Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB.

In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to finetune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.

Integrated conjugal plasmid pLS20 in the Bacillus subtilis genome produced 850-kbp circular subgenomes transmissible to another B. Subtilis.

Bacillus subtilis was engineered to produce circular subgenomes that are directly transmittable to another B. subtilis. The conjugational plasmid pLS20 integrated into the B. subtilis genome supported not only subgenome replication but also transmission to another B. subtilis species. The subgenome system developed in this study completes a streamlined platform from the synthesis to the transmission of giant DNA by B. subtilis.

Bacillus subtilis stress-associated mutagenesis and developmental DNA repair.

SUMMARYThe metabolic conditions that prevail during bacterial growth have evolved with the faithful operation of repair systems that recognize and eliminate DNA lesions caused by intracellular and exogenous agents. This idea is supported by the low rate of spontaneous mutations (10-9) that occur in replicating cells, maintaining genome integrity. In contrast, when growth and/or replication cease, bacteria frequently process DNA lesions in an error-prone manner. DNA repairs provide cells with the tools needed for maintaining homeostasis during stressful conditions and depend on the developmental context in which repair events occur. Thus, different physiological scenarios can be anticipated. In nutritionally stressed bacteria, different components of the base excision repair pathway may process damaged DNA in an error-prone approach, promoting genetic variability. Interestingly, suppressing the mismatch repair machinery and activating specific DNA glycosylases promote stationary-phase mutations. Current evidence also suggests that in resting cells, coupling repair processes to actively transcribed genes may promote multiple genetic transactions that are advantageous for stressed cells. DNA repair during sporulation is of interest as a model to understand how transcriptional processes influence the formation of mutations in conditions where replication is halted. Current reports indicate that transcriptional coupling repair-dependent and -independent processes operate in differentiating cells to process spontaneous and induced DNA damage and that error-prone synthesis of DNA is involved in these events. These and other noncanonical ways of DNA repair that contribute to mutagenesis, survival, and evolution are reviewed in this manuscript.

Bacillus phage phi18-2 is a novel temperate virus with an unintegrated genome present in the cytoplasm of lysogenic cells as a linear phage-plasmid.

Bacillus subtilis is a Gram-positive bacterium that is widely used in fermentation and in the pharmaceutical industry. Phage contamination occasionally occurs in various fermentation processes and causes significant economic loss. Here, we report the isolation and characterization of a temperate B. subtilis phage, termed phi18-2, from spore powder manufactured in a fermentation plant. Transmission electron microscopy showed that phi18-2 has a symmetrical polyhedral head and a long noncontractile tail. Receptor analysis showed that phi18-2 recognizes wall teichoic acid (WTA) for infection. The phage virions have a linear double-stranded DNA genome of 64,467 bp with identical direct repeat sequences of 309 bp at each end of the genome. In lysogenic cells, the phage genome was found to be present in the cytoplasm without integration into the host cell chromosome, and possibly as a linear phage-plasmid with unmodified ends. Our data may provide some insight into the molecular basis of the unique lysogenic cycle of phage phi18-2.

Expression of Multicopy Tandem Recombinant Ginseng Hexapeptide in Bacillus subtilis and the Evaluation of Antiaging Activity.

Ginseng oligopeptides are naturally occurring small-molecule peptides extracted from ginseng that exhibit positive effects on health and longevity. However, the current industrial production of ginseng oligopeptides primarily relies on plant extraction and chemical synthesis. In this study, we proposed a novel genetic engineering approach to produce active ginseng peptides through multicopy tandem insertion (5 and 15 times). The recombinant ginseng peptides were successfully produced from engineered Bacillus subtilis with an increasing yield from 356.55 to 2900 mg/L as the repeats multiple. Additionally, an oxidative stress-induced aging model caused by H2O2 was established to evaluate whether the recombinant ginseng peptides, without enzymatic hydrolysis into individual peptides, also have positive effects on antiaging. The results demonstrated that all two kinds of recombinant ginseng peptides could also delay cellular aging through various mechanisms, such as inhibiting cell cycle arrest, suppressing the expression of pro-inflammatory factors, and enhancing cellular antioxidant capacity.

