Growth of Bacillus amyloliquefaciens as influence by Si nutrition.

The aim of study was to determine the influence of soluble and solid forms of Si on the growth of B. amyloliquefaciens. The experiment was conducted at two regimes: under sterile conditions (without B. amyloliquefaciens) and infected conditions (with B. amyloliquefaciens). New formed silica gel, diatomite and monosilicic acid at 1 mM Si and 2 mM Si were used as source of Si. The concentration of monosilicic acid in the solution was measured on second and tenth days of experiment. The total carbon in the solution before and after centrifugation was determined on day 10 of the experiment. The experiment has demonstrated a significant positive effect (by 4.7-41.2%) on B. amyloliquefaciens growth in water system. The presence of B. amyloliquefaciens in Si-rich solution reduced the concentration of monosilicic acid in the solution up to 16.2%. About 13.5-30.7% of B. amyloliquefaciens can be attached to the Si-rich surface without formation of cell clusters. Si can be classified as a beneficial nutrient for B. amyloliquefaciens. The tested strain of Bacillus can form channels in silica gel. The presence of monosilicic acid resulted in the formation of an aligned positioning of cells in water-based solution. This study is the first to demonstrate the direct influence of active Si forms on bacteria growth. The research showed that monosilicic acid or Si-rich solid substances with high solubility on Si can be recommended to increase B. amyloliquefaciens growth in soil, water or reactors.

Differential expression of bio-active metabolites produced by chitosan polymers-based Bacillus amyloliquefaciens fermentation.

Bacillus amyloliquefaciens strain PPL shows a potential for the control of phytopathogenic fungi. In the present study, upon growing the strain PPL on various forms of chitosan (0.5 % powder, 0.1 % soluble, and 0.15 % colloidal) as the carbon source, the antifungal activity on tomato Fusarium wilt correlated with the activity of chitosanase and ß-1,3-glucanase. The colloidal substrate-based strain PPL fermentation displayed the highest degree of spore germination inhibition (79.5 %) and biocontrol efficiency (76.0 %) in tomato by increased biofilm formation. The colloidal culture upregulated the expression of chitosanase gene (5.9-fold), and the powder attributed to the expression of cyclic lipopeptides-genes (2.5-5.7 fold). Moreover, the three chitosan cultures induced the morphological changes of Fusarium oxysporum. These results suggest that the choice of growth substrate synergistically affects the production of secondary metabolites by PPL strain, and consequently its antifungal activity.

Bioutilization of Chicken Feather Waste by Newly Isolated Keratinolytic Bacteria and Conversion into Protein Hydrolysates with Improved Functionalities.

Chicken feathers are major poultry waste that is difficult to process in its native form due to highly resistant keratin protein in large amounts. In this study, a novel feather-degrading bacterium, Bacillus amyloliquefaciens KB1, was screened from a chicken farm bed (CFB) using morphological and biochemical tests followed by 16s rDNA analysis. Among observed isolates, bacterial isolate (KB1) showed the highest degree of feather degradation (74.78 ± 2.94%) and total soluble protein (205 ± 0.03 mg/g). The optimum fermentation conditions obtained were at 40 °C (temperature), pH 9, and 1% (w/v) feather concentration using response surface methodology in a Box-Behnken design. It produced 260 mg/g of soluble protein and bioactive peptides with 86.16% feather degradation. The amino acid profile showed an increase in the concentration of essential amino acids compared with the feather meal broth. The selection of a safe screening source for this new bacterium in CFB produced hydrolysates with enhanced bioactivity applicable for feed, and cosmetic applications, along with environmental bioremediation.

Application of ultrasound technology in the field of solid-state fermentation: increasing peptide yield through ultrasound-treated bacterial strain.

