Licheniformis Isolates Research Articles

Evaluation of antibacterial and anticancer properties of secondary metabolites isolated from soil Bacillus spp focusing on two strains of Bacillus licheniformis and Bacillus siamensis

BackgroundBacillus strains are well recognized for their inherent production of bioactive compounds that exhibit antibacterial and anticancer properties. This study aims to evaluate the antimicrobial and anticancer effects of the secondary metabolite isolated from Bacillus licheniformis and Bacillus siamensis strain.Material and methodWe developed and purified a new soil-derived Bacillus strain to study its metabolites on cancer cells and bacteria. After evaluating the antimicrobial effects of the selected strains’ secondary metabolites by well diffusion, growth conditions and temperature optimised using liquid-liquid extraction, secondary metabolites isolated, and active compounds identified using GC-MS. Evaluation of PC-3 and HPrEpC cytotoxicity. AV/PI staining and comet assay assessed necrosis and apoptosis. Real-time PCR measured apoptotic gene expression. Finally, the scratch test measured cell movement.ResultsBacillus strain metabolites exhibit dual-purpose antimicrobial and anticancer properties. Bacillus licheniformis isolate 56 and S2-G12 isolate 60 demonstrated the greatest antibacterial activity. Among all Bacillus isolates, isolates 56 (Bacillus licheniformis) and 60 (Bacillus siamensis strain) had the highest antibacterial activity. Crude extracts obtained from strains 56 and 60 decreased PC-3 cell viability in a dose-dependent manner. At 200 µg/mL, the survival rate of cells treated with strain 56 and 60 crude extract was 23% and 25%, respectively (p < 0.001). The treatment of PC-3 cells with strains 56 and 60 crude extract led to considerable apoptosis (46.2% and 50.09%, respectively) compared to the control group. After treatment with the crude extract from strains 56 and 60 at an IC50 concentration, a significant number of PC-3 cells showed comet formation, indicating DNA fragmentation. Metabolites extracted from strain 56 and 60 enhanced caspase 3, caspase 8, and Bax genes expression and reduced Bcl-2 expression (p < 0.001). Cell migration was also prevented.ConclusionOur findings show that the secondary metabolites of B. licheniformis and B. siamensis have antibiotic and anticancer properties. However in vivo studies are necessary to confirm these findings.

Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives

In this study levels and types of microbial contaminants were investigated in 88 different plant-based ingredients including many that are used to manufacture dairy alternatives. Studied ingredients encompassed samples of pulses (pea, faba bean, chickpea, and mung bean), cereals/pseudocereals (oat, rice, amaranth and quinoa) and drupes (coconut, almond and cashew). The microbial analysis included: i) total viable count (TVC), ii) total aerobic mesophilic spore count (TMS), iii) heat resistant aerobic thermophilic spore count (HRTS), iv) anaerobic sulfite reducing Clostridium spore count (SRCS), and v) Bacillus cereus spore count (BCES). Microorganisms isolated from the counting plates with the highest sample dilutions were identified using 16S rRNA and MALDI-TOF MS analyses.Many of the investigated ingredients showed a high proportion of spores as part of their total aerobic mesophilic counts. In 63 % of the samples, the difference between TVC and TMS counts was 1 Log10 unit or less. This was particularly the case for the majority of pea isolates and concentrates, faba bean isolates, oat kernels and flakes, and for single samples of chickpea isolate, almond, amaranth, rice, quinoa, and coconut flours. Concentrations of TVC ranged between <1.0 and 5.3 Log10 CFU/g in different samples, and TMS varied between <1.0 and 4.1 Log10 CFU/g. Levels of HTRS, BCES and SRCS were generally low, typically around or below the LOD of 1.0 Log10 CFU/g.In total, 845 individual bacterial colonies were isolated belonging to 33 different genera. Bacillus licheniformis and B. cereus group strains were most frequently detected among Bacillus isolates, and these species originated primarily from pea and oat samples. Geobacillus stearothermophilus was the main species encountered as part of the HRTS. Among the Clostridium isolates, Clostridum sporogenes/tepidum were predominant species, which were mostly found in pea and almond samples. Strains with potential to cause foodborne infection or intoxication were typed using the PCR-based method for toxin genes detection. In the B. cereus group, 9 % of isolates contained the ces gene, 28 % contained hbl, 42 % cytK, and 69 % were positive for the nhe gene. Absence of the boNT-A and -B genes was confirmed for all isolated C. sporogenes/tepidum strains. Nearly all (98 %) B. licheniformis isolates were positive for the lchAA gene.Insight into the occurrence of microbial contaminants in plant-based ingredients, combined with knowledge of their key inactivation and growth characteristics, can be used for the microbial risk assessment and effective design of plant-based food processing conditions and formulations to ensure food safety and prevent spoilage.