Antimicrobial Activity of Polycaprolactone Nanofiber Coated with Lavender and Neem Oil Nanoemulsions against Airborne Bacteria.

The development of efficient, eco-friendly antimicrobial agents for air purification and disinfection addresses public health issues connected to preventing airborne pathogens. Herein, the antimicrobial activity of a nanoemulsion (control, 5%, 10%, and 15%) containing neem and lavender oils with polycaprolactone (PCL) was investigated against airborne bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Various parameters such as the physicochemical properties of the nanoemulsion, pH, droplet size, the polydispersity index (PDI), the minimum inhibitory concentration (MIC), the minimum bacterial concentration (MBC), and the color measurement of the emulsion have been evaluated and optimized. Our results showed that the antimicrobial activity of PCL combined with neem and lavender oil was found to be the highest MIC and MBC against all tested bacteria. The droplet sizes for lavender oil are 21.86-115.15 nm, the droplet sizes for neem oil are 23.92-119.15 nm, and their combination is 25.97-50.22 nm. The range of pH and viscosity of nanoemulsions of various concentrations was found to be 5.8 to 6.6 pH and 0.372 to 2.101 cP. This study highlights the potential of nanotechnology in harnessing the antimicrobial properties of natural essential oils, paving the way for innovative and sustainable solutions in the fight against bacterial contamination.

Insight into effects of terbium on cell growth, sporulation and spore properties of Bacillus subtilis.

Recovery of rare earth elements (REEs) from wastewater with Bacillus subtilis (B. subtilis) during culture is promising due to its environmental benefits. However, the effects of REEs in the culture media on B. subtilis are poorly understood. This study aims to investigate the effects of the terbium (Tb(III)), a typical rare earth element, on the cell growth, sporulation, and spore properties of B. subtilis. Tb(III) can suppress bacterial growth while enhancing spore tolerance to wet heat. Spore germination and content of dipicolinic acid (DPA) were promoted at low concentrations of Tb(III) while inhibited at a high level, but an inverse effect on initial sporulation appeared. Scanning electron microscope and energy dispersive spectrometer detection indicated that Tb(III) complexed cells or spores and certain media components simultaneously. The germination results of the spores after elution revealed that Tb(III) attached to the spore surface was a key effector of spore germination. In conclusion, Tb(III) directly or indirectly regulated both the nutrient status of the media and certain metabolic events, which in turn affected most of the properties of B. subtilis. Compared to the coat-deficient strain, the wild-type strain grew faster and was more tolerant to Tb(III), DPA, and wet heat, which in turn implied that it was more suitable for the recovery of REEs during cultivation. These findings provide fundamental insights for the recovery of rare earths during the culture process using microorganisms.

B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.

Different levels of single-strain probiotic (Bacillus subtilis) with proteolytic enzyme (serratiopeptidase) can be used as an alternative to antibiotic growth promoters in broiler.

In the current study, the proteolytic enzyme (serratiopeptidase) was used to enhance the efficacy of Bacillus subtilis (B. subtilis) probiotic as a growth promotor in broiler chicken. The effects of serratiopeptidase on the efficacy of different levels of B. subtilis as a growth promotor in broiler chicks were evaluated regarding growth performance traits, villus histomorphometric characterization, and intestinal microbiota count. Day-old broiler chicks (n = 120) were allocated into 4 groups having 3 replicates/group. In the control group (C), the basal diet was kept without supplementation. In treatment groups (P100, P150, and P200), the basal diet was supplemented with 100, 150, and 200 mg probiotics, respectively besides 30 mg proteolytic enzyme in the 3 treated groups for 4 wk. The performance parameters were significantly affected by the supplementation of serratiopeptidase to the B. subtilis treatment groups. Feed intake (FI), body weight gain (WG), feed conversion ratio (FCR), and dressing percent were significantly improved in the treatment groups as compared to the control group. Significantly, the lowest feed intake was recorded for the P200 group. The highest body weight gain and dressing percentage were recorded for the P200 group. An improved FCR was recorded in the P200 group (1.7) as compared to the control group. The different levels of B. subtilis supplemented with serratiopeptidase revealed significant improvements (P<0.05) in the morphology of the intestine by showing increases in villus height and width and crypt depth of the small intestine. The microbial count revealed that E. coli and salmonella colonies were significantly reduced in the P200 group as compared to the control and other treatment groups. In conclusion, the supplementation of B. subtilis with serratiopeptidase as a growth promoter in broiler chicks significantly improved the overall performance, and intestinal health and reduced microbial load contributing to optimizing the performance of broiler chickens. The greatest improvement was observed in the P200 group fed with B. subtilis as a probiotic and serratiopeptidase enzyme (200 mg:30 mg).

