[Effects of Different Microbial Fertilizers on Soil Quality and Maize Yield in Coastal Saline Soil].

Microbial fertilizers have the characteristics of high efficiency and environmental protection in improving saline soils, and the application of functional microbial fertilizers is of great significance for the green abatement of saline barriers and the improvement of soil quality in coastal areas. The experiment was based on moderately saline soil in the coastal area of Hebei Province, with corn as the indicator crop, on the basis of conventional chemical fertilizer application. Different microbial fertilizer treatments, namely, T1 （conventional chemical fertilizer 750 kg·hm-2 + compound microbial agent 75 kg·hm-2）, T2 （conventional chemical fertilizer 750 kg·hm-2 + Bacillus megaterium 300 kg·hm-2）, T3 （conventional chemical fertilizer 750 kg·hm-2 + B. mucilaginosus 300 kg·hm-2）, T4 （conventional chemical fertilizer 750 kg·hm-2 + organic silicon fertilizer 600 kg·hm-2）, T5 （conventional chemical fertilizer 750 kg·hm-2 + bio-organic fertilizer 600 kg·hm-2）, T6 （conventional fertilizer 750 kg·hm-2 + active microalgae 15 kg·hm-2）, and CK （only fertilizer 750 kg·hm-2）, were used for these seven treatments, to study the effects of different microbial fertilizers on soil nutrients, salinity, bacterial community, and corn yield and economic efficiency during two critical periods （V12 stage and maturity stage） of corn. The results showed that compared with that in CK, T1 significantly increased soil total nitrogen （TN） and available phosphorus （AP） contents during the whole growth period. Over the whole reproductive period, soil organic matter （OM） at maturity increased by 10.35% over the V12 stage compared to that in CK, but there was no significant difference between treatments. Compared with that in CK, T5 and T6 significantly reduced soil total salinity and Ca2+ content during the whole growth period by an average of 14.51%-18.48% and 24.25%-25.51%. T1 significantly increased the bacterial diversity index over the whole growth period by 45.16% compared to that in CK. The dominant soil phyla were Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi, and the dominant genera were Bacillus and Geminicoccaceae. The most abundant functions of the bacterial community in the study area were chemoheterotrophy and aerobic chemoheterotrophy, with average relative abundances of 28.89% and 27.11%, and T3 and T6 significantly improved soil N cycling function. The results of redundancy analysis （RDA） indicated that Na+, SO42-, pH, and EC were important factors driving the structure of the bacterial community, and correlation heatmaps showed that Na+, SO42-, pH, and EC were significantly and positively correlated mainly with the phylum Planctomycetota, whereas soil OM and TN were significantly and positively correlated with Cyanobacteria. Compared with that in CK, T6 increased the relative abundance of Cyanobacteria and optimized the bacterial community structure during the whole growth period. Using recommended dosages of bacterial fertilizers T1 and T6 increased maize yield by 7.31%-24.83% and economic efficiency by 9.05%-23.23%, respectively. The preliminary results of soil chemical properties and yield correlation analysis revealed that EC, AP, HCO3-, and Mg2+ were the obstacle factors limiting soil productivity in coastal areas. In conclusion, the use of the compound bacterial agent （T1） and active microalgae （T6） at the recommended dosage can significantly enhance soil nutrients, reduce salinity, and improve the structural diversity of soil bacterial communities, which not only ensures the increase in maize yield and efficiency but also realizes the efficient use of microbial fertilizers and the improvement of soil quality.

Protein Gas Vesicles of Bacillus megaterium as Enhancers of Ultrasound-Induced Transcriptional Regulation.

Gas vesicles (GVs) are large cylindrical gas-filled protein assemblies found in diverse aquatic bacteria that enable their adaptation of buoyancy. GVs have already been used as ultrasound contrasting agents. Here, we investigate GVs derived from Bacillus megaterium, aiming to minimize the number of accessory Gvps within the GV gene cluster and demonstrate the use of GVs as enhancers of acoustic radiation force administered by ultrasound. Three (GvpR, GvpT, and GvpU) out of 11 genes in the cluster were found to be dispensable for functional GV formation, and their omission resulted in narrower GVs. Two essential proteins GvpJ and GvpN were absent from recently determined GV structures, but GvpJ was nevertheless found to be tightly bound to the cylindrical part of GVs in this study. Additionally, the N-terminus of GvpN was observed to play an important role in the formation of mature GVs. The binding of engineered GvpC fromAnabaena flos-aquae to HEK293 cells via integrins enhanced the acoustic force delivered by ultrasound and resulted in an increased Ca2+ influx into cells. Coupling with a synthetic Ca2+-dependent signaling pathway GVs efficiently enhanced cell stimulation by ultrasound, which expands the potentials of noninvasive sonogenetics cell stimulation.

