Lipopeptides from Bacillus velezensis ZLP-101 and their mode of action against bean aphids Acyrthosiphon pisum Harris.

BACKGROUND: Natural products are important sources for the discovery of new biopesticides to control the worldwide destructive pests Acyrthosiphon pisum Harris. Here, insecticidal substances were discovered and characterized from the secondary metabolites of the bio-control microorganism Bacillus velezensis strain ZLP-101, as informed by whole-genome sequencing and analysis. RESULTS: The genome was annotated, revealing the presence of four potentially novel gene clusters and eight known secondary metabolite synthetic gene clusters. Crude extracts, prepared through ammonium sulfate precipitation, were used to evaluate the effects of strain ZLP-101 on Acyrthosiphon pisum Harris aphid pests via exposure experiments. The half lethal concentration (LC50) of the crude extract from strain ZLP-101 against aphids was 411.535 mg/L. Preliminary exploration of the insecticidal mechanism revealed that the crude extract affected aphids to a greater extent through gastric poisoning than through contact. Further, the extracts affected enzymatic activities, causing holes to form in internal organs along with deformation, such that normal physiological activities could not be maintained, eventually leading to death. Isolation and purification of extracellular secondary metabolites were conducted in combination with mass spectrometry analysis to further identify the insecticidal components of the crude extracts. A total of 15 insecticidal active compounds were identified including iturins, fengycins, surfactins, and spergualins. Further insecticidal experimentation revealed that surfactin, iturin, and fengycin all exhibited certain aphidicidal activities, and the three exerted synergistic lethal effects. CONCLUSIONS: This study improved the available genomic resources for B. velezensis and serves as a foundation for comprehensive studies of the insecticidal mechanism by Bacillus velezensis ZLP-101 in addition to the active components within biological control strains.

Mechanism of oxalate decarboxylase Oxd_S12 from Bacillus velezensis BvZ45-1 in defence against cotton verticillium wilt.

Verticillium wilt, a soilborne vascular disease caused by Verticillium dahliae, strongly affects cotton yield and quality. In this study, an isolated rhizosphere bacterium, designated Bacillus velezensis BvZ45-1, exhibited >46% biocontrol efficacy against cotton verticillium wilt under greenhouse and field conditions. Moreover, through crude protein extraction and mass spectrometry analyses, we found many antifungal compounds present in the crude protein extract of BvZ45-1. The purified oxalate decarboxylase Odx_S12 from BvZ45-1 inhibited the growth of V. dahliae Vd080 by reducing the spore yield, causing mycelia to rupture, spore morphology changes, cell membrane rupture, and cell death. Subsequently, overexpression of Odx_S12 in Arabidopsis significantly improved plant resistance to V. dahliae. Through studies of the resistance mechanism of Odx_S12, V. dahliae was shown to produce oxalic acid (OA), which has a toxic effect on Arabidopsis leaves. Odx_S12 overexpression reduced Arabidopsis OA content, enhanced tolerance to OA, and improved resistance to verticillium wilt. Transcriptomics and quantitative real-time PCR analysis revealed that Odx_S12 promoted a reactive oxygen species burst and a salicylic acid- and abscisic acid-mediated defence response in Arabidopsis. In summary, this study not only identified B. velezensis BvZ45-1 as an efficient biological control agent, but also identified the resistance gene Odx_S12 as a candidate for cotton breeding against verticillium wilt.

The beneficial rhizobacterium Bacillus velezensis SQR9 regulates plant nitrogen uptake via an endogenous signaling pathway.

Nitrogen fertilizer is widely used in agriculture to boost crop yields. Plant growth-promoting rhizobacteria (PGPRs) can increase plant nitrogen use efficiency through nitrogen fixation and organic nitrogen mineralization. However, it is not known whether they can activate plant nitrogen uptake. In this study, we investigated the effects of volatile compounds (VCs) emitted by the PGPR strain Bacillus velezensis SQR9 on plant nitrogen uptake. Strain SQR9 VCs promoted nitrogen accumulation in both rice and Arabidopsis. In addition, isotope labeling experiments showed that strain SQR9 VCs promoted the absorption of nitrate and ammonium. Several key nitrogen-uptake genes were up-regulated by strain SQR9 VCs, such as AtNRT2.1 in Arabidopsis and OsNAR2.1, OsNRT2.3a, and OsAMT1 family members in rice, and the deletion of these genes compromised the promoting effect of strain SQR9 VCs on plant nitrogen absorption. Furthermore, calcium and the transcription factor NIN-LIKE PROTEIN 7 play an important role in nitrate uptake promoted by strain SQR9 VCs. Taken together, our results indicate that PGPRs can promote nitrogen uptake through regulating plant endogenous signaling and nitrogen transport pathways.