Investigating macroecological patterns in coarse-grained microbial communities using the stochastic logistic model of growth.

The structure and diversity of microbial communities are intrinsically hierarchical due to the shared evolutionary history of their constituents. This history is typically captured through taxonomic assignment and phylogenetic reconstruction, sources of information that are frequently used to group microbes into higher levels of organization in experimental and natural communities. Connecting community diversity to the joint ecological dynamics of the abundances of these groups is a central problem of community ecology. However, how microbial diversity depends on the scale of observation at which groups are defined has never been systematically examined. Here, we used a macroecological approach to quantitatively characterize the structure and diversity of microbial communities among disparate environments across taxonomic and phylogenetic scales. We found that measures of biodiversity at a given scale can be consistently predicted using a minimal model of ecology, the Stochastic Logistic Model of growth (SLM). This result suggests that the SLM is a more appropriate null-model for microbial biodiversity than alternatives such as the Unified Neutral Theory of Biodiversity. Extending these within-scale results, we examined the relationship between measures of biodiversity calculated at different scales (e.g. genus vs. family), an empirical pattern previously evaluated in the context of the Diversity Begets Diversity (DBD) hypothesis (Madi et al., 2020). We found that the relationship between richness estimates at different scales can be quantitatively predicted assuming independence among community members, demonstrating that the DBD can be sufficiently explained using the SLM as a null model of ecology. Contrastingly, only by including correlations between the abundances of community members (e.g. as the consequence of interactions) can we predict the relationship between estimates of diversity at different scales. The results of this study characterize novel microbial patterns across scales of organization and establish a sharp demarcation between recently proposed macroecological patterns that are not and are affected by ecological interactions.

Structural assembly of the bacterial essential interactome.

The study of protein interactions in living organisms is fundamental for understanding biological processes and central metabolic pathways. Yet, our knowledge of the bacterial interactome remains limited. Here, we combined gene deletion mutant analysis with deep-learning protein folding using AlphaFold2 to predict the core bacterial essential interactome. We predicted and modeled 1402 interactions between essential proteins in bacteria and generated 146 high-accuracy models. Our analysis reveals previously unknown details about the assembly mechanisms of these complexes, highlighting the importance of specific structural features in their stability and function. Our work provides a framework for predicting the essential interactomes of bacteria and highlight the potential of deep-learning algorithms in advancing our understanding of the complex biology of living organisms. Also, the results presented here offer a promising approach to identify novel antibiotic targets.

Effects of bacterial direct-fed microbial mixtures offered to beef cattle consuming finishing diets on intake, nutrient digestibility, feeding behavior, and ruminal kinetics/fermentation profile.