Drought tolerance of Aspergillus violaceofuscus and Bacillus licheniformis and their influence on tomato growth and potassium uptake in mica amended tropical soils under water-limiting conditions.

Drought is a significant abiotic stress that alters plant physiology and ultimately affects crop productivity. Among essential plant nutrients, potassium (K) is known to mitigate the deleterious effect of drought on plant growth. If so, K addition or inoculation of potassium solubilizing microorganisms (KSMs) that are tolerant to drought should promote plant growth during water stress. Therefore, in this study, K solubilizing Aspergillus violaceofuscus and Bacillus licheniformis, isolated from saxicolous environments, were tested for their capacity to tolerate drought using different molecular weights (~4000, 6000, and 8000 Da), and concentrations (0, 250, 500, 750, 1000, and 1250 mg/L) of polyethylene glycol (PEG) under in vitro conditions. The results showed that high concentrations (750 and 1000 mg/L) of PEG with different molecular weight considerably improved bacterial cell numbers/fungal biomass and catalase (CAT) and proline activities. Moreover, the ability of KSMs alone or in combination to impart drought tolerance and promote plant growth in the presence and absence of mica (9.3% K2O) supplementation was tested in Alfisol and Vertisol soil types under greenhouse conditions. The results revealed that the tomato plants inoculated with KSMs individually or dually with/without mica improved the physiological and morphological traits of the tomato plants under drought. Generally, tomato plants co-inoculated with KSMs and supplemented with mica were taller (2.62 and 3.38-fold) and had more leaf area (2.03 and 1.98-fold), total root length (3.26 and 8.86-fold), shoot biomass (3.87 and 3.93-fold), root biomass (9.00 and 7.24-fold), shoot K content (3.08 and 3.62-fold), root K content (3.39 and 2.03-fold), relative water content (1.51 and 1.27-fold), CAT activity (2.11 and 2.14-fold), proline content (3.41 and 3.28-fold), and total chlorophyll content (1.81 and 1.90-fold), in unsterilized Alfisol and Vertisol soil types, respectively, than uninoculated ones. Dual inoculation of the KSMs along with mica amendment, also improved the endorrhizal symbiosis of tomato plants more than their individual inoculation or application in both soil types. These findings imply that the A. violaceofuscus and B. licheniformis isolates are promising as novel bioinoculants for improving crop growth in water-stressed and rainfed areas of the tropics in the future.

Bacterial community analysis of infant foods obtained from Chinese markets by combining culture-dependent and high-throughput sequence methods

In this study, twenty-two baby foods including cereal-based products and powdered infant formula (PIF) obtained from local markets were comprehensively investigated for their bacterial contamination using culture-dependent and high-throughput sequence (HTS) methods. In addition, the genetic diversity and biofilm-forming capacity of the most abundant species were analyzed using random amplified polymorphic DNA (RAPD) and crystal violet staining assay, respectively. Results showed that 170 mesophilic isolates collected from 22 samples were clustered into 15 genera and 41 species. Bacillus (77.65%) was the most prevalent genus, followed by Paenibacillus (7.06%), Alkalibacillus (3.53%), and Lysinibacillus (2.35%). Bacillus licheniformis (49.41%) proved to be the most dominant species in infant foods, and a high genetic diversity with six different RAPD profiles was observed. A total of 87.5% of B. licheniformis isolates were identified as strong biofilm formers, and heterogeneous biofilm-forming ability was observed among the isolates sharing the same RAPD pattern. HTS analysis revealed an 18-fold higher biodiversity at the genus level, and a significantly different bacterial community of infant foods was dominated by Lactococcus, Streptococcus, and Bifidobacterium. Foodborne pathogens including Bacillus cereus, and potentially pathogenic microorganisms such as Acinetobacter baumannii, were identified in infant foods by HTS. The current results could expand the crucial information about bacterial contamination of baby foods.