BACKGROUND: The increase of peptide yield contributed to reducing the usage of antibiotics in solid-state fermented feed. Ultrasound technology is used in the field of liquid-state fermentation to improve yield of fermented products but has not been utilized in the field of solid-state fermentation (SSF). The main objective of this study was to investigate the feasibility of improving peptide yield in SSF products through ultrasound-treated bacterial strain. RESULTS: The highest peptides content in soybean meal SSF products reached 153.28 mg g-1 , which increased by 15.05% compared with the control. This content value was acquired through treating the bacteria of Bacillus amyloliquefaciens by ultrasound before inoculating into soybean meal under the optimized mode and parameters (simultaneous dual-frequency ultrasound mode, frequency combination of 40/60 kHz, total power density of 40 W L-1 , time of 20 min, pulse-on and pulse-off times of 40 and 60 s, delayed inoculation time of 0 h). Fermenting with ultrasound-treated bacterial strain can effectively increase peptide yield, biomass and protease activity of soybean meal fermented products during the SSF prophase. After treating by ultrasound, the latent phase and logarithmic phase of the bacterial strain shortened by 1 and 3 h while the generation time reduced by 23.64%. In qualitative test of protease activity, diameter ratio (DR) value of ultrasound-treated bacterial cells enlarged by 12.0% compared with the control. CONCLUSION: Peptide yield of soybean meal SSF products can be improved through ultrasound-treated bacterial inoculum, which attributed to the promoting effect of ultrasound treatment on growth activity and protease production capability of bacterial cells. © 2021 Society of Chemical Industry.

Bacillus amyloliquefaciens B10 can alleviate liver apoptosis and oxidative stress induced by aflatoxin B1.

Aflatoxin B1 (AFB1) is a mycotoxin often found in food and livestock feed. It can affect human and animal health and is especially damaging to the liver. This study aims to evaluate whether Bacillus amyloliquefaciens (hereafter referred to as B. amyloliquefaciens) B10 can alleviate the toxic effects of AFB1 and, if so, what mechanism is responsible for its action. Specific pathogen-free (SPF) Kunming mice (5-6 weeks old) were divided into four groups (Control, AFB1, B10 strain, and AFB1 + B10 strain) and conducted continuously via gavage for 28 days. Oxidation indices (MDA, T-AOC, SOD, GSH-Px, and CAT) were then measured using their liver tissues and liver coefficient were calculated. Apoptosis was determined using the TUNEL method. Gene expression was determined for Bax, Bcl-2, BIP, CHOP, JNK, Caspase-12, Caspase-9, and Caspase-3, and protein expression was detected for Bax, Bcl-2, and Caspase-3. Our results showed that AFB1 induced the oxidative damage and apoptosis in the livers of mice. However, for mice given B. amyloliquefaciens B10, the biochemical indices, pathological changes, the expressions of genes and proteins related to oxidative stress and apoptosis were significantly reversed. The results indicate that B. amyloliquefaciens B10 antagonizes oxidative damage and apoptosis induced by AFB1 in the livers of mice. The results of this study are of significance for the future use of this strain to reduce the harm of AFB1 to human health and animal reproductive performance.

Effects of integrated biocontrol on bacterial wilt and rhizosphere bacterial community of tobacco.

Bacterial wilt as a soil-borne disease was caused by Ralstonia solanacearum, and seriously damages the growth of tobacco. Integrated biocontrol method was explored to control bacterial wilt. Nevertheless, the long-term effects of the integrated biocontrol method on soil bacterial community, soil physicochemical properties and the incidence of bacterial wilt are not well understood. In this study, B. amyoliquefaciens ZM9, calcium cyanamide and rice bran were applied to tobacco fields in different ways. The disease index and incidence of tobacco bacterial wilt (TBW), soil physicochemical properties, colonization ability of B. amyoliquefaciens ZM9, and rhizopshere bacterial community were investigated. The results showed that the integrated application of B. amyoliquefaciens ZM9, rice bran and calcium cyanamide had the highest control efficiency of TBW and bacteria community diversity. Additionally, the integrated biocontrol method could improve the colonization ability of B. amyoliquefaciens ZM9. Furthermore, the integrated biocontrol method could effectively suppress TBW by regulating soil physicochemical properties, promoting beneficial bacteria and antagonistic bacteria of rhizopshere soil. This strategy has prospect of overcoming the defects in application of a single antagonistic bacteria and provides new insights to understand how to improve the colonization capacity of antagonistic bacteria and control efficacy for TBW.