Effects of bacterial direct-fed microbial (DFM) mixtures on intake, nutrient digestibility, feeding behavior, ruminal fermentation profile, and ruminal degradation kinetics of beef steers were evaluated. Crossbred Angus ruminally cannulated steers (n = 6; body weight [BW] = 520 ±â€…30 kg) were used in a duplicated 3 × 3 Latin square design and offered a steam-flaked corn-based finisher diet to ad libitum intake for 3, 28-d periods. Treatments were 1) Control (no DFM, lactose carrier only); 2) Treat-A (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis), at 1:1:1:3 ratio, respectively; totaling 6 × 109 CFU (50 mg)/animal-daily minimum; and 3) Treat-B, the same DFM combination, but doses at 1:1:3:1 ratio. Bacterial counts were ~30% greater than the minimum expected. Data were analyzed using the GLIMMIX procedure of SAS with the model including the fixed effect of treatment and the random effects of square, period, and animal (square). For repeated measure variables, the fixed effects of treatment, time, and their interaction, and the random effects of square, period, animal (square), and animal (treatment) were used. Preplanned contrasts comparing Control × Treat-A or Treat-B were performed. Intake and major feeding behavior variables were not affected (P ≥ 0.17) by treatments. Steers offered Treat-A had an increased (P = 0.04) ADF digestibility compared with Control. Steers offered Treat-A experienced daily 300 min less (P = 0.04) time under ruminal pH 5.6, a greater (P = 0.04) ruminal pH average and NH3-N concentration (P = 0.05) and tended (P = 0.06) to have a lower ruminal temperature compared to Control. Ruminal VFA was not affected (P ≥ 0.38) by treatments. Steers offered Treat-A increased (P = 0.02) and tended (P = 0.08) to increase the ruminal effective degradable NDF and ADF fractions of the diet-substrate, respectively. When the forage-substrate (low quality) was incubated, steers offered Treat-A tended (P = 0.09) to increase the effective degradable hemicellulose fraction compared to Control. In this experiment, the bacterial combinations did not affect intake and feeding behavior, while the combination with a greater proportion of B. licheniformis (Treat-A) elicited an improved core-fiber digestibility and a healthier ruminal pH pattern, in which the ruminal environment showed to be more prone to induce the effective degradability of fiber fractions, while also releasing more NH3-N.

During the finishing phase, a high-energy diet offers benefits related to beef cattle growth and development. However, it is essential to acknowledge that finisher diets are energy-dense and can pose digestive challenges, such as subacute ruminal acidosis. Digestive disturbances negatively affect animal well-being, growth performance, and economic returns. To address digestive challenges endured by animals on high-energy diets, the current experiment focused on the addition of bacterial direct-fed microbial (DFM) mixtures. A unique combination of bacterial DFM containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis was evaluated. These bacteria have been individually reported to improve cattle nutrient utilization, digestibility, ruminal function, and maintain ruminal pH. The study aimed to investigate the effects of this specific microbial combination and doses when added to beef cattle finisher diets. The DFM mixtures offered seemed to not affect intake and major feeding behavior variables. The DFM combination containing a greater proportion of B. licheniformis (Treat-A) seemed to elicit an improved total tract core-fiber digestibility, and a safer ruminal pH pattern. The ruminal environment was shown to be more prone to improve the ruminal effective degradability of fiber fractions, while also releasing more NH3­N.

Mechanical constraints to unbound expansion of B. subtilis on semi-solid surfaces.

IMPORTANCE: How bacterial cells colonize new territory is a problem of fundamental microbiological and biophysical interest and is key to the emergence of several phenomena of biological, ecological, and medical relevance. Here, we demonstrate how bacteria stuck in a colony of finite size can resume exploration of new territory by aquaplaning and how they fine tune biofilm viscoelasticity to surface material properties that allows them differential mobility. We show how changing local interfacial forces and colony viscosity results in a plethora of bacterial morphologies on surfaces with different physical and mechanical properties.

Ultrasound-assisted fermentation for antioxidant peptides preparation from okara: Optimization, stability, and functional analyses.

This study investigated the effects of ultrasound-assisted fermentation (UAF) on the preparation of antioxidant peptides (UAFP) from okara and examined their content, chemical structures, and antioxidant activity. After the optimal ultrasonic processing (time, 20 min; frequency, 45 KHz; power, 120 W/L), the peptide content yield reached the maximum of 12.36 ± 0.02 mg/mL, and their DPPH free radical scavenging rate was 65.15 ± 0.32 %. UAF increased the number of globular aggregates with deeper gullies, a looser structure, and higher porosity. The experiments conducted using the oxidative stress injury model of HepG2 cells showed that okara UAFP promoted cell growth and exerted a protective effect. Moreover, ultrasonic treatment remarkably improved the environmental stability (NaCl, glucose, sodium benzoate, temperature, pH, metal ions) and antioxidant activity of UAFP. Concisely, optimal ultrasonic processing can aid the fermentation of agroindustrial by-products to prepare antioxidant peptides, such as natural food antioxidant peptides from soybean waste.