Lichenysin Production by Bacillus licheniformis Food Isolates and Toxicity to Human Cells.

Bacillus licheniformis can cause foodborne intoxication due to the production of the surfactant lichenysin. The aim of this study was to measure the production of lichenysin by food isolates of B. licheniformis in LB medium and skimmed milk and its cytotoxicity for intestinal cells. Out of 11 B. licheniformis isolates tested, most showed robust growth in high salt (1M NaCl), 4% ethanol, at 37 or 55°C, and aerobic and anaerobic conditions. All strains produced lichenysin (in varying amounts), but not all strains were hemolytic. Production of this stable compound by selected strains (high producers B4094 and B4123, and type strain DSM13T) was subsequently determined using LB medium and milk, at 37 and 55°C. Lichenysin production in LB broth and milk was not detected at cell densities < 5 log10 CFU/ml. The highest concentrations were found in the stationary phase of growth. Total production of lichenysin was 4–20 times lower in milk than in LB broth (maximum 36 μg/ml), and ∼10 times lower in the biomass obtained from milk agar than LB agar. Under all conditions tested, strain B4094 consistently yielded the highest amounts. Besides strain variation and medium composition, temperature also had an effect on lichenysin production, with twofold lower amounts of lichenysin produced at 55°C than at 37°C. All three strains produced lichenysin A with varying acyl chain lengths (C11–C18). The relative abundance of the C14 variant was highest in milk and the C15 variant highest in LB. The concentration of lichenysin needed to reduce cell viability by 50% (IC50) was 16.6 μg/ml for Caco-2 human intestinal epithelial cells and 16.8 μg/ml for pig ileum organoids. Taken together, the presence of low levels (<5 log10 CFU/ml) of B. licheniformis in foods is unlikely to pose a foodborne hazard related to lichenysin production. However, depending on the strain present, the composition, and storage condition of the food, a risk of foodborne intoxication may arise if growth to high levels is supported and such product is ingested.

Diverse thermophilic Bacillus species with multiple biotechnological activities are associated within the Egyptian soil and compost samples.

Thermophilic strains of Bacillus can express enzymes of higher thermal stability, which allows carrying out industrial fermentations under higher temperatures. This lowers the contamination potential, accelerates mixing rates and facilitates the recovery of fermentation end products. The present study was thus designed to isolate and characterize thermophilic Bacillus cultures from soil and compost samples. Forty-two thermophilic Bacillus isolates could be identified employing morphological, physiological and the 16S rRNA gene sequencing analyses. The isolates showed a high degree of biological diversity involving 13 Bacillus species and 1 subspecies but were dominated by Bacillus licheniformis. Phylogenetic analysis of B. licheniformis isolates based on the DNA sequencing of gyrA and rpoB genes presented them in two main genetic groups. Isolates of five thermophilic species including B. licheniformis, Bacillus altitudinis, Bacillus paralicheniformis, Bacillus subtilis and Bacillus thermoamylovorans showed multiple activities to degrade all of cellulose, hemicellulose and lignin. Those multifunctional thermophilic Bacillus isolates can be harnessed in the degradation of plant wastes for the production of biofuels and compost.

CRISPR-Cas9 mediated metabolic engineering of a mucoid Bacillus licheniformis isolate for mass production of 2,3-butanediol

Bacillus licheniformis is a bacterial strain generally recognized as safe (GRAS) with a tremendous potential for 2,3-butanediol (BDO) synthesis. Naturally isolated B. licheniformis usually secretes mucoid exopolymers, which hinder the strain development and metabolite production. A non-mucoid strain was generated by deleting both the pgsBCAE operon encoding polyglutamate synthase and the sacB gene encoding levansucrase using the CRISPR-Cas9 system. In addition, lactate, glycerol and ethanol were identified as the major byproducts of B. licheniformis. The corresponding genes including ldhA encoding lactate dehydrogenase, dgp encoding D-α-glycerophosphatase, and adhE encoding alcohol dehydrogenase were deleted, resulting in the generation of a byproduct-free strain via flask culture. The fed-batch fermentation yielded more than 120 g/L of 2,3-BDO with negligible amounts of other byproducts using a corn steep liquor (CSL)-based medium. This study is the first of its kind to demonstrate the development of non-mucoid and byproduct-free strains for mass production of 2,3-BDO using a naturally isolated B. licheniformis strain.