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production.

BACKGROUND: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. RESULTS: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. CONCLUSIONS: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

Development of a Strain-Specific Quantification Method for Monitoring Bacillus amyloliquefaciens TF28 in the Rhizospheric Soil of Soybean.

Bacillus amyloliquefaciens TF28 can be used to control soybean root disease. To assess its commercial potential as a biocontrol agent, it is necessary to develop a strain-specific quantification method to monitor its colonization dynamics in the rhizospheric soil of soybean under field conditions. Based on genomic comparison with the same species in NCBI databases, a strain-unique gene ukfpg was used as molecular marker to develop strain-specific PCR assay. Among three primer pairs, only primer pairs (F2/R2) could specifically differentiate TF28 from other strains of B. amyloliquefaciens with the detection limit of 10 fg and 100 CFU/g for DNA extracted from pure culture and dry soil, respectively. Then, a colony count coupled with PCR assay was used to monitor the population of TF28 in the rhizospheric soil of soybean in the field. The results indicated that TF28 successfully colonized in the rhizospheric soil of soybean. The colonization population of TF28 changed dynamically within the 120-day growth period with high population at the branching (V6) and flowering stages (R2). This study provides an efficient method to quantitatively monitor the colonization dynamics of TF28 in the rhizospheric soil of soybean in the field and demonstrates the potential of TF28 as a biocontrol agent for commercial development.

Enhancement of S-adenosylmethionine production by deleting thrB gene and overexpressing SAM2 gene in Bacillus amyloliquefaciens.

OBJECTIVES: To improve the S-adenosylmethionine (SAM) production in methionine-free medium, effects of deleting genes of SAM decarboxylase (speD) and homoserine kinase (thrB) on SAM titers were investigated, and the SAM synthetase gene (SAM2) was also overexpressed. RESULTS: In B. amyloliquefaciens HSAM2, deleting speD to block the SAM utilization pathway significantly reduced the SAM titer. After knockout of thrB to block the branched pathway, the resulted mutant HSAM4 produced 143.93 mg/L SAM, increasing by 42% than HSAM2. Further plasmid-based expression of SAM2 improved the SAM titer to 226.92 mg/L, and final optimization of key fermentation parameters resulted in the maximum SAM titer of 412.01 mg/L in flasks batch fermentation. CONCLUSIONS: Deleting thrB and overexpressing SAM2 gene were efficient for enhanced SAM production in B. amyloliquefaciens. The maximum SAM titer in flasks batch fermentation was much higher than that of previous reports.

Botrydial confers Botrytis cinerea the ability to antagonize soil and phyllospheric bacteria.

The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with Bacillusamyloliquefaciens strain MEP218 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP218 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP218. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea.

Evaluation of an Industrial Soybean Byproduct for the Potential Development of a Probiotic Animal Feed Additive with Bacillus Species.

Probiotics are gaining public attention for their application in animal husbandry due to their ability to promote growth and prevent infections. Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 are two spore-forming probiotic microorganisms that have been demonstrated to provide health benefits for poultry when supplemented into their diet. These strains can be propagated on a wide range of substrates, including soybean-derived byproducts from the food processing industry. Soybean-derived byproducts are often incorporated into animal feeds, but the value of an additive could potentially be increased by the addition of probiotic microorganisms, which may decrease production costs and reduce environmental impact. In this study, a soybean byproduct and a desalted version of this byproduct were evaluated as potential substrates for the growth of two probiotic bacilli species. Chemical analysis of these byproducts showed favorable carbohydrate, fat, and amino acid profiles, which were not affected by the desalting process. The desalted byproduct was further evaluated as a substrate for the growth of B. subtilis KATMIRA1933 and B. amyloliquefaciens B-1895 under solid-state conditions, and samples from this experiment were visualized by scanning electron microscopy. The results of this study indicate that the desalted soybean byproduct is a suitable substrate for the propagation of the two Bacillus species, which grew to numbers sufficient for the formulation of a probiotic animal feed additive.

Root-Secreted Spermine Binds to Bacillus amyloliquefaciens SQR9 Histidine Kinase KinD and Modulates Biofilm Formation.

The signal molecules in root exudates that are sensed by plant growth-promoting rhizobacteria (PGPR) are critical to regulate their root colonization. Phosphorylated Spo0A is an important global transcriptional regulator that controls colonization and sporulation in Bacillus species. In this study, we found that deletion of kinD from PGPR strain Bacillus amyloliquefaciens SQR9, encoding an original phosphate donor of Spo0A, resulted in reduced biofilm formation in root exudates compared with the wild-type strain, indicating that KinD is responsible for sensing root exudates. Ligands of B. amyloliquefaciens SQR9 KinD in cucumber root exudates were determined by both the nontargeted ligand fishing method and the targeted surface plasmon resonance detection method. In total, we screened 80 compounds in root exudates for binding to KinD and found that spermine and guanosine could bind to KinD with dissociation constant values of 213 and 51 µΜ, respectively. In addition, calcium l-threonate, N-acetyl-l-aspartic acid, sodium decanoic acid, and parabanic acid could also bind weakly to KinD. The three-dimensional binding models were then constructed to demonstrate the interactions between the root-secreted signals and KinD. It was observed that exogenous spermine reduced the wrinkles of biofilm when kinD was deleted, indicating that KinD might be involved in sensing root-secreted spermine and stabilizing biofilm in response to this negative effector. This study provided a new insight of interaction between a rhizobacterial sensor and root-secreted signals.

Simultaneous and sequential based co-fermentations of Trichoderma asperellum GDFS1009 and Bacillus amyloliquefaciens 1841: a strategy to enhance the gene expression and metabolites to improve the bio-control and plant growth promoting activity.

BACKGROUND: The consequence of simultaneous and sequential inoculation of T. asperellum and B. amyloliquefaciens cultures with respect to growth rate, differential expression of vital genes and metabolites were examined. RESULTS: The competition was observed between T. asperellum and B. amyloliquefaciens under co-cultivation. The proliferation of Trichoderma was reduced in the simultaneous inoculation (TB1) method, possibly due to the fastest growth of Bacillus. Both T. asperellum and B. amyloliquefaciens were proliferated in sequential inoculation method (TB2). The sequential inoculation method (TB2) upregulated the expression of metabolites and vital genes (sporulation, secondary metabolites, mycoparasitism enzymes and antioxidants) in Trichoderma and downregulated in Bacillus and vice versa in co-inoculation method (TB1). The metabolic changes in the co-culture promoted the maize plant growth and defense potential under normal and biotic stress conditions. CONCLUSION: The metabolites produced by the co-culture of T. asperellum and B. amyloliquefaciens improved the maize plant growth and defense potential under normal and biotic stress conditions.

Establishment of a rapeseed meal fermentation model for iturin A production by Bacillus amyloliquefaciens CX-20.

Iturin A is an important broad-spectrum antifungal cyclic lipopeptide used as an ideal potential biological control agent. However, its application is limited mainly due to the producer strains' low productivity and the high production costs. Here, a potentially industrial strain Bacillus amyloliquefaciens CX-20 was proved to use low-cost rapeseed meal (RSM) as the sole source of all nutrients except the carbon source for the high productivity of iturin A. A fermentation model was first established to analyse the specific roles of different RSM components on iturin A production. Proteins and minerals in RSM were confirmed to play positive role, whereas fibre had negative effect. And the maximal concentration of iturin A was predicted to be more than 1.64 g l-1 by the established evaluation model. Moreover, submerged fermentation of B. amyloliquefaciens CX-20 demonstrated a strong ability to hydrolyse RSM and release water-soluble nutrients. This fermentation broth, a mixture of Bacillus, iturin A and RSM hydrolysate, could simultaneously combat clubroot disease and promote the growth of Brassica napus. In conclusion, this study provides a promising strategy to achieve full utilization of RSM for the production of a combination of value-added biological control agent and biofertilizer.