LPA3 agonist-producing Bacillus velezensis ADS024 is efficacious in multiple neuroinflammatory disease models.

Neuroinflammation is a common component of neurological disorders. In the gut-brain-immune axis, bacteria and their metabolites are now thought to play a role in the modulation of the nervous and immune systems which may impact neuroinflammation. In this respect, commensal bacteria of humans have recently been shown to produce metabolites that mimic endogenous G-protein coupled receptor (GPCR) ligands. To date, it has not been established whether plant commensal bacteria, which may be ingested by animals including humans, can impact the gut-brain-immune axis via GPCR agonism. We screened an isopropanol (IPA) extract of the plant commensal Bacillus velezensis ADS024, a non-engrafting live biotherapeutic product (LBP) with anti-inflammatory properties isolated from human feces, against a panel of 168 GPCRs and identified strong agonism of the lysophosphatidic acid (LPA) receptor LPA3. The ADS024 IPA extracted material (ADS024-IPA) did not agonize LPA2, and only very weakly agonized LPA1. The agonism of LPA3 was inhibited by the reversible LPA1/3 antagonist Ki16425. ADS024-IPA signaled downstream of LPA3 through G-protein-induced calcium release, recruitment of ß-arrestin, and recruitment of the neurodegeneration-associated proteins 14-3-3γ, ε and ζ but did not recruit the ß isoform. Since LPA3 agonism was previously indirectly implicated in the reduction of pathology in models of Parkinson's disease (PD) and multiple sclerosis (MS) by use of the nonselective antagonist Ki16425, and since we identified an LPA3-specific agonist within ADS024, we sought to examine whether LPA3 might indeed be part of a broad underlying mechanism to control neuroinflammation. We tested oral treatment of ADS024 in multiple models of neuroinflammatory diseases using three models of PD, two models of MS, and a model each of amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and chemo-induced peripheral neuropathy (CIPN). ADS024 treatment improved model-specific functional effects including improvements in motor movement, breathing and swallowing, and allodynia suggesting that ADS024 treatment impacted a universal underlying neuroinflammatory mechanism regardless of the initiating cause of disease. We used the MOG-EAE mouse model to examine early events after disease initiation and found that ADS024 attenuated the increase in circulating lymphocytes and changes in neutrophil subtypes, and ADS024 attenuated the early loss of cell-surface LPA3 receptor expression on circulating white blood cells. ADS024 efficacy was partially inhibited by Ki16425 in vivo suggesting LPA3 may be part of its mechanism. Altogether, these data suggest that ADS024 and its LPA3 agonism activity should be investigated further as a possible treatment for diseases with a neuroinflammatory component.

Analysis of antimicrobial biological activity of a marine Bacillus velezensis NDB.

A new strain of Bacillus velezensis NDB was isolated from Xiangshan Harbor and antibacterial test revealed antibacterial activity of this strain against 12 major pathogenic bacteria. The whole genome of the bacterium was sequenced and found to consist of a 4,214,838 bp circular chromosome and a 7410 bp circular plasmid. Furthermore, it was predicted by AntiSMASH and BAGEL4 to have 12 clusters of secondary metabolism genes for the synthesis of the inhibitors, fengycin, bacillomycin, macrolactin H, bacillaene, and difficidin, and there were also five clusters encoding potentially novel antimicrobial substances, as well as three bacteriocin biosynthesis gene clusters of amylocyclicin, ComX1, and LCI. qRT-PCR revealed significant up-regulation of antimicrobial secondary metabolite synthesis genes after 24 h of antagonism with pathogenic bacteria. Furthermore, MALDI-TOF mass spectrometry revealed that it can secrete surfactin non-ribosomal peptide synthase and polyketide synthase to exert antibacterial effects. GC-MS was used to analyze methanol extract of B. velezensis NDB, a total of 68 compounds were identified and these metabolites include 16 amino acids, 17 acids, 3 amines, 11 sugars, 11 alcohols, 1 ester, and 9 other compounds which can inhibit pathogenic bacteria by initiating the antibiotic secretion pathway. A comparative genomic analysis of gene families showed that the specificity of B. velezensis NDB was mainly reflected in environmental adaptability. Overall, this research on B. velezensis NDB provides the basis for elucidating its biocontrol effect and promotes its future application as a probiotic.