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis.

BACKGROUND: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. RESULTS: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration. CONCLUSION: Recombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Efficacy of DAP coated with bacterial strains and their metabolites for soil phosphorus availability and maize growth.

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P2O5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.

The role and mechanism of Bacillus megaterium strain A14 in inhibiting the cadmium uptake by peanut plants in acidic red soil.

AIMS: This study explores the potential of cadmium (Cd)-resistant bacteria, specifically Bacillus megaterium A14, to decrease Cd accumulation in peanuts, a crop susceptible to metal uptake from contaminated soils, by understanding the bacterium's impact on plant Cd absorption mechanisms. METHODS AND RESULTS: Through pot experiments, we observed that A14 inoculation significantly increased peanut biomass under Cd stress conditions, primarily by immobilizing the metal and reducing its bioavailability. The bacterium effectively changed the distribution of Cd, with a notable 46.53% reduction in the exchangeable fraction, which in turn limited the expression of genes related to Cd transport in peanuts. Additionally, A14 enhanced the plant's antioxidant response, improving its tolerance to stress. Microbial analysis through 16S sequencing demonstrated that A14 inoculation altered the peanut rhizosphere, particularly by increasing populations of Firmicutes and Proteobacteria, which play crucial roles in soil remediation from heavy metals. CONCLUSION: The A14 strain effectively counters Cd toxicity in peanuts, promoting growth through soil Cd sequestration, root barrier biofilm formation, antioxidant system enhancement, suppression of Cd transport genes, and facilitation of Cd-remediating microorganisms.

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Production and secretion of recombinant spider silk in Bacillus megaterium.

BACKGROUND: Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. RESULTS: In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. CONCLUSION: It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Dissolving mechanism of strain P17 on insoluble phosphorus of yellow-brown soil

Strain P17 was a bacterial strain identified as Bacillus megaterium isolated from ground accumulating phosphate rock powder. The fermentation broth of strain P17 and the yellow-brown soil from Nanjing Agricultural University garden were collected to conduct this study. The simulation of fixed insoluble phosphorous forms after applying calcium superphosphate into yellow-brown soil was performed in pots, while available P and total P of soil were extremely positive correlative with those of groundwater. Then the dissolving effect of strain P17 on insoluble P of yellow-brown soil was studied. Results showed that Bacillus megaterium strain P17 had notable solubilizing effect on insoluble phosphates formed when too much water-soluble phosphorous fertilizer used. During 100 days after inoculation, strain P17 was dominant. Until the 120th day, compared with water addition, available P of strain P17 inoculation treated soil increased by 3 times with calcium superphosphate addition. Besides available P, pH, activity of acid and alkaline phosphatase and population of P-solubilizing microbes were detected respectively. P-solubilizing mechanism of P-solubilizing bacteria strain P17 seems to be a synergetic effect of pH decrease, organic acids, phosphatase, etc.

Enriquecimiento en mutantes de Bacillus megaterium deficientes en la síntesis de poli-ß-hidroxibutirato (PHB) / Enrichment for mutants of Bacillus megaterium deficient in the synthesis of poly-ß-hydroxybutyrate (PHB)

La centrifugación en gradientes de sacarosa permite la separación de células según su densidad boyante de acuerdo al contenido en poli-ß-hidroxibutirato (PHB). En este trabajo este método se evaluó y se adaptó para detectar mutantes deficientes en la síntesis de PHB de B. megaterium analizando un bajo porcentaje de la población mutagenizada (AU)

Enriquecimiento en mutantes de Bacillus megaterium deficientes en la síntesis de poli-ß-hidroxibutirato (PHB) / Enrichment for mutants of Bacillus megaterium deficient in the synthesis of poly-ß-hydroxybutyrate (PHB)

La centrifugación en gradientes de sacarosa permite la separación de células según su densidad boyante de acuerdo al contenido en poli-ß-hidroxibutirato (PHB). En este trabajo este método se evaluó y se adaptó para detectar mutantes deficientes en la síntesis de PHB de B. megaterium analizando un bajo porcentaje de la población mutagenizada