Biochemical and genomic identification of novel thermophilic Bacillus licheniformis strains YNP1-TSU, YNP2-TSU, and YNP3-TSU with potential in 2,3-butanediol production from non-sterile food waste fermentation

In the U.S., 30–40% of all food produced ends up as waste. By taking advantage of biological processes this waste nutrients can be converted into value-added 2,3-butanediol (2,3-BDO). However, food waste contains live bacteria and fungi which must first be inactivated, a process typically done with autoclaving under high pressure steam. If contaminants are not removed they can out-compete added industrial microbes for nutrients, resulting in lower yields of bio-products. Unfortunately, sterilization methods such as autoclaving greatly increase production time, and increase energy consumption. This creates a demand for novel microorganisms that can out-compete food waste microbiota by natural means without the need for sterilization of food waste. For this study, several Bacillus licheniformis isolates from Yellowstone National Park were investigated for their potential use in the 2,3-BDO industry. B. licheniformis species are a strain of industrial importance as of late, due to the wide metabolic functions and sustainable growth conditions above 45 °C. Whole genomic sequencing data and biochemical classification of B. licheniformis YNP1-TSU, B. licheniformis YNP2-TSU, and B. licheniformis YNP3-TSU were assessed and revealed a wide assortment of anti-microbial peptides and antibodies as well as catabolic genes for assimilation of glucose, fructose, galactose, xylose, arabinose, mannose, starch and sucrose, and linoleic acid. All three B. licheniformis strains had an optimal growth temperature of 50 °C, and were identified as high 2,3-BDO producers. Each strain YNP1-TSU, YNP2-TSU, and YNP3-TSU yielded 0.44 g/g, 0.45 g/g, and 0.43 g/g from mixed sugars found in non-sterilized food waste, respectively.

Sequence Analysis of the Complete Gene for Endoglucanase from Strains of Thermotolerant Bacillus

Three thermotolerant Bacillus isolates from the Philippines – identified as Bacillus subtilis (1280),Bacillus velezensis (1573), and Bacillus amyloliquefaciens (10105) – exhibited cellulase activity in carboxymethylcellulose (CMC) agar plate. Moreover, the expected size (approximately 1,500 bp) putative endoglucanase gene was amplified from all three local isolates. The endoglucanase gene amplified from a Bacillus licheniformis isolate (1320) was also used in the gene sequence analysis. A colorimetric dinitrosalicylic (DNS) acid assay revealed that the total endoglucanase activity of B. velezensis 1573 at 0.168 µM reducing sugar/min is significantly higher than the endoglucanase activity of the B. subtilis 1280 at 0.0673 µM rs/min and B. amyloliquefaciens 10105 at 0.0579 µM rs/min. Endoglucanase gene sequence analysis showed the highest pairwise identity between the endoglucanase gene of B. velezensis 1573 and B. amyloliquefaciens 10105 at 97.4%. The endoglucanase gene of B. licheniformis 1320 has the lowest pairwise identity at 74.6%, 76.2%, and 75.4% with B. amyloliquefaciens 10105, B. subtilis 1280, and B. velezensis 1573, respectively. Predicted amino acid sequences of the endoglucanases revealed variations at the signal peptide, catalytic core (CC) domain, linker, and carbohydrate-binding module (CBM). The endoglucanase of B. subtilis 1280 has a 506D insertion at the CBM domain. None of these variations, however, are located in regions implicated in catalytic reaction and substrate binding. Although the predicted protein structures using MODELLER revealed endoglucanases with good fit and high homology, variations in amino acid sequences may have resulted in the differences in enzyme activity and stability. The study generated gene and protein sequences as well as amplified genes that can be used as a starting template for enzyme improvement by rational design and site-specific mutagenesis or directed evolution using DNA shuffling.

Antibiotic resistance profile of local thermophilic Bacillus licheniformis isolated from Maysan province soil

The key concern for public health is that bacterial strains isolated from various ecosystems are immune to antibiotics used in human medicine, thus dramatically limiting therapeutic options and threatening the lives of infected people. The present study aims to reveal the antibiotics profile of fiftysix isolates of local thermophilic Bacillus licheniformis isolated from different environmental soil sites in Maysan city, Iraq. The antimicrobial agent resistance profile of B. licheniformis isolates was performed using the disc diffusion assay according to Kirby-Bauer susceptibility test protocol. The results showed that isolates were resistance against cefepime (n=56; 100%), amoxicillin (n=13; 23.3%) and ampicillin (n=52; 92.9%); and intermediate (n=56; 100%) against cephalothin and naldixic acid. The percentage resistance was low for aztreonam (n=4; 7%), chloramphenicol (n=3; 5%), clotrimazole (n=6; 10%), novobiocin (n=2; 3.5%) and ticarcillin (n=3; 5%). On the other hand, all isolates were sensitive (n=56; 100%) towards the following antibiotics: amikacin, ceftazidime, ciprofloxacin, clindamycin, imipenem, netilmycin, gentamicin, nitrofurantion, rifampin, trimethoprim and vancomycin. The results of this study suggest that the Iraqi thermophilic B. licheniformis isolates are variable in their susceptibility towards the standards antimicrobial agents. Furthermore, the presence of cefepime, amoxicillin, ampicillin, cephalothin and naldixic resistant isolates of B. licheniformis in Iraqi soils is of concern about how resistance could spread to other bacteria, and ultimately to humans.

Experimental and bioinformatics study for production of l-asparaginase from Bacillus licheniformis: a promising enzyme for medical application

A Bacillus licheniformis isolate with high l-asparaginase productivity was recovered upon screening two hundred soil samples. This isolate produces the two types of bacterial l-asparaginases, the intracellular type I and the extracellular type II. The catalytic activity of type II enzyme was much higher than that of type I and reached about 5.5 IU/ml/h. Bioinformatics analysis revealed that l-asparaginases of Bacillus licheniformis is clustered with those of Bacillus subtilis, Bacillus haloterans, Bacillus mojavensis and Bacillus tequilensis while it exhibits distant relatedness to l-asparaginases of other Bacillus subtilis species as well as to those of Bacillus amyloliquefaciens and Bacillus velezensis species. Upon comparison of Bacillus licheniformisl-asparaginase to those of the two FDA approved l-asparaginases of E. coli (marketed as Elspar) and Erwinia chrysanthemi (marketed as Erwinaze), it observed in a cluster distinct from- and with validly predicted antigenic regions number comparable to those of the two mentioned reference strains. It exhibited maximum activity at 40 °C, pH 8.6, 40 mM asparagine, 10 mM zinc sulphate and could withstand 500 mM NaCl and retain 70% of its activity at 70 °C for 30 min exposure time. Isolate enzyme productivity was improved by gamma irradiation and optimized by RSM experimental design (Box–Behnken central composite design). The optimum conditions for maximum l-asparaginase production by the improved mutant were 39.57 °C, 7.39 pH, 20.74 h, 196.40 rpm, 0.5% glucose, 0.1% ammonium chloride, and 10 mM magnesium sulphate. Taken together, Bacillus licheniformisl-asparaginase can be considered as a promising candidate for clinical application as antileukemic agent.

Spoilage potential of spore-forming bacteria from refrigerated raw milk

Aerobic bacterial spores are an important group of microorganisms in raw milk. These microbes are thermoduric, whereas the vegetative forms are thermophilic, thermoduric and psychrotrophic and reduce the shelf life of pasteurized milk. In Brazil, there are a lack of studies on the load of aerobic spores in raw milk; thus, little is known about the spoilage activity of these organisms. The aim the present study was to quantify the aerobic spores in Brazilian refrigerated raw milk of dairy region of Castro, Paraná state, assess the potential proteolytic and/or lipolytic isolates and identify the microorganisms derived from the germination. Twenty milk samples were evaluated, and the aerobic spore count was performed after plating the samples following heat treatment at 80°C for 12 min. The activity proteolytic and lipolytic isolates were evaluated through subculture on milk agar and tributyrin agar, respectively, and these microorganisms were identified using partial 16S rRNA gene sequences that were compared through GenBank. The aerobic spore counts ranged from 1 to 3.7 log CFU.mL-1, with a mean of 1.75 (± 0.59) log CFU.mL-1. After spore germination, 137 aerobic bacterial isolates were obtained, 40 of which (29.2%) showed milk spoilage activity. Among these, 31 isolates (77.5%) were proteolytic and lipolytic, seven isolates (17.5%) were exclusively lipolytic and two isolates (5%) were only proteolytic. Based on the 16S rRNA gene analysis, Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) and Brevibacillus spp. (2.5%) were identified. Studies of Brazilian raw milk microbiota have not yet described B. circulans which are frequently detected in milk from other countries. Among the 22 B. licheniformis isolates, 21 microbes (95.5%) showed proteolytic and lipolytic activity, and one isolate (4.5%) exhibited only proteolytic activity. The two B. pumilus isolates were proteolytic and lipolytic, whereas the B. circulans isolate was only lipolytic. Among the 11 Bacillus spp. isolates, eight isolates (72.7%) were proteolytic and lipolytic, one isolate (9.1%) was proteolytic and the other two isolates (18.2%) were lipolytic. The three Paenibacillus spp. and Brevibacillus spp. isolates were primarily lipolytic. Therefore, to extend the shelf life of pasteurized milk, preventive measures must be adopted to reduce contamination with spores because one-third of these microorganisms exhibited proteolytic and/or lipolytic activity.

Calcite-forming Bacillus licheniformis Thriving on Underwater Speleothems of a Hydrothermal Cave

The underwater environment of Grotta Giusti (Monsummano Terme, Italy) is a suggestive setting with different types of speleothems including “leafy” and “cauliflower” concretions along the walls and roof, and conical pseudo-stalagmites on the floor. Very high calcium and dissolved CO2 levels, and massive calcium carbonate precipitation characterize this cave environment. Yet, life thrives on the leafy concretion surfaces with loads of cultivable heterotrophic microorganisms around 105 colony-forming units per cm2. Bacillus licheniformis appeared to be the prevalent cultivable microorganism on a low-nutrient medium that was used for screening. 16S rRNA gene-based polymerase chain reaction–single strand conformation polymorphism profiling indicated that Group VI Bacillaceae species was well represented in the bacterial community of underwater speleothems. Interpretation of X-ray diffraction spectra and Raman spectroscopy data indicated that the B. licheniformis isolate produced in vitro abundant calcite microcrystals that were also characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Production of calcite microcrystals was analyzed in different media (Christensen’s urea agar and B4 calcium carbonate precipitation medium) and incubation conditions, and it was found to be enhanced by nitrate supplement in B4 medium under low-oxygen conditions. B4 and B4-nitrate media also stimulated antibiotic production by the B. licheniformis isolate, which was analyzed by microbiological assays.

Multiplex PCR identification and culture-independent quantification of Bacillus licheniformis by qPCR using specific DNA markers

Probiotics benefits in fish farming have been usually inferred appraising the effects observed on the host and not through the direct assessment of probiotic dynamics in the host gut microbiota. To overcome this gap, quantitative PCR (qPCR) can be a powerful approach to study the bacterial dynamics in fish gut microbiota. The presented work proposes four B. licheniformis-specific DNA markers and details a qPCR method to track putative probiotics B. licheniformis on fish gut. The four B. licheniformis-specific DNA markers - BL5B (hypothetical protein BL00303), BL8A (serA2), BL13C (rfaB) and BL18A (ligD) - were selected and validated by PCR and multiplex-PCR with 20 B. licheniformis isolates and a broad range of non-target bacteria. To assess the dynamics of B. licheniformis in the digesta of farmed fish, a qPCR was validated using markers BL8A and BL18A and calibration curves obtained for both markers with digesta samples spiked with B. licheniformis cells showed a high correlation (R2 > 0.99) over 6 log units (CFU/reaction), and a limit of detection (LOD) as low as 247 CFUs/reaction. Furthermore, the consistent qPCR repeatability and reproducibility underline the specificity and reliability of the qPCR proposed. Ultimately, the possibility to monitor the dynamics of B. licheniformis probiotics in the gut microbiota of farmed fish might be instrumental to optimize best practices in aquaculture.

Assessment of bacterial communities and activities of thermotolerant enzymes produced by bacteria indigenous to oil-bearing sandstone cores for potential application in Enhanced Oil Recovery

Enhanced Oil Recovery (EOR) is a potential approach to improve oil yield in petroleum production. In this research, indigenous bacterial communities residing in oil-bearing sandstone cores, taken from oil wells in Fang oil field in Northern Thailand, were investigated using the Ion Torrent PGM sequencing method. Based on 16rRNA gene sequences, Proteobacteria and Firmicutes were found to be the predominant phyla, and Bacillus, Sinomonas, Paenibacillus and Hydrogenophaga were the major genera. Thermotolerant lipase-esterase and urease producing bacteