Search Results (11)

Reprocessing lignocellulosic waste to obtain new products for industrial purposes is a vital part of circular economy. This paper reports the cellulase production by newly isolated Bacillus methylotrophicus cultured on lignocellulosic agro-industrial by-products, out of which brewer’s spent grain (BSG) was selected as most beneficial. Plackett–Burman design was used for screening medium components, while Box–Behnken design was further applied to model the impact of the three most influential variables. The maximum approximated cellulase activity was 0.469 U/mL (1 U = 1 µmol of reducing sugars/1 min), at 48.6 g/L substrate, 5.3 g/L ammonium sulfate, pH 6.1. The partially purified cellulase was characterized, which demonstrated broad range of optimal pH (6.5–9.4), temperature (50–60 °C), and sensitivity to metals. Changes in lignin and pentosans content was demonstrated as a result of BSG hydrolysis with a cell-free cellulase preparation. The produced enzyme was used for hydrolysis of various chemically pretreated (NaOH and H₂SO₄) cellulosic substrates, where for reused alkali-pretreated BSG (after microbial enzyme production) the saccharification efficiency was at a level of 25%. The cellulolytic potential of the bacterial strain, along with its resistance to ethanol, present a beneficial combination, potentially applicable to aid saccharification of lignocellulosic by-products for biofuel production. Full article

The effects of Bacillus spp. (7 Log CFU g⁻¹ feed) and fructooligosaccharide (FOS, 1%) as functional feed additives, either alone or in combination, were evaluated in a study on rohu, Labeo rohita fingerlings. The fish were fed different diets for 90 days, including a control diet and diets supplemented with FOS, B. licheniformis, B. methylotrophicus or synbiotic formulations of these. The results showed that the combination of B. licheniformis and FOS significantly improved weight gain, feed utilisation and protease activity compared to the other groups. Overall, the groups supplemented with probiotics and synbiotics (B. licheniformis + FOS or B. methylotrophicus + FOS) showed improvements in haematology, serum biochemistry and immune parameters compared to the control group. After 90 days of experimental feeding, the fish were challenged with pathogenic Aeromonas hydrophila, and data on haematology, immunity and stress parameters were collected. The results indicated that the application of Bacillus spp. and FOS boosted immunity and resistance to physiological stress in the fish. The highest post-challenge survival rate was observed in fish fed a diet with B. licheniformis and FOS, indicating the potential of this particular combination of functional feed additives to enhance growth, immunity and disease resistance in L. rohita. Full article

Low-temperature stress seriously affects the growth, development, yield, and quality of tomato production. Bacillus methylotrophicus is an important plant growth promoting rhizobacteria (PGPR). However, the role of B. methylotrophicus under low-temperature stress is poorly understood. Accordingly, the effects of B. methylotrophicus (‘VL-10′) on tomato cold stress (15 °C/8 °C, 12 h/12 h, and day/night) were studied. B. methyltrophicus ‘VL-10′ was added into the substrate at the time of sowing, and the plants were treated at a low temperature for 2 weeks after 40 days of growth. We found that the low temperature reduced the spatial distribution of the aboveground and underground sections of tomatoes and the leaf SPAD and photochemical efficiency of PS II (Fv/Fm). After low-temperature stress, the tomato flowering was delayed, the vitamin C and lycopene content in fruit decreased, and the nitrate content increased. However, inoculated with B. methyltrophicus ‘VL-10′ during sowing promoted the growth of tomato seedlings, enhanced the native defense ability of the tomatoes, and effectively reduced the cold shock response of the roots to cold damage and the adverse effects of low temperature on leaf SPAD and Fv/Fm. After the cultivation at normal temperature, the inoculat B. methyltrophicus ‘VL-10′ could rapidly regain plant growth levers, and eliminate the delay of low temperature on flowering. TOPSIS analysis showed that the nutritional quality of tomatoes could be effectively improved by inoculation with B. methyltrophicus ‘VL-10′ regardless of normal cultivation or low-temperature stress. Full article

The present study aimed to characterize the potential plant growth-promoting rhizobacteria (PGPR) based on biochemical tests based on eight bacterial isolates, and to identify potential PGPR based on the 16S rRNA sequencing molecular method. Eight potential PGPR strains (UPMC1166, UPMC1168, UPMC1254, UPMC1376, UPMC1389, UPMC1393, UPMC703 and UPMC704) isolated from the soils in the oil palm (Elaeis guineensis) estates across Malaysia were selected because of their most PGPR activities. They were screened for nitrogen fixation, phosphate and potassium solubilization, and production of indole-3-acetic acid (IAA). All isolates showed the ability to grow between pH 2 to 9 and survive from 2 to 15% (w/v) of the salt medium. Among the isolated PGPRs, four PGPRs (UPMC1166, UPMC1168, UPMC1254 and UPMC1389) showed the ability to fix nitrogen and had the potential to produce IAA. Furthermore, two PGPRs (UPMC1393 and UPMC1376) demonstrated the ability to solubilize phosphate, while three PGPRs (UPMC703, UPMC704, and UPMC1393) showed the ability to solubilize potassium. Therefore, all the above eight PGPR isolates can benefit the oil palm cultivation industry. The molecular identification based on 16S rRNA gene sequence revealed that UPMC1166 was identified as Bacillus methylotrophicus; UPMC1168 as B. siamensis; UPMC1254 as B. subtilis; UPMC1389 as B. albus; UPMC1376 as Lactobacillus plantarum; UPMC1393 as B. marisflavi; UPMC703 as Burkhoderiaanthina and UPMC704 as B. metallica. These novel strains can be further investigated for their viability and effectiveness for bio-organic fertilizer production and application in the immature stage of oil palm growth. Full article

This study assessed the effects of Lactiplantibacillus plantarum (ZZU203), cellulase-producing Bacillus methylotrophicus (CB), or their combination (ZZU203_CB) on the fermentation parameters of alfalfa after 10 and 60 days of ensiling. Additionally, the bacterial community compositions were analyzed using absolute quantification 16S-seq (AQS). The results showed that CB silage displayed a higher lactic acid (LA) concentration at 10 d, a higher abundance of Lactobacillus, and lower abundance of Pediococcus, Enterococcus, and Weissella than those in the control (CK) silage. Compared with CK silage, the ZZU203 silage increased LA concentration, fructose and rhamnose concentrations, and the abundance of Lactobacillus, and decreased pH value, ammoniacal nitrogen, acetic acid, neutral detergent fiber and acid detergent fiber concentrations, and the abundance of Pediococcus, Enterococcus, Weissella, Hafnia, and Garciella after 60 days of ensiling. In addition, ZZU203 and ZZU203_CB silage had a similar silage quality and bacterial community, while the inoculation of ZZU203_CB significantly promoted LA accumulation and the numbers of Lactobacillus at 10 d compared with ZZU203 silage. Therefore, ZZU203 or a combination of ZZU203 and CB can be used as potential silage additives to improve the silage quality of alfalfa. Full article

Tomato production is gradually shifting to modern production, which requires the factorization of tomato seedlings to shorten the seedling cycle and improve the seedling quality. Bacillus methylotrophicus, as a biofertilizer for plant growth-promoting rhizobacteria, can promote plant growth and enhance native plant defenses. However, reports on the role of this type of bacterial agent in horticultural crop seedlings are limited. We investigated the effects of different dosages of Bacillus methylotrophicus (0.00, 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50 g/strain) on tomato plug seedlings and aimed to screen out the suitable dosage of Bacillus methylotrophicus for tomato seedlings in 50-hole cavity trays. In this experiment, with the increase in Bacillus methylotrophicus, the number of leaves, plant height, stem thickness, leaf area, dry matter accumulation in each organ, growth function (G value), and seedling strength index of tomato seedlings showed an increasing trend, followed by a decreasing one. The appropriate dosage (0.50–1.25 g/strain) of bacterial agent increased the activities of the substrates urease, sucrase, and catalase, thus forming a good microbial community to maintain the balance of organic and inorganic carbon and guaranteeing the normal development of the root system. Meanwhile, under the treatment of 1.00 and 1.25 g/strain of inoculum, the absorption range of tomato roots increased, more nitrogen, phosphorus, and potassium were absorbed from the substrate, and more nutrients were transported from the underground to the above-ground parts, which promoted shoot elongation and thickening of the shoots, increased the leaf number and dry matter accumulation, and improved the seedling quality. In this study, the mechanism of action of this microbial product on tomato seedlings was studied from the perspective of nutrient uptake and supply, and a sowing root application of 1.00 g/strain of Bacillus methylotrophicus in 50-hole cavity trays can improve the quality of tomato seedlings. Full article

(1) Background: Ilyonectria robusta can cause ginseng to suffer from rusty root rot. Secondary metabolites (SMs) produced by Bacillus methylotrophicus NJ13 can inhibit the mycelial growth of I. robusta. However, the molecular mechanism of the inhibition and response remains unclear. (2) Methods: Through an in vitro trial, the effect of B. methylotrophicus NJ13’s SMs on the hyphae and conidia of I. robusta was determined. The change in the physiological function of I. robusta was evaluated in response to NJ13’s SMs by measuring the electrical conductivity, malondialdehyde (MDA) content, and glucose content. The molecular interaction mechanism of I. robusta’s response to NJ13’s SMs was analyzed by using transcriptome sequencing. (3) Results: NJ13’s SMs exhibited antifungal activity against I. robusta: namely, the hyphae swelled and branched abnormally, and their inclusions leaked out due to changes in the cell membrane permeability and the peroxidation level; the EC₅₀ value was 1.21% (v/v). In transcripts at 4 dpi and 7 dpi, the number of differentially expressed genes (DEGs) (|log₂(fold change)| > 1, p adj ≤ 0.05) was 1960 and 354, respectively. NJ13’s SMs affected the glucose metabolism pathway, and the sugar-transporter-related genes were downregulated, which are utilized by I. robusta for energy production. The cell wall structure of I. robusta was disrupted, and chitin-synthase-related genes were downregulated. (4) Conclusions: A new dataset of functional responses of the ginseng pathogenic fungus I. robusta was obtained. The results will benefit the development of targeted biological fungicides for I. robusta and the study of the molecular mechanisms of interaction between biocontrol bacteria and phytopathogenic fungi. Full article

Vibriosis in farmed animals is a serious threat to aquaculture worldwide. Using probiotics and anti-Vibrio antimicrobial substances in aquaculture systems can be a means of preventing Vibrio infections. Therefore, we aimed to characterize and compare 16 potential anti-Vibrio probiotics (Vi+) isolated from marine sponges and fish intestines collected from the Vietnam Sea, as well as an anti-Vibrio bacteriocin to fully explore their application potentials. 16S rRNA sequencing confirmed all Vi+ to be Bacillus species with different strain variants across two sample types. An obvious antimicrobial spectrum toward Gram-negative bacteria was observed from intestinal Vi+ compared to sponge-associated Vi+. The reason was the higher gene frequency of two antimicrobial compounds, non-ribosomal peptides (NRPS) and polyketide type-I (PKS-I) from intestinal Vi+ (66.7%) than sponge-associated Vi+ (14.3% and 0%, respectively). Additionally, a three-step procedure was performed to purify an anti-Vibrio bacteriocin produced by B. methylotrophicus NTBD1, including (i) solvent extraction of bacteriocin from cells, (ii) hydrophobic interaction chromatography, and (iii) reverse-phase HPLC. The bacteriocin had a molecular weight of ~2–5 kDa, was sensitive to proteolysis and thermally stable, and showed a broad antimicrobial spectrum, all of which are essential properties for promising feed additives. This study provides necessary information of the potential of probiotic Bacillus species with anti-Vibrio antimicrobial properties to study their further use in sustainable aquaculture. Full article

Agriculture is in need of alternative products to conventional phytopharmaceutical treatments from chemical industry. One solution is the use of natural microorganisms with beneficial properties to ensure crop yields and plant health. In the present study, we focused our analyses on a bacterium referred as strain B25 and belonging to the species Bacillus velezensis (synonym B. amyloliquefaciens subsp. plantarum or B. methylotrophicus), a promising plant growth promoting rhizobacterium (PGPR) and an inhibitor of pathogenic fungi inducing crops diseases. B25 strain activities were investigated. Its genes are well preserved, with their majority being common with other Bacillus spp. strains and responsible for the biosynthesis of secondary metabolites known to be involved in biocontrol and plant growth-promoting activities. No antibiotic resistance genes were found in the B25 strain plasmid. In vitro and in planta tests were conducted to confirm these PGPR and biocontrol properties, showing its efficiency against 13 different pathogenic fungi through antibiosis mechanism. B25 strain also showed good capacities to quickly colonize its environment, to solubilize phosphorus and to produce siderophores and little amounts of auxin-type phytohormones (around 13,051 µg/mL after 32 h). All these findings combined to the fact that B25 demonstrated good properties for industrialization of the production and an environmental-friendly profile, led to its commercialization under market authorization since 2018 in several biostimulant preparations and opened its potential use as a biocontrol agent. Full article

Loquat fruit is one of the most perishable fruits in China, and has a very limited shelf life because of mechanical injury and microbial decay. Due to an increasing concern about human health and environmental security, antagonistic microorganisms have been a potential alternative for fungicides to control postharvest diseases. In this work, the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus methylotrophicus BCN2 and Bacillus thuringiensis BCN10 against five postharvest pathogens isolated from loquat fruit, Fusarium oxysporum, Botryosphaeria sp., Trichoderma atroviride, Colletotrichum gloeosporioides, and Penicillium expansum were evaluated by in vitro and in vivo experiments. As a result, the VOCs released by BCN2 and BCN10 were able to suppress the mycelial growth of all targeted pathogens according to inhibition ratio in the double petri-dish assay as well as disease incidence and disease diameter on loquat fruits. The main volatile compounds were identified by solid-phase microextraction (SPME)-gas chromatography. These VOCs produced by the two strains played complementary roles in controlling these five molds and enabled loquat fruits to keep fresh for ten days, significantly. This research will provide a theoretic foundation and technical support for exploring the functional components of VOCs applicable in loquat fruit preservation. Full article

Endophyte Bacillus methylotrophicus NJ13 was isolated from Panax ginseng. Its sterile fermentation liquid showed a significant inhibitory effect against Ilyonectria robusta, causing the rusty root rot of P. ginseng and P. quinquefolius. The antifungal protein was obtained after precipitation by 20% saturated ammonium sulfate, desalted by Sephadex G-25, weak anion exchange chromatography, and gel filtration chromatography. SDS-PAGE showed that the purified protein was approximately 29 KDa. The antifungal protein after desalting was not resistant to temperatures higher than 100 °C, resistant to acid conditions, and did not tolerate organic solvents and protease K. The amino acid sequence of purified antifungal protein had an identity of 76% to flagellin from Bacillus velezensis. The isoelectric point of the protein was 4.97 and its molecular mass was 27 KDa. Therefore, a specific primer G1 was designed based on the flagellin gene sequence, and a 770 bp gene sequence was cloned in NJ13 genomic DNA, which shared the same size of flagellin. There were ten base differences between the gene sequences of flagellin and the cloned gene, however, the amino acid sequence encoded by the cloned gene was identical to the flagellin. In conclusion, the antifungal protein produced by biocontrol agent NJ13 contained a flagellin protein. Full article