Bacillus Toyonensis Research Articles

Combined Use of a Bacterial Consortium and Early-Colonizing Plants as a Treatment for Soil Recovery after Fire: A Model Based on Los Guájares (Granada, Spain) Wildfire.

During 2022, intense heat waves, together with particularly extreme dry conditions, created a propitious scenario for wildfires, resulting in the area of vegetation consumed in Europe doubling. Mediterranean countries have been particularly affected, reaching 293,155 hectares in Spain, the worst data in the last 15 years. The effects on the vegetation and the soil are devastating, so knowing the recovery factors is essential for after-fire management. Resilient microorganisms play a fundamental role in rapid nutrient recycling, soil structure, and plant colonization in fire-affected soils. In this present work, we have studied emergent microbial communities in the case of the Los Guájares (Granada, Spain) fire, one of the most extensive of the year, to evaluate their role in the recovery of soil and vegetation cover. We aim to discern which are the main actors in order to formulate a new treatment that helps in the ecosystem recovery. Thus, we have found the relevant loss in phosphorous and potassium solubilizers, as well as siderophores or biofilm producers. Here, we decided to use the strains Pseudomonas koreensis AC, Peribacillus frigoritolerans CB, Pseudomonas fluorescens DC, Paenibacillus lautus C, Bacillus toyonensis CD, and Paenarthrobacter nitroguajacolicus AI as a consortium, as they showed most of the capacities required in a regenerative treatment. On the other hand, the microcosm test showed an enhanced pattern of germination of the emerging model plant, Bituminaria bituminosa, as well as a more aggregated structure for soil. This new approach can create a relevant approach in order to recover fire-affected soils in the future.

Polychlorinated Biphenyl Transformation, Peroxidase and Oxidase Activities of Fungi and Bacteria Isolated from a Historically Contaminated Site.

Causing major health and ecological disturbances, polychlorinated biphenyls (PCBs) are persistent organic pollutants still recovered all over the world. Microbial PCB biotransformation is a promising technique for depollution, but the involved molecular mechanisms remain misunderstood. Ligninolytic enzymes are suspected to be involved in many PCB transformations, but their assessments remain scarce. To further inventory the capabilities of microbes to transform PCBs through their ligninolytic enzymes, we investigated the role of oxidase and peroxidase among a set of microorganisms isolated from a historically PCB-contaminated site. Among 29 isolated fungi and 17 bacteria, this work reports for the first time the PCB-transforming capabilities from fungi affiliated to Didymella, Dothiora, Ilyonectria, Naganishia, Rhodoturula, Solicoccozyma, Thelebolus and Truncatella genera and bacteria affiliated to Peribacillus frigotolerans, Peribacillus muralis, Bacillus mycoides, Bacillus cereus, Bacillus toyonensis, Pseudarthrobacter sp., Pseudomonas chlororaphis, Erwinia aphidicola and Chryseobacterium defluvii. In the same way, this is the first report of fungal isolates affiliated to the Dothiora maculans specie and Cladosporium genus that displayed oxidase (putatively laccase) and peroxidase activity, respectively, enhanced in the presence of PCBs (more than 4-fold and 20-fold, respectively, compared to controls). Based on these results, the observed activities are suspected to be involved in PCB transformation.

Isolation and Molecular Identification of Cellulase producing bacteria from soil and litter in Veerakkal forest area, The Western Ghats, India

The cellulolytic bacterial strains were screened from both soil and litter samples in different study sites of Veerakkal forest, Manar beat, The Western Ghats, India. In the current investigation, out of 64 and 48 differentiated total purified isolates, 53 soil isolates and 34 litter isolates were screened based on the CMC method. The percent wise calculation indicated that 82 % were cellulase positive while 18 % were negative in soil isolates. Likewise, 70 % of them were positive while 30 % were negative cellulase strains in litter isolates. The average hydrolysis capacity (HC) values ranged between 1.67 to 3.25 cm. The highly potential cellulolytic bacterial isolates viz. Bacillus thuringiensis, Bacillus licheniformis, Bacillus cereus, Bacillus sporothermodurans, Bacillus circulans, Bacillus albus, Bacillus amyloliquefaciens, Lysinibacillus sphaericus and Bacillus mobilis were identified from soil. Likewise, the litter cellulolytic bacterial species such as Bacillus sp., Lysinibacillus macroides, Bacillus paramycoides, Solibacillus silvestris, Pseudomonas stutzeri, Bacillus toyonensis, Bacillus subtilis, Bacillus pacificus, Bacillus flexus, Bacillus paranthracis and Pseudomonas putida were identified. The cellulolytic bacterial population was higher during the monsoon because of the presence of higher moisture content in leaf litter. However, the cellulolytic bacterial population of soil was higher during the pre-monsoon season because of the continuous breakdown of litter which inhaled more cellulolytic strains in the soil. Finally, it was concluded that corresponding with seasonal changes, the cellulolytic bacterial community also changed and it plays a significant role in the mineralization process.

Effect of a new probiotic on Artemia cysts determined by a convolutional neural network

One of the problems in sea farming is infections that cause mass mortality of crustaceans. To fight infections and improve sanitary conditions, farmers are actively using probiotic preparations. We aimed to study the effect of a new probiotic based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains on the incubation of Artemia franciscana cysts. Another purpose was to test a possibility of using a convolutional neural network for fast automatic counting of cysts, nauplii, and embryos.
 A pilot batch of the probiotic was prepared at the Prombiotech Engineering Center, Altai State University, from two strains of spore bacteria from the Center’s collection: B. toyonensis B-13249 and B. pumilus B-13250.
 The recommended amount of the probiotic was experimentally determined as 0.1 per 2 g of cysts. This concentration increased the number of hatched cysts by 1.4 and 10% in the batches from Lake Bolshoye Yarovoye (Z29.04) and from Lake Kuchuk (C9). It also increased the biomass yield to 7.40 ± 0.69 and 6.80 ± 0.43 g in these two batches, respectively, compared to the control samples where the yields were 5.30 ± 0.60 and 4.60 ± 0.50 g, respectively. The robot counter reduced the sample processing time 15 times and saved the data for further use.
 The probiotic based on B. toyonensis B-13249 and B. pumilus B-13250 had a positive effect on the hatching rate and biomass yield of A. franciscana. The new method for rapid counting of Artemia, which was based on the convolutional neural network and developed as an application of the Artemeter-1 robot, reduced the processing time and lowered labor costs.

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress.

Bacillus toyonensis SFC 500-1E is a member of the consortium SFC 500-1 able to remove Cr(VI) and simultaneously tolerate high phenol concentrations. In order to elucidate mechanisms utilized by this strain during the bioremediation process, the differential expression pattern of proteins was analyzed when it grew with or without Cr(VI) (10mg/L) and Cr(VI) + phenol (10 and 300mg/L), through two complementary proteomic approaches: gel-based (Gel-LC) and gel-free (shotgun) nanoUHPLC-ESI-MS/MS. A total of 400 differentially expressed proteins were identified, out of which 152 proteins were down-regulated under Cr(VI) and 205 up-regulated in the presence of Cr(VI) + phenol, suggesting the extra effort made by the strain to adapt itself and keep growing when phenol was also added. The major metabolic pathways affected include carbohydrate and energetic metabolism, followed by lipid and amino acid metabolism. Particularly interesting were also ABC transporters and the iron-siderophore transporter as well as transcriptional regulators that can bind metals. Stress-associated global response involving the expression of thioredoxins, SOS response, and chaperones appears to be crucial for the survival of this strain under treatment with both contaminants. This research not only provided a deeper understanding of B. toyonensis SFC 500-1E metabolic role in Cr(VI) and phenol bioremediation process but also allowed us to complete an overview of the consortium SFC 500-1 behavior. This may contribute to an improvement in its use as a bioremediation strategy and also provides a baseline for further research.

Sequential use of nutritional additives in diets for finishing Nellore steers in confinement.

The objective was to evaluate the effect of using prebiotics (Saccharomyces cerevisiae boulardii) or Monensin in the confinement initial phase and replacing monensin with probiotics (Bacillus toyonensis) in the final phase. Forty-eight Nellore steers were used, with an initial mean body weight of 356.2 ± 17.98kg, distributed in a completely randomized design. Two animals per pen were confined in 80 m2 pens. The experiment was divided into two stages. The first phase lasted from day 1 to the 30th day, during which the animals were divided into two groups of 24 animals each. The treatments were the nutritional additives added to the diet: monensin or prebiotics (Saccharomyces cerevisiae boulardii). In the second phase, each group was subdivided into 12 animals by treatment, which received monensin or probiotics (Bacillus toyonensis). Dry matter intake (DMI), animal performance, and economic evaluation of the use of additives were evaluated. There was no additive effect on DMI, average daily gain, and total weight gain of the animals in the first experimental stage (0-30th day). Likewise, in the second stage (31st-100th day), there was no treatment effect for the variables of intake and performance. There was no effect of the use of different nutritional additives on carcass characteristics. The use of prebiotics sequentially to probiotics promoted gross and net yield that was superior to that of the animals that consumed monensin. Yeasts and bacteria respectively in the first and second phases of confinement can replace monensin in confinement diets.

Mercury-resistant biofilm-forming bacteria and local plants in phytoremediation of small-scale gold mine tailings in Lombok Island, Indonesia

Small-scale gold mining is one of the sectors that contribute to the world's largest mercury contamination through the tailings it produces. Many efforts have been made to reduce mercury concentrations from tailings, one of which is by utilizing a combination of plants and bacteria. This study aims to analyze the combination of mercury-resistant biofilm-forming bacteria and local plants in the phytoremediation of small-scale gold mine tailings. This study used ten plant species divided into three groups and three biofilm-forming mercury-resistant bacteria (<em>Bacillus toyonensis</em>, <em>Burkholderia cepacia</em>, and <em>Microbacterium chocolatum</em>). Parameters observed included plant biomass, total chlorophyll, plant mercury content and media. The results showed that adding bacteria to each plant in the treatment had a different effect. Some plants with the addition of biofilm-forming bacteria had a higher wet weight than others. However, the addition of bacteria was not effective in increasing plant dry weight. The combination of biofilm-forming bacteria in the first and second plant groups reduced tailings mercury concentrations better than without the addition of bacteria. The combination of plants and bacteria in the third group gave higher media and plant mercury concentrations. This study shows that the addition of biofilm-forming bacteria can lead to increased remediation by plants. The second plant group treatment with a combination of <em>P. indica, P. conjugatum</em>, and <em>S. sesban</em> plants was the most effective in reducing tailings mercury content.

Characterization of the biosurfactant production and enzymatic potential of bacteria isolated from an oil-contaminated saline soil.

Biosurfactants are amphiphilic compounds with extensive applications in oily contaminated environments to remove hydrocarbons. Moreover, enzymes such as laccase and manganese peroxidase are responsible for the oxidation of a variety of phenolic compounds and aromatic amines. Therefore, in the present study, bacteria with the potential to produce biosurfactants and enzymes (namely, laccase, manganese peroxidase, and endoglucanase carboxymethyl cellulose (CMCase)) were isolated from petroleum oil-contaminated soil. From 15 isolated bacteria, three isolates were selected as the best producers of biosurfactants according to the related tests, such as tests for surface tension reduction. These three bacteria indicated tolerance to a salinity test and were classified as resistant and very resistant. The isolates 3, 12, 13, and 14 showed positive results for the degradation of guaiacol, phenol red, and carboxymethylcellulose, as well as the decoloration of methylene blue by the creation of a clear halo around the bacterial colony. Upon the quantitation of the laccase and manganese peroxidase activities, 22.58U/L and 21.81U/L, respectively, were measured by isolate 13. Furthermore, CMCase activity was recorded with 0.057436U/ml belonging to isolate 14. Bacterial strains with appreciable laccase, peroxidase, CMCase activity, and biosurfactant production potentials were identified through 16S rDNA sequence analysis as Bacillus sp. (isolate 3), Bacillus toyonensis (isolate 12), Bacillus cereus (isolate 13), and Bacillus tropicus (isolate 14), and their nucleotide sequences were deposited in the GenBank. The potentials for the industrial applicability of the biosurfactants and enzymes abound, and production needs to be optimized by the selected bacterial strains.

Caracterização bioquímica de micro-organismos promotores de crescimento de plantas individuais e combinados

ABSTRACT The increasing demand for using microorganisms in agriculture to improve food production requires constantly assessing microbial diversity. This study aimed to investigate the biochemical properties of individual and combined multifunctional microorganisms, as well as to identify potential applications in biotechnology or agriculture. The experiment comprised 29 treatments, with 7 single and 21 combined microorganisms: M01 (Serratia marcescens), M02 (Bacillus toyonensis), M03 (Phanerochaete australis), M04 (Trichoderma koningiopsis), M05 (Azospirillum brasilense), M06 (Azospirillum sp.), M07 (Bacillus sp.), M08 to M28 (combination among these microorganisms) and M29 (control - no microorganisms). All the single and combined treatments assimilated nitrogen, produced siderophores and indoleacetic acid and solubilized phosphate. Only the treatments M04, M13 and M26 produced HCN. Additionally, all treatments, except for M03, produced biofilm. Only M03, M07, M09, M10, M12 and M13 solubilized potassium.

POME Processing with Bioremediation Using Indigenous Bacteria (Bacillus toyonensis and Stenotrophomonas rhizophila), Electrocoagulation, and Bioremediation assisted with Electrocoagulation Methods

Palm oil mill effluent (POME) is wastewater containing dissolved materials with concentrations above the threshold. Generally, POME processing in Indonesia uses an open pond system. It is less efficient because it requires large areas and causing greenhouse gas effects. This research aims to improve the POME processing, three methods were studied; bioremediation using enrich indigent lypholitics bacteria (Bacillus toyonensis and Stenotrophomonas rhizophila.), electrocoagulation system, and combination of those methods. The parameters were measured were oil and fat contents, COD, pH, TSS, TDS, Fe and Cu metals. In the electrocoagulation method, the electrodes were used iron electrodes with a variation of electrical voltage 1.5, 3, 4.5 and 6 V. The bioremediation using indigent bacteria results in the removal of oil and fat -74.62%, COD -52.44%, TSS -43.11% and pH 6.38 at the incubation time for 6 days, TDS 21.42%, Fe -5.3% and Cu -64.70%. The electrocoagulation methods results oil and fat removal were -93.27%, COD was -85.71%, TDS -39%, TSS -84%, Cu -46%, Fe +92.35%, and pH 11.39 at a voltage of 6 V for 6 h. The combination of the two methods can reduce oil and fat levels by -87.39%, COD -56.70%, TDS -0.42%, TSS +15.88%, Cu -23.33%, Fe +93.74% and pH 8.82. The three methods were studied can improve the POME processing significantly.

272 Effects of a Probiotic Bacillus Strain on Ileal Digestibility of Crude Protein, Starch, Energy and fat and Total Tract Digestibility of Energy and Dietary Fiber in Diets fed to Weanling Pigs

Abstract An experiment was conducted to test the hypothesis that probiotic Bacillus toyonensis M15750 improve the apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of energy and nutrients when included in diets fed by weanling pigs. A control diet was formulated based on corn, soybean-meal, and distillers dried grains with solubles and a second diet was formulated by supplementing the probiotic Bacillus toyonensis M15750 (1 × 109 cfu/kg feed) to the control diet. Titanium dioxide (0.50 %) was added to the diets as an indigestible marker. Sixteen weanling barrows (10.7 ± 0.7 kg) that had a T-cannula in the distal ileum were allotted to the two diets with 8 replicate pigs per diet in a randomized complete block design with body weight being the blocking factor. Pigs were adapted to the diets and to the metabolism crates for 7 days, urine and fecal materials were collected during the following 4 days according to standard procedures using the marker-to-marker approach, whereas ileal digesta were collected for 8 h during the following 2 days. The AID and ATTD of energy and nutrients were calculated for each diet. The AID of dry matter and gross energy was greater (P < 0.05) and the AID of starch tended to be greater (P = 0.05) in the diet supplemented with the probiotic Bacillus toyonensis M15750 compared with the control diet (Table 1). However, no differences were observed for the ATTD of energy or nutrients between the 2 diets, which indicates that the increased digestibility in the small intestine of pigs fed the diet supplemented with Bacillus toyonensis M15750 was offset by a reduction in hindgut fermentation. These results indicate that inclusion of probiotic Bacillus toyonensis M15750 in diets for weanling pigs have the potential to improve the AID of dry matter, gross energy and starch.

Administration of some probiotic strains in the rearing water enhances the water quality, performance, body chemical analysis, antioxidant and immune responses of Nile tilapia, Oreochromis niloticus

The performance, efficiency of consumed feed, body chemical composition, survival rate, antioxidant and immunity parameters of Nile tilapia (Oreochromis niloticus) reared in probiotic-treated water were studied. Two hundred apparently healthy Nile tilapia (20 ± 0.3 g) juveniles were reared for 70 days in five different treatments, with five replications as the control group (clean water) and four test groups with two probiotics strains (Bacillus toyonensis and Geobacillus stearothermophilus) at two different levels (1 or 2 × 105 CFU ml−1) applied in rearing water. Fish reared in water supplemented with G. stearothermophilus at low level demonstrated significantly enhanced (p < 0.05) growth performances in terms of final body weight (FBW), weight gain (WG), daily weight gain (DWG), specific growth rate (SGR), and relative growth rate (RGR) compared to the control group. In the same context, supplied fish rearing water a lower amount of G. stearothermophilus (GS1) remarkedly reduced feed conversion ratio values when compared to the control group. In contrary, all other feed efficiency parameters increased significantly when G. stearothermophilus (GS1) water was added at low amount in the compartment with the untreated group. Moreover, probiotic water additives significantly reduced the range and median levels of unionized ammonia (NH3) in water when compared to the untreated group. According to the findings of the body chemical composition, treated tilapia water with a high level of B. toyonensis had significantly higher crude protein and fat levels, as well as lower ash levels, than the control group. When compared to the control group, probiotic-water supplementation significantly improved oxidative status and immunological activity at all bacterial dosage levels, with the fish group enriched with a high level of G. stearothermophilus recording the maximum values of both antioxidant and immune activity. Finally, results reveal that water treated with B. toyonensis or G. stearothermophilus as a probiotic promoted Nile tilapia growth and health status, and this technology may be applied to stimulate tilapia productivity in culture farms.

Assessment of Bacillus aerius and Bacillus toyonensis extracts as biocontrol agents against Biomphalaria alexandrina snails

Schistosomiasis is one of the most deleterious parasitic diseases in the world and it is transmitted by aquatic snails. Consequently, there is a continuous need to search for new molluscicides to control the snails. The present study aimed to evaluate the molluscicidal activity of two bacterial diethyl ether extracts against adult Biomphalaria alexandrina snails. The present study indicated that LC50 and LC90 values were 193.734 and 280.670 mg/L for Bacillus aerius extract and 88.814 and 103.653 mg/L for Bacillus toyonensis extract against B. alexandrina snails after 48 h of exposure. After a 21-day chronic exposure period, the results showed that the sublethal concentrations (LCo and LC10) of both B. aerius and B. toyonensis extracts significantly decreased the survival rates of B. alexandrina snails. Likewise, the sublethal concentration (LC10) caused biochemical changes, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase enzymes, total protein, albumin, globulin, urea and creatinine and induced histopathological alterations in the digestive and hermaphrodite glands of exposed snails after 21 days. Despite negative effects on snails, we did not observe substantial morality in Artemia salina, a crustacean, after exposure to both extracts emphasizing that these extracts may be safe on other non-target aquatic organisms. Ultimately, our research suggests that B. aerius and B. toyonensis extracts can be used as promising biocontrol agents of B. alexandrina snails.

OPTIMIZATION OF THE GROWTH OF PROTEOLYTIC BACTERIA Bacillus toyonensis AT DIFFERENT SALINITY AND CONCENTRATION OF TOFU WASTEWATER

Proteolythic bacteria, including Bacillus toyonensis, have potencial to be used in feed industry and in processing of industrial waste. Cultivation of this bacteria requires appropriate enviromental condition and substrate for the growth. The objective of this research was to observe the growth of B. toyonensis at different salinity and tofu wastewater concentration. This experimental research used completely randomized design consisted of two factors. Those were salinity (0‰, 15‰, dan 30‰) and different concentrations of tofu wastewater (8%, 10%, and 12%). Bacterial cell growth was observed by the spectrophotometric method and total plate count (TPC) method. The bacterial cell growth was observed at 0, 24, 48, 72 and 96 hours-incubation. The results indicated that the best medium for the growth B. toyonensis was in tofu wastewater concentration of 12% at salinity of 15‰. The optimum growth occurred at 48 hours-incubation.

Effect of Plant Growth-Promoting Bacteria on Biometrical Parameters and Antioxidant Enzymatic Activities of Lupinus albus var. Orden Dorado Under Mercury Stress.

Heavy metal contamination of soils is a large-scale environmental problem. It leads to significant disqualification of the territory, in addition to being a source of the potential risk to human health. The exposure of plants to mercury (Hg) generates responses in its growth and their oxidative metabolism. The impact of increasing concentrations of Hg on the development of Lupinus albus var. Orden Dorado seedlings has been studied, as well as the plant’s response to the maximum concentration of Hg that allows its development (16 μg ml–1). The result shows that only the inoculum with plant growth promoting bacteria (PGPB) allows the biometric development of the seedling (root length, weight, and number of secondary roots) and prevents the toxic effects of the heavy metal from aborting the seedlings. Specifically, treatments with strains 11, 20 (Bacillus toyonensis), 48 (not determined), and 76 (Pseudomonas syringae) are interesting candidates for further PGPB-assisted phytoremediation trials as they promote root biomass development, through their PGPB activities. The plant antioxidant response has been analyzed by quantifying the catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) enzyme activity in the root, under 16 μg ml–1 of HgCl2 and different PGPB treatments. Results show that, although Hg stress generally induces enzyme activity, strains 31 and 69I (Pseudomonas corrugata) and 18 and 43 (Bacillus toyonensis) can keep SOD and APX levels close to those found in control without Hg (p < 0.01). Strain 18 also shows a significant reduction of GR to control levels without Hg. The present work demonstrates the benefit of PGPB treatments in situations of high Hg stress. These findings may be a good starting point to justify the role of PGPB naturally isolated from bulk soil and the rhizosphere of plants subjected to high Hg pressure in plant tolerance to such abiotic stress conditions. More studies will be needed to discover the molecular mechanisms behind the phytoprotective role of the strains with the best results, to understand the complex plant-microorganism relationships and to find effective and lasting symbioses useful in bioremediation processes.

Rhizospheric Bacillus-Facilitated Effects on the Growth and Competitive Ability of the Invasive Plant Ageratina adenophora.

The rhizospheric microbial community affects the population establishment of invasive plants in introduced areas, among which Bacillus has numerous functions in promoting plant growth. This study isolated and enriched the Bacillus community in the rhizospheric soil of the invasive plant Ageratina adenophora and the native accompanying plant Rabdosia amethystoides. The effects of these rhizospheric Bacillus communities on the growth and competition of A. adenophora and R. amethystoides were evaluated in pot experiments. The results showed that the number and diversity of Bacillus in the rhizospheric soil of A. adenophora were higher than those of R. amethystoides (A. adenophora: 122 strains in soil, 16 Bacillus taxa; R. amethystoides: 88 strains in soil, 9 Bacillus taxa). After Bacillus inoculation of A. adenophora in a pot experiment, Bacillus idriensis, Bacillus toyonensis and Bacillus cereus were accumulated in the rhizospheric of A. adenophora, which significantly increased the nitrate nitrogen (NO3–-N) content in the soil and the total carbon and nitrogen concentrations in A. adenophora in the mixed treatment. The selective accumulation of Bacillus enhanced the competitive advantage of A. adenophora over the native accompanying plant; the corrected index of relative competition intensity of A. adenophora-inoculated Bacillus reached double that of the uninoculated treatment, and the growth of native plants was greatly suppressed under mixed planting. Our study confirmed that invasion of A. adenophora can lead to the accumulation of specific Bacillus taxa in the rhizospheric soil, which in turn can increase the competitive advantage of A. adenophora.

Isolation of Bacteria Capable of Degrading Various AHLs for Biofouling Control in Membrane Bioreactors

Membrane bioreactors (MBRs) are widely used to treat wastewater, mainly due to the production of high-quality effluent. However, biofilm forming on the surface of membranes can cause many problems, which remains one of the major limitations of this technique. Bacterial quorum quenching (QQ) has been proven to be a successful strategy to control biofouling in MBRs. However, for many QQ bacterial isolates, the detailed degradation rates of acyl homoserine lactones (AHLs) have rarely been reported. Therefore, this study aimed to isolate potential QQ bacteria and investigate their degradation rates against eight different AHLs. Results showed that four isolates (A9, A12, B11, and D3) exhibited consistent C8-HSL–(N-octanoyl-L-homoserine lactone) removal capabilities. These four isolates removed at least 70% of all AHLs tested within 180 min. They might have different QQ enzymes, based on our observation that the locations of enzyme activities differed. The bacteria most closely related to A9, A12, and B11 were Brucella anthropic, Bacillus cereus, and Bacillus toyonensis, respectively. Bacillus species have shown QQ activity in many studies, but AHL-reducing Brucella species have not been previously reported. Overall, this study extends our current knowledge of QQ bacteria that could be used to mitigate biofilm formation on MBR membranes.

Calcium carbonate precipitation by cave bacteria isolated from Kashmir Cave, Khyber Pakhtunkhwa, Pakistan.

The participation of numerous physicochemical and biological functions maintains the evolution and expansion of the remarkable nature. Due to its vast applicability in several engineering disciplines, naturally occurring bio-mineralization or microbially induced calcium carbonate (MICP) precipitation is attracting more interest. Cave bacteria contribute to the precipitation of calcium carbonate (CaCO3 ). In the present study, soil sediments were collected from Kashmir cave, KPK, Pakistan, and plated on B4 specific nutrients limited medium for bacterial isolation and the viable bacterial count was calculated. Three bacterial strains named GSN-11, TFSN-14, and TFSN-15 were capable of precipitating CaCO3 . These bacterial isolates were identified through 16S rRNA gene sequencing and strain GSN-11 was identified as Bacillus toyonensis, TFSN-14 as Paracoccus limosus and TFSN-15 as Brevundimonas diminuta. Enhanced CaCO3 precipitation potential of these bacteria strains was observed at 25°C and pH5. The precipitated CaCO3 was confirmed by scanning electron microscopy, X-ray powder diffraction, and Fourier transform infra-red spectroscopy. The findings showed that the precipitates were dominated by calcite, aragonite, and nanosize vaterite. Current research suggests that precipitation of CaCO3 by proteolytic cave bacteria is widespread in Kashmir cave and these bacterial communities can actively contribute to the formation of CaCO3 by enhancing the pH of the microenvironment. RESEARCH HIGHLIGHTS: Kashmir cave inhabit potentially active bacteria in terms of biogeochemical processes. Cave bacteria significantly precipitated CaCO3 . Calcite, aragonite, and nanosize vaterite were dominant in precipitates.

Effect of bactofugation of raw milk on total bacterial and aerobic spore counts and on microbial diversity of sporeformers

This study describes the effect of bactofugation (10,000 × g in a continuous flow of 10,000 L/h) of three batches of raw milk on total bacterial count (TBC) and aerobic spore count, and it also shows the effect on the microbial diversity of spore-forming bacteria by analysing their genetic variability through molecular approaches. Given that milk must be preheated to approximately 55 °C before being bactofuged, for comparison, the three batches were evaluated at different stages as refrigerated raw, preheated, and bactofuged milk. For preheated milk, it was found that bactofugation caused a significant reduction (p &lt; 0.05) of 99.52% (from 4.5 × 106 to 2.1 × 104 CFU/mL) in the mean TBC and of 95% (from 333 to 17 CFU/mL) in the aerobic mesophilic spore count. Due to the effect of bactofugation on preheated milk, a reduction of 82% was observed in both TBC and aerobic spore count. With respect to diversity, 107 isolates from raw milk, prior to bactofugation, and 16 isolates from bactofuged milk were recovered and grouped into 40 and 8 clusters, respectively. The predominant species detected in raw and preheated milk were Bacillus toyonensis (63% - 20 clusters) and Lysinibacillus fusiformis (15% - 8 clusters). Proportionally, B. toyonensis (69% - 6 clusters) and L. fusiformis (25% - 1 cluster) were predominant in bactofuged milk. B. pumilus, L. varians, B. flexus, B. invictae, and B. megaterium, bacteria with a known milk spoilage potential, were isolated from milk prior to bactofugation, and they reduced to undetectable levels in bactofuged milk. Bactofugation of milk, therefore, reduces the TBC and aerobic spore count, with a significant effect in reducing the microbial diversity of spore-forming bacteria, proportional to their incidence in raw milk. Therefore, bactofugation can be an alternative to increase the shelf life and technological potential of milk.

Molecular assessment of microbial diversity on eggshell of chukar partridges (Alectoris chukar) under semi-captive conditions

Due to human activity, climatic changes and habitat damage, the Chukar partridge (Alectoris chukar) is experiencing a population decrease. The goal of this work was to isolate and identify the inner eggshell microflora in order to learn more about their function in A. chukar embryo mortality and hatchability in semi-captive settings. During the egg-laying season, 204 eggs were gathered from the Balkasar Research Complex in Chakwal, Pakistan. These eggshells were divided into four categories after incubation: un-pricked with dead embryo, pricked with dead embryo, hatched, and unfertilized eggs. A total of 47 (23.03%) samples tested positive for gram-positive bacteria, which were then identified using 16S rRNA sequence analysis. Bacillus subtilis, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus toyonensis, Bacillus thuringiensis and Staphylococcus sciuri were among the identified species. Hatched eggshells had the highest percentage of bacterial flora (36.17%), followed by un-pricked with dead embryo (23.40%) and pricked with death embryo (21.27%), and unfertilized eggshells had the lowest percentage (19.14%). As a result, the findings of this study revealed that microbial contamination of eggshells could be a cause of early embryonic stage degeneration and bird death.