A new alkaline pectin lyase with novel thermal and pH stability from Bacilus velezensis.

Pectin lyases are important in various industries, including tobacco leaves processing. In this paper, a novel pectin lyase Pel04 from Bacillus velezensis was characterized. Pel04 molecular weight (Mw) and isoelectric point (pI) of the protein sequence after removing the signal peptide are 43.0 kDa. The optimal temperature and pH of Pel04 is 50 °C and 9.0, respectively. Pel04 was stable in the range of 30-50 °C, and pH 9.5-11. Ca2+ can significantly stimulate the enzyme activity, while Cu2+, Co2+, Fe3+, and Mn2+ have inhibitory effects on Pel04. By Pel04 treatment, the overall content of acids, alcohols, esters and other aromas in tobacco leaves increased, while the contents of phenolic and heterocyclic substances decreased. Pel04 has important potential for industrial application particularly in improving quality of tobacco leaves.

Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum.

Previous studies have demonstrated the presence of chitinase in Bacillus velezensis through extensive genomic sequencing and experimental analyses. However, the detailed structure, functional roles, and antifungal activity of these chitinases remain poorly characterized. In this study, genomic screening identified three genes-chiA, chiB, and lpmo10-associated with chitinase degradation in B. velezensis S161. These genes encode chitinases ChiA and ChiB, and lytic polysaccharide monooxygenase LPMO10. Both ChiA and ChiB contain two CBM50 binding domains and one catalytic domain, whereas LPMO10 includes a signal peptide and a single catalytic domain. The chitinases ChiA, its truncated variant ChiA2, and ChiB were heterologously expressed in Escherichia coli. The purified enzymes efficiently degraded colloidal chitin and inhibited the spore germination of Penicillium digitatum. Notably, even after losing one CBM50 domain, the resultant enzyme, consisting of the remaining CBM50 domain and the catalytic domain, maintained its colloidal chitin hydrolysis and antifungal activity, indicating commendable stability. These results underscore the role of B. velezensis chitinases in suppressing plant pathogenic fungi and provide a solid foundation for developing and applying chitinase-based biocontrol strategies.

Exploring the protective role of Bacillus velezensis BV1704-Y in zebrafish health and disease resistance against Aeromonas hydrophila infection.

Bacillus genus, particularly Bacillus velezensis, is increasingly considered as viable alternatives to antibiotics in aquaculture due to their safety and probiotic potential. However, the specific mechanisms through which probiotic B. velezensis confers protection against Aeromonas hydrophila infection in fish remain poorly understood. This study delved into the multifaceted impacts of B. velezensis BV1704-Y on diverse facets of zebrafish health, including gut barrier function, immune response, oxidative stress, gut environment, microbiome composition, and disease resistance. Our findings demonstrate that supplementation with B. velezensis BV1704-Y significantly alleviated symptoms and reduced mortality in zebrafish infected with A. hydrophila. Furthermore, a notable reduction in the expression of pivotal immune-related genes, such as IL-1ß, IL6, and TNF-α, was evident in the gut and head kidney of zebrafish upon infection. Moreover, B. velezensis BV1704-Y supplementation resulted in elevated activity levels of essential antioxidant enzymes, including SOD, CAT, and GSH, in gut tissue. Notably, B. velezensis BV1704-Y positively modulated the structure and function of the intestinal microbiome, potentially enhancing immune response and resilience in zebrafish. Specifically, supplementation with B. velezensis BV1704-Y promoted the relative abundance of beneficial bacteria, such as Cetobacterium, which showed a noteworthy negative correlation with the expression of pro-inflammatory genes and a positive correlation with gut barrier-related genes. Altogether, our study suggests that B. velezensis BV1704-Y holds promise as an effective probiotic for protecting zebrafish against A. hydrophila infection, offering potential benefits for the aquaculture industry.

Dietary Bacillus velezensis T23 fermented products supplementation improves growth, hepatopancreas and intestine health of Litopenaeus vannamei.

This study aimed to elucidate the effects of dietary fermented products of Bacillus velezensis T23 on the growth, immune response and gut microbiota in Pacific white shrimp (Litopenaeus vannamei). Shrimp were fed with diets containing fermentation products of B. velezensis T23 at levels of (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 g/kg) for 4 weeks, to assess the influence on shrimp growth. The results showed that 0.3 and 0.4 g/kg T23 supplementation improved shrimp growth and feed utilization. Based on these results we selected these three diets (Control, 0.3T23 and 0.4T23) to assess the effect on immune response and gut microbiota of shrimp. Compared with the control, the 0.3T23 and 0.4T23 groups enhanced lipase and α-amylase activities in the gut significantly. Moreover, the 0.4T23 group decreased TAG and MDA levels in hepatopancreas, ALT and AST levels of serum significantly (P < 0.05). In hepatopancreas, CAT and SOD activities were improved observably and the MDA content was reduced markedly in both T23 groups. The expressions of antimicrobial related genes, Cru and peroxinectin in the 0.3T23 group, and proPO and peroxinectin in the 0.4T23 group were up-regulated remarkably (P < 0.05). Moreover, hepatopancreas of shrimp fed with a diet amended with T23 showed a significant down-regulated expression of nf-kb and tnf-α genes, while expressions of tgf-ß was considerably up-regulated. Furthermore, serum LPS and LBP contents were reduced markedly in T23 groups. Intestinal SOD and CAT were noteworthy higher in T23 groups (P < 0.05). In the intestine of shrimp fed on the diet enriched with T23 the expression of nf-κb and tnf-α exhibited markedly down-regulated, whereas hif1α was up-regulated (P < 0.05). Besides, in the intestine of shrimp grouped under T23, Cru and peroxinectin genes were markedly up-regulated (P < 0.05). Dietary 0.3 g/kg T23 also upregulated the ratio of Rhodobacteraceae to Vibrionaceae in the gut of the shrimp. Taken together, the inclusion of B. velezensis T23 in the diet of shrimp enhanced the growth and feed utilization, enhanced hepatopancreas and intestine health.

Bacillus velezensis: a versatile ally in the battle against phytopathogens-insights and prospects.

The escalating interest in Bacillus velezensis as a biocontrol agent arises from its demonstrated efficacy in inhibiting both phytopathogenic fungi and bacteria, positioning it as a promising candidate for biotechnological applications. This mini review aims to offer a comprehensive exploration of the multifaceted properties of B. velezensis, with particular focus on its beneficial interactions with plants and its potential for controlling phytopathogenic fungi. The molecular dialogues involving B. velezensis, plants, and phytopathogens are scrutinized to underscore the intricate mechanisms orchestrating these interactions. Additionally, the review elucidates the mode of action of B. velezensis, particularly through cyclic lipopeptides, highlighting their importance in biocontrol and promoting plant growth. The agricultural applications of B. velezensis are detailed, showcasing its role in enhancing crop health and productivity while reducing reliance on chemical pesticides. Furthermore, the review extends its purview in the industrial and environmental arenas, highlighting its versatility across various sectors. By addressing challenges such as formulation optimization and regulatory frameworks, the review aims to chart a course for the effective utilization of B. velezensis. KEY POINTS: • B. velezensis fights phytopathogens, boosting biotech potential • B. velezensis shapes agri-biotech future, offers sustainable solutions • Explores plant-B. velezensis dialogue, lipopeptide potential showcased.

Bacillus velezensis BE2 controls wheat and barley diseases by direct antagonism and induced systemic resistance.

Wheat and barley rank among the main crops cultivated on a global scale, providing the essential nutritional foundation for both humans and animals. Nevertheless, these crops are vulnerable to several fungal diseases, such as Septoria tritici blotch and net blotch, which significantly reduce yields by adversely affecting leaves and grain quality. To mitigate the effect of these diseases, chemical fungicides have proven to be genuinely effective; however, they impose a serious environmental burden. Currently, biocontrol agents have attracted attention as a sustainable alternative to fungicides, offering an eco-friendly option. The study aimed to assess the efficacy of Bacillus velezensis BE2 in reducing disease symptoms caused by Zymoseptoria tritici and Pyrenophora teres. This bacterium exhibited significant antagonistic effects in vitro by suppressing fungal development when pathogens and the beneficial strain were in direct confrontation. These findings were subsequently confirmed through microscopic analysis, which illustrated the strain's capacity to inhibit spore germination and mycelial growth in both pathogens. Additionally, the study analysed the cell-free supernatant of the bacterium using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry). The results revealed that strain BE2 produces, among other metabolites, different families of cyclic lipopeptides that may be involved in biocontrol. Furthermore, the beneficial effects of strain BE2 in planta were assessed by quantifying the fungal DNA content directly at the leaf level after bacterization, using two different application methods (foliar and drenching). The results indicated that applying the beneficial bacterium at the root level significantly reduced pathogens pressure. Finally, gene expression analysis of different markers showed that BE2 application induced a priming effect within the first hours after infection. KEY POINTS: • BE2 managed Z. tritici and P. teres by direct antagonism and induced systemic resistance. • Strain BE2 produced seven metabolite families, including three cyclic lipopeptides. • Application of strain BE2 at the root level triggered plant defense mechanisms.

Rhizosphere competent inoculants modulate the apple root-associated microbiome and plant phytoalexins.

Modulating the soil microbiome by applying microbial inoculants has gained increasing attention as eco-friendly option to improve soil disease suppressiveness. Currently, studies unraveling the interplay of inoculants, root-associated microbiome, and plant response are lacking for apple trees. Here, we provide insights into the ability of Bacillus velezensis FZB42 or Pseudomonas sp. RU47 to colonize apple root-associated microhabitats and to modulate their microbiome. We applied the two strains to apple plants grown in soils from the same site either affected by apple replant disease (ARD) or not (grass), screened their establishment by selective plating, and measured phytoalexins in roots 3, 16, and 28 days post inoculation (dpi). Sequencing of 16S rRNA gene and ITS fragments amplified from DNA extracted 28 dpi from different microhabitat samples revealed significant inoculation effects on fungal ß-diversity in root-affected soil and rhizoplane. Interestingly, only in ARD soil, most abundant bacterial amplicon sequence variants (ASVs) changed significantly in relative abundance. Relative abundances of ASVs affiliated with Enterobacteriaceae were higher in rhizoplane of apple grown in ARD soil and reduced by both inoculants. Bacterial communities in the root endosphere were not affected by the inoculants but their presence was indicated. Interestingly and previously unobserved, apple plants responded to the inoculants with increased phytoalexin content in roots, more pronounced in grass than ARD soil. Altogether, our results indicate that FZB42 and RU47 were rhizosphere competent, modulated the root-associated microbiome, and were perceived by the apple plants, which could make them interesting candidates for an eco-friendly mitigation strategy of ARD. KEY POINTS: • Rhizosphere competent inoculants modulated the microbiome (mainly fungi) • Inoculants reduced relative abundance of Enterobacteriaceae in the ARD rhizoplane • Inoculants increased phytoalexin content in roots, stronger in grass than ARD soil.

Effects of dietary supplementation with Bacillus velezensis on the growth performance, body composition, antioxidant, immune-related gene expression, and histology of Pacific white shrimp, Litopenaeus vannamei.

In recent decades, probiotics have become an acceptable aquaculture strategy for shrimp growth promotion and immune modulation. This study aimed to evaluate the effect of Bacillus velezensis on Litopenaeus vannamei following a 60-day trial. L. vannamei (3 ± 0.4 g) were distributed into four groups with three replicates per group and fed an isonitrogenous diet supplemented with B. velezensis at 0, 1 × 107, 1 × 108, and 1 × 109 CFU/g, which were defined as the control, G1, G2, and G3 groups, respectively. B. velezensis significantly improved the growth, survival rate, and proximate body composition of L. vannamei (P < 0.05). All groups fed the B. velezensis diet showed significant increases in digestive enzymes (lipase, amylase, and protease), superoxide dismutase (SOD; G3), catalase (CAT; G3, G2, and G1), lysozyme activity (G3 and G2), immunoglobulin M (IgM), bactericidal activity BA%, alkaline phosphatase (AKP), and acid phosphatase (ACP) compared with the control group (P < 0.05). Malondialdehyde (MDA), triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were significantly decreased in all groups fed B. velezensis diet compared with the control group (P < 0.05). The expression levels of SOD (G3), LZM, and serine proteinase genes were significantly higher in L. vannamei fed diets containing B. velezensis than in the control group (P < 0.05). This is the first study to address the effects of B. velezensis on the expression of the LZM and serine proteinase genes in L. vannamei. L. vannamei fed diet containing B. velezensis had more B and R cells in its hepatopancreas than did the control group. In conclusion, B. velezensis is a promising probiotic that can be safely added to the diet of L. vannamei with 1 × 109 CFU/g. Its application had a positive influence on the health status, survival rate, nutritional value, and immunity of L. vannamei.

Genome analysis of a newly isolated Bacillus velezensis-YW01 for biodegrading acetaldehyde.

Acetaldehyde (AL), a primary carcinogen, not only pollutes the environment, but also endangers human health after drinking alcohol. Here a promising bacterial strain was successfully isolated from a white wine cellar pool in the province of Shandong, China, and identified as Bacillus velezensis-YW01 with 16 S rDNA sequence. Using AL as sole carbon source, initial AL of 1 g/L could be completely biodegraded by YW01 within 84 h and the cell-free extracts of YW01 has also been detected to biodegrade the AL, which indicate that YW01 is a high-potential strain for the biodegradation of AL. The optimal culture conditions and the biodegradation of AL of YW01 are at pH 7.0 and 38 °C, respectively. To further analyze the biodegradation mechanism of AL, the whole genome of YW01 was sequenced. Genes ORF1040, ORF1814 and ORF0127 were revealed in KEGG, which encode for acetaldehyde dehydrogenase. Furthermore, ORF0881 and ORF052 encode for ethanol dehydrogenase. This work provides valuable information for exploring metabolic pathway of converting ethanol to AL and subsequently converting AL to carboxylic acid compounds, which opened up potential pathways for the development of microbial catalyst against AL.

Analysis of the Genomic Sequences and Metabolites of Bacillus velezensis YA215.

Discovering more novel antimicrobial compounds has become a keen research problem. In this study, YA215 genome was sequenced by the Illumina HiSeq + PacBio sequencing platform. Genome assembly was performed by Unicycler software and the gene clusters responsible for secondary metabolite biosynthesis were predicted by antiSMASH. The genome comprised 3976514 bp and had a 46.56% G + C content. 3809 coding DNA sequences, 27 rRNAs, 86 tRNAs genes, and 79 sRNA were predicted. Strain YA215 was re-identified as Bacillus velezensis based on ANI and OrthoANI analysis. In the COG database, 23 functional groups from 3090 annotations were predicted. In the GO database, 2654 annotations were predicted. 2486 KEGG annotations linked 41 metabolic pathways. Glycosyl transferases, polysaccharide lyases, auxiliary activities, glycoside hydrolases, carbohydrate esterases, and carbohydrate-binding modules were predicted among the 127 annotations in the CAZy database. AntiSMASH analysis predicted that B. velezensis YA215 boasted 13 gene clusters involved in synthesis of antimicrobial secondary metabolites including surfactin, fengycin, macrolactin H, bacillaene, difficidin, bacillibactin, bacilysin, and plantazolicin. Three of the gene clusters (gene cluster 5, gene cluster 9, and gene cluster 10) have the potential to synthesize unknown compounds. The research underscore the considerable potential of secondary metabolites, identified in the genomic composition of B. velezensis YA215, as versatile antibacterial agents with a broad spectrum of activity against pathogenic bacteria.

Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production.

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

Enhanced low-cost lipopeptide biosurfactant production by Bacillus velezensis from residual glycerin.

Biosurfactants (BSFs) are molecules produced by microorganisms from various carbon sources, with applications in bioremediation and petroleum recovery. However, the production cost limits large-scale applications. This study optimized BSFs production by Bacillus velezensis (strain MO13) using residual glycerin as a substrate. The spherical quadratic central composite design (CCD) model was used to standardize carbon source concentration (30 g/L), temperature (34 °C), pH (7.2), stirring (239 rpm), and aeration (0.775 vvm) in a 5-L bioreactor. Maximum BSFs production reached 1527.6 mg/L of surfactins and 176.88 mg/L of iturins, a threefold increase through optimization. Microbial development, substrate consumption, concentration of BSFs, and surface tension were also evaluated on the bioprocess dynamics. Mass spectrometry Q-TOF-MS identified five surfactin and two iturin isoforms produced by B. velezensis MO13. This study demonstrates significant progress on BSF production using industrial waste as a microbial substrate, surpassing reported concentrations in the literature.

ccdC Regulates Biofilm Dispersal in Bacillus velezensis FZB42.

Bacillus velezensis FZB42 is a plant growth-promoting rhizobacterium (PGPR) and a model microorganism for biofilm studies. Biofilms are required for the colonization and promotion of plant growth in the rhizosphere. However, little is known about how the final stage of the biofilm life cycle is regulated, when cells regain their motility and escape the mature biofilm to spread and colonize new niches. In this study, the non-annotated gene ccdC was found to be involved in the process of biofilm dispersion. We found that the ccdC-deficient strain maintained a wrinkled state at the late stage of biofilm formation in the liquid-gas interface culture, and the bottom solution showed a clear state, indicating that no bacterial cells actively escaped, which was further evidenced by the formation of a cellular ring (biofilm pellicle) located on top of the preformed biofilm. It can be concluded that dispersal, a biofilm property that relies on motility proficiency, is also positively affected by the unannotated gene ccdC. Furthermore, we found that the level of cyclic diguanylate (c-di-GMP) in the ccdC-deficient strain was significantly greater than that in the wild-type strain, suggesting that B. velezensis exhibits a similar mechanism by regulating the level of c-di-GMP, the master regulator of biofilm formation, dispersal, and cell motility, which controls the fitness of biofilms in Pseudomonas aeruginosain. In this study, we investigated the mechanism regulating biofilm dispersion in PGPR.

Strengthening the microbial community and flavor structure of jiupei by simulating strong-aroma baijiu fermentation with Bacillus velezensis DQA21.

BACKGROUND: Bacillus velezensis DQA21 is a functional strain used in the fermentation process of strong-aroma baijiu; however, its specific role in the process is still unclear. RESULTS: In this study, specific roles of B. velezensis DQA21 in the fermentation process were explored. Bioaugmentation of B. velezensis DQA21 increased the diversity and abundance of the bacterial community during the first 32 days of fermentation and significantly inhibited the diversity and richness of the fungal community during days 12 to 32. According to cluster analysis, changes in the microbial community structure were observed during fermentation, and the fermentation process could be divided into two stages: stage I, days 0-12; and stage II, days 12-45. Additionally, the microbial community structures during the two fermentation stages were significantly different. Co-occurrence analysis showed that bioaugmentation with Bacillus increased the correlation between microorganisms in jiupei and had a significant impact on the overall network structure of the microbial community. In addition, Bacillus significantly increased the production of flavor substances in jiupei, causing the total esters, total alcohols, and total acids contents to increase by 19.1%, 81.1%, and 25.9% respectively. CONCLUSION: The results suggested that bioaugmentation with B. velezensis DQA21 had a strong impact on the microbial community structure in strong-aroma baijiu, enhancing the volatile flavor components. Additionally, the work also provides a better understanding on the effect of augmentation on the microbial community in jiupei, which could help better regulation of solid-state fermentation in strong-aroma baijiu. © 2024 Society of Chemical Industry.

Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215.

Antibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, a cyclic lipopeptide with a C12-C17 ß-hydroxy fatty acid chain, exhibits significant antibacterial activity and resists resistance, making it a research focus. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin's structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by Bacillus velezensis YA215. Process optimization shows that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximized surfactin production to 18.59%, 19.23%, and 20.64%, respectively. Surfactin content peaked at 36 h with L-Val and L-Ile, yielding 19.72% and 11.37%. In contrast, L-Leu addition peaked at 24 h, yielding 11.33%. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against Escherichia coli and Staphylococcus aureus, with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 ß-hydroxy fatty acids from 13.3% and 4.216-23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds.