter, and Serratia, are represented only by the leaves, while four genera such as Enterobacter, Escherichia, Kosakonia, and Pseudomonas are represented only by the shoots. Five genera including, Klebsiella, Enterobacter, Pseudomonas, Burkholderia, and Bacillus are represented only by the roots of rice plants. All these strains demonstrated cell wall hydrolytic enzyme activities, except pectinase. All treatments, either individual strains or consortia of strains, enhanced rice plant growth at germination, seedling, vegetative and reproductive stages. Among four (I-IV) consortia, consortium-III generated the maximum rice yield under 70% lower doses of urea compared to that of control (treated with only fertilizer). The decoded genome of Klebsiella sp. HSTU-F2D4R revealed nif-cluster, chemotaxis, phosphates, biofilm formation, and organophosphorus insecticide-degrading genes. Sufficient insecticide-degrading proteins belonging to strain HSTU-F2D4R had interacted with diazinon, confirmed in molecular docking and formed potential catalytic triads, suggesting the strains have bioremediation potential with biofertilizer applications in rice cultivation.

Unveiling lignocellulolytic trait of a goat omasum inhabitant Klebsiella variicola strain HSTU-AAM51 in light of biochemical and genome analyses.

Klebsiella variicola is generally known as endophyte as well as lignocellulose-degrading strain. However, their roles in goat omasum along with lignocellulolytic genetic repertoire are not yet explored. In this study, five different pectin-degrading bacteria were isolated from a healthy goat omasum. Among them, a new Klebsiella variicola strain HSTU-AAM51 was identified to degrade lignocellulose. The genome of the HSTU-AAM51 strain comprised 5,564,045 bp with a GC content of 57.2% and 5312 coding sequences. The comparison of housekeeping genes (16S rRNA, TonB, gyrase B, RecA) and whole-genome sequence (ANI, pangenome, synteny, DNA-DNA hybridization) revealed that the strain HSTU-AAM51 was clustered with Klebsiella variicola strains, but the HSTU-AAM51 strain was markedly deviated. It consisted of seventeen cellulases (GH1, GH3, GH4, GH5, GH13), fourteen beta-glucosidase (2GH3, 7GH4, 4GH1), two glucosidase, and one pullulanase genes. The strain secreted cellulase, pectinase, and xylanase, lignin peroxidase approximately 76-78 U/mL and 57-60 U/mL, respectively, when it was cultured on banana pseudostem for 96 h. The catalytically important residues of extracellular cellulase, xylanase, mannanase, pectinase, chitinase, and tannase proteins (validated 3D model) were bound to their specific ligands. Besides, genes involved in the benzoate and phenylacetate catabolic pathways as well as laccase and DiP-type peroxidase were annotated, which indicated the strain lignin-degrading potentiality. This study revealed a new K. variicola bacterium from goat omasum which harbored lignin and cellulolytic enzymes that could be utilized for the production of bioethanol from lignocelluloses.

Neither glutamate nor alanine but arginine sensitizes BY-2 cells to arsenate.

Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

Guard Cell Salicylic Acid Signaling Is Integrated into Abscisic Acid Signaling via the Ca2+/CPK-Dependent Pathway.

The phenolic hormone salicylic acid (SA) induces stomatal closure. It has been suggested that SA signaling is integrated with abscisic acid (ABA) signaling in guard cells, but the integration mechanism remains unclear. The Ca2+-independent protein kinase Open Stomata1 (OST1) and Ca2+-dependent protein kinases (CPKs) are key for ABA-induced activation of the slow-type anion channel SLAC1 and stomatal closure. Here, we show that SA-induced stomatal closure and SA activation of slow-type anion channel are impaired in the CPK disruption mutant cpk3-2 cpk6-1 but not in the OST1 disruption mutant ost1-3 We also found that the key phosphorylation sites of SLAC1 in ABA signaling, serine-59 and serine-120, also are important for SA signaling. Chemiluminescence-based detection of superoxide anion revealed that SA did not require CPK3 and CPK6 for the induction of reactive oxygen species production. Taken together, our results suggest that SA activates peroxidase-mediated reactive oxygen species signal that is integrated into Ca2+/CPK-dependent ABA signaling branch but not the OST1-dependent signaling branch in Arabidopsis (Arabidopsis thaliana) guard cells.

Exogenous proline enhances the sensitivity of Tobacco BY-2 cells to arsenate.

Arsenic causes physiological and structural disorders in plants. Proline is accumulated as a compatible solute in plants under various stress conditions and mitigates stresses. Here, we investigated the effects of exogenous proline on tobacco Bright Yellow-2 (BY-2) cultured cells under [Formula: see text] stress. Arsenate did not inhibit BY-2 cell growth at 40 and 50 µM but did it at 60 µM. Proline at 0.5 to 10 mM did not affect the cell growth but delayed it at 20 mM. At 40 µM [Formula: see text], neither 0.5 mM nor 1 mM proline affected the cell growth but 10 mM proline inhibited it. In the presence of [Formula: see text], 10 mM proline increased the number of Evans Blue-stained (dead) cells and decreased the number of total cells. Together, our results suggest that exogenous proline does not alleviate arsenate toxicity but enhances the sensitivity of BY-2 cells to arsenate.

Chitosan signaling in guard cells requires endogenous salicylic acid.

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.