Engineered Bacillus subtilis Biofilm@Biochar living materials for in-situ sensing and bioremediation of heavy metal ions pollution.

The simultaneous sensing and remediation of multiple heavy metal ions in wastewater or soil with microorganisms is currently a significant challenge. In this study, the microorganism Bacillus subtilis was used as a chassis organism to construct two genetic circuits for sensing and adsorbing heavy-metal ions. The engineered biosensor can sense three heavy metal ions (0.1-75 µM of Pb2+ and Cu2+, 0.01-3.5 µM of Hg2+) in situ real-time with high sensitivity. The engineered B. subtilis TasA-metallothionein (TasA-MT) biofilm can specifically adsorb metal ions from the environment, exhibiting remarkable removal efficiencies of 99.5% for Pb2+, 99.9% for Hg2+and 99.5% for Cu2+ in water. Furthermore, this engineered strain (as a biosensor and absorber of Pb2+, Cu2+, and Hg2+) was incubated with biochar to form a hybrid biofilm@biochar (BBC) material that could be applied in the bioremediation of heavy metal ions. The results showed that BBC material not only significantly reduced exchangeable Pb2+ in the soil but also reduced Pb2+ accumulation in maize plants. In addition, it enhanced maize growth and biomass. In conclusion, this study examined the potential applications of biosensors and hybrid living materials constructed using sensing and adsorption circuits in B. subtilis, providing rapid and cost-effective tools for sensing and remediating multiple heavy metal ions (Pb2+, Hg2+, and Cu2+).

Marvels of Bacilli in soil amendment for plant-growth promotion toward sustainable development having futuristic socio-economic implications.

Microorganisms are integral components of ecosystems, exerting profound impacts on various facets of human life. The recent United Nations General Assembly (UNGA) Science Summit emphasized the critical importance of comprehending the microbial world to address global challenges, aligning with the United Nations Sustainable Development Goals (SDGs). In agriculture, microbes are pivotal contributors to food production, sustainable energy, and environmental bioremediation. However, decades of agricultural intensification have boosted crop yields at the expense of soil health and microbial diversity, jeopardizing global food security. To address this issue, a study in West Bengal, India, explored the potential of a novel multi-strain consortium of plant growth promoting (PGP) Bacillus spp. for soil bioaugmentation. These strains were sourced from the soil's native microbial flora, offering a sustainable approach. In this work, a composite inoculum of Bacillus zhangzhouensis MMAM, Bacillus cereus MMAM3), and Bacillus subtilis MMAM2 were introduced into an over-exploited agricultural soil and implications on the improvement of vegetative growth and yield related traits of Gylcine max (L) Meril. plants were evaluated, growing them as model plant, in pot trial condition. The study's findings demonstrated significant improvements in plant growth and soil microbial diversity when using the bacterial consortium in conjunction with vermicompost. Metagenomic analyses revealed increased abundance of many functional genera and metabolic pathways in consortium-inoculated soil, indicating enhanced soil biological health. This innovative bioaugmentation strategy to upgrade the over-used agricultural soil through introduction of residual PGP bacterial members as consortia, presents a promising path forward for sustainable agriculture. The rejuvenated patches of over-used land can be used by the small and marginal farmers for cultivation of resilient crops like soybean. Recognizing the significance of multi-strain PGP bacterial consortia as potential bioinoculants, such technology can bolster food security, enhance agricultural productivity, and mitigate the adverse effects of past agricultural activities.

Increased Expression Levels of Thermophilic Serine Protease TTHA0724 through Signal Peptide Screening in Bacillus subtilis and Applications of the Enzyme.

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications.

Effect of a Bacillus-Based Probiotic on Performance and Nutrient Digestibility When Substituting Soybean Meal with Rapeseed Meal in Grower-Finisher Diets.