Enhanced tomato plant growth in soil under reduced P supply through microbial inoculants and microbiome shifts.

Soil microbial communities interact with roots, affecting plant growth and nutrient acquisition. In the present study, we aimed to decipher the effects of the inoculants Trichoderma harzianum T-22, Pseudomonas sp. DSMZ 13134, Bacillus amyloliquefaciens FZB42 or Pseudomonas sp. RU47 on the rhizosphere microbial community and their beneficial effects on tomato plants grown in moderately low phosphorous soil under greenhouse conditions. We analyzed the plant mass, inoculant colony forming units and rhizosphere communities on 15, 22, 29 and 43 days after sowing. Selective plating showed that the bacterial inoculants had a good rhizocompetence and accelerated shoot and root growth and nutrient accumulation. 16S rRNA gene fingerprints indicated changes in the rhizosphere bacterial community composition. Amplicon sequencing revealed that rhizosphere bacterial communities from plants treated with bacterial inoculants were more similar to each other and distinct from those of the control and the Trichoderma inoculated plants at harvest time, and numerous dynamic taxa were identified. In conclusion, likely both, inoculants and the rhizosphere microbiome shifts, stimulated early plant growth mainly by improved spatial acquisition of available nutrients via root growth promotion. At harvest, all tomato plants were P-deficient, suggesting a limited contribution of inoculants and the microbiome shifts to the solubilization of sparingly soluble soil P.

Improving poly-(γ-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing.

To excavate the application of Jerusalem artichoke on poly(γ-glutamic acid) (γ-PGA) production, a γ-PGA producing strain Bacillus amyloliquefaciens NX-2S154 was obtained through atmospheric and room temperature plasma mutagenesis, which produced 14.83 ± 0.31 g/L of γ-PGA in batch fermentation with raw inulin extract. Simultaneous saccharification and fermentation (SSF) by adding commercial inulinase were further investigated for γ-PGA fermentation. Results showed SSF could eliminate the ineffective utilization of inulin while avoiding inhibition effect of high concentration substrate, which made γ-PGA concentration reach 18.54 ± 0.39 g/L with the process being shortened by 17%. Finally, an immobilized column for reducing inulinase cost was introduced to γ-PGA production. Repeated batch cultures showed the novel bioreactor exhibited higher stability and simplicity and gave average γ-PGA concentration and productivity of 19.40 ± 0.37 g/L and 0.27 ± 0.008 g/L/h, respectively. This work proposes a productive method for efficient γ-PGA production using Jerusalem artichoke feedstock.

Glucose limitation and glucose uptake rate determines metabolite production and sporulation in high cell density continuous cultures of Bacillus amyloliquefaciens 83.

Bacillus amyloliquefaciens spores have been used as the principal ingredient of biocontrol products. However, during the process of spore production, wild-type strains produce poly-γ-glutamic acid (γ-PGA), an undesirable byproduct that increases broth viscosity and hinders recovery and drying. This work examined the influence of specific glucose uptake rates (qGluc) in glucose-controlled overflow metabolism. Diverse scenarios, from glucose limitation to glucose sufficiency, were evaluated in continuous cultures to control qGluc. Cell yields of glucose were higher at low qGluc, while the opposing trend was found for γ-PGA and other overflow metabolic byproducts yields. However, γ-PGA production was still detectable in cultures with the highest glucose limitation (D = 0.06 h-1), even though high sporulation incidence was observed in these cultures. Indeed, in such conditions, nonsporulating vegetative cells seem to maintain glucose overflow metabolism, allowing limited γ-PGA production. These findings can be used to establish fed-batch culture strategies for high cell density Bacillus amyloliquefaciens cultures where γ-PGA production (and apparent viscosity) is significantly reduced. This is the first time that the dependence of qGluc on growth, sporulation and carbon overflow metabolism of a spore and biofilm producer, Bacillus amyloliquefaciens strain, has been reported.

Potential of Bacillus amyloliquefaciens for biocontrol of bacterial canker of tomato incited by Clavibacter michiganensis ssp. michiganensis.

A total of 150 rhizobacteria and endorhizobacteria previously isolated from three different horticultural crops; strawberry, apple and apricot were screened for antagonistic activitiy against Clavibacter michiganensis ssp. michiganensis. Among them strain S1, exhibiting significantly higher antagonistic and plant growth promoting ability was characterized as Bacillus amyloliquefaciens based on morphological, biochemical and partial gene sequence analysis of 16S rRNA. B. amyloliquefaciens strain S1 showed maximum growth inhibition of C. michiganensis (12 mm). Moreover, B. amyloliquefaciens strain S1 exhibit significant phosphorus solubilization (94.16 %SEl) and indole acetic acid (27 µg ml-1) production under in vitro conditions. Antagonistic activity of Bacillus amyloliquefaciens strain S1 was compared with other four strains KU2S1, R2S(1), RG1(3) and AG1(7) against bacterial canker of tomato under net house conditions. Minimum bacterial canker disease incidence (30.0%) was recorded in B. amyloliquefaciens S1 followed by RG1(3) after 30 days of inoculation. The bio-control efficacy was higher in B. amyloliquefaciens S1 treated plants, followed by RG1(3).

An investigation of inactivation mechanisms of Bacillus amyloliquefaciens spores in non-thermal plasma of ambient air.

BACKGROUND: To utilize the potential of non-thermal plasma technologies for food safety control and sanitation, the inactivation mechanisms of Bacillus amyloliquefaciens spores by non-thermal plasma of ambient air (NTP-AA) were investigated using scanning electron microscopy, atomic force microscopy, attenuated total reflectance Fourier transform infrared spectroscopy with chemometric analysis and proton nuclear magnetic resonance spectroscopy, aiming to probe both the morphological and biochemical changes occurring in spores during the kinetic inactivation process. RESULTS: Kinetic analysis indicates that there is no intrinsic D-value (i.e. time required to inactivate 90% of the spores) in spore inactivation by NTP-AA because we observed non-linear (biphasic) inactivation kinetics and, in addition, the inactivation rate depended on the initial spore concentration and how the spores were exposed to the reactive species in the NTP-AA. The presence of suitable amount of water in the NTP-AA field accelerates spore inactivation. CONCLUSION: Progressive erosion of spore surface by NTP-AA with ensuing or concomitant biochemical damage, which includes the alteration of structural proteins, internal lipids and the loss of dipicolinic acid content from the spore core, represent the main mechanisms of inactivation, and there is evidence that reactive NTP-AA species could penetrate the cortex and reach the core of spores to cause damage. © 2018 Society of Chemical Industry.

Improved Production of Spores and Bioactive Metabolites from Bacillus amyloliquefaciens in Solid-state Fermentation by a Rapid Optimization Process.

A dipicolonic acid fluorimetry assay was used instead of plate counting for the assessment of spore yields for enhanced optimization efficiency. The associated parameters, including the ratio of solid substrates, composition of liquid substrates, and cultivation conditions, were systematically optimized in a shake-flask culture. The maximum spore yield of 7.24 × 1010 CFU/g of wet substrate was achieved. The optimization process produced a 25.7-fold increase in spore yields compared with those before optimization. In addition, the maximum release of bioactive metabolites during spore accumulation was subsequently obtained with 573.0 U/g of protease, 188.8 U/g of amylase, 186.8 U/g of cellulase, and 3.45 mg/g of acid-soluble protein. The experiment provides a methodological basis for the rapidly optimized production of Bacillus spores in pure solid-state fermentation.