The objective of the present study was to test the hypothesis of B. subtilis and B. licheniformis supplementation to a negative control diet in comparison to a standard control diet, had the potential to improve the performance and nutrient digestibility of growing-finishing pigs. For this purpose, 384 fattening pigs of 85 d of age were allotted to three treatments: a standard diet, a negative control (NC) diet (5% soybean meal replaced by 5% rapeseed meal), or a NC diet + probiotic. After reaching a body weight of approximately 110 kg, all animals going to the slaughterhouse (87% of total pigs) were selected to measure carcass quality. Moreover, the apparent total tract digestibility of protein was evaluated at the end of the grower period. The results of this study indicate that supplementation of the tested Bacillus-based probiotic significantly improved average daily gain (ADG, +14.6%) and Feed:gain ratio (F:G, -9.9%) during the grower phase compared to the NC diet. The improvement observed during the grower phase was maintained for the whole fattening period (ADG, +3.9%). Probiotic supplementation significantly improved the total apparent faecal digestibility of dry matter and crude protein in pigs at the end of the grower period. The improvements observed with the additive tested could indicate that supplementation of the Bacillus-based probiotic was able to counteract the lower level of crude protein and standardised ileal digestible amino acids in the NC diet by means of improved protein digestibility.

Evaluation of the lead and chromium removal capabilities of Bacillus subtilis-induced food waste compost-based biomedia.

Food waste compost (FWC) is a sustainable recycling approach employed in soil media, offering extensive advantages to urban areas by promoting resource circulation and effectively managing water pollution. To improve value, Bacillus subtilis (B. subtilis)-induced FWC-based biomedia (BIBMFWCs) was produced via a secondary treatment involving selective meso-thermophilic stages. During the production of BIBMFWCs, physicochemical properties were found to have favorable characteristics for the efficient removal of metal ions. The produced organic-carbonate complex structure demonstrated the synergistic effect involving simultaneous sorption/precipitation mechanisms for the removal of Pb(II) and Cr(III). Also, the dose of B. subtilis has an impact on the pseudo-second-order (PSO) and intra-particle diffusion (IPD) reaction, leading to distinct removal capacities for Pb(II) and Cr(III) [24.26-24.74 mg g-1 in Pb(II) and 12.7-23.93 mg g-1 in Cr(III)]. Furthermore, B. subtilis, an inducing mediator for microbial metabolites, exhibits the potential to facilitate the removal of Pb(II) and Cr(III) through biological modification of raw materials, which are transformed, facilitating the presence of hydroxyl groups, immobilizing metal ions, and enabling ion exchange via biogenic carbonate formation processes. Finally, the developed BIBMFWCs could be used as a nature-based solution (NBS) material without in-situ pH control.

Aptamer-based technology for detecting Bacillus subtilis in soil.

The uncertainty associated with the impact of a bioinoculant on soil microbial community and, as a consequence, on soil quality, as well as the need to define its persistence, has prompted the demand for an accurate detection and tracking of the presence and the quantification of a target microbial inoculant in soil. Although DNA or RNA-based molecular detection are well established and commonly applied in this regard, alternative ligands such as DNA-aptamers have several advantages over them, such as low cost, ease of modification, ease of immobilisation on lab-on-chip or nanosensors, high stability and not thermolability. In this study, we used a toggle-cell SELEX method to isolate, select and characterise ssDNA (single-strand DNA) aptamers to detect a Bacillus subtilis strain which is being tested as a plant growth promoting rhizobacteria (PGPR) formulation. Two ssDNA aptamers (patenting application n.102022000022590) showed strong affinity and specificity for B. subtilis strains, with values of the kinetic parameters Kd (dissociation constant) in the nanomolar range and Bmax (maximum intensity of binding) around 1. Validation of the suitability of the aptamers was validated on three inoculated soils characterised by different chemical-physical features and in soil from a field trial with the formulated B. subtilis PCM/B 00105 strain. These are considered significant features to monitor B. subtilis strains in soil, practical to optimise bioinoculant application methods, support regulatory processes and foster the shift of agricultural production toward more sustainable cropping systems. KEY POINTS: • First DNA aptamers binding a B. subtilis strain included in a bioinoculum formulation. • First DNA aptamer binding B. subtilis in soil. • Aptamer may be a method for microbial inoculant detection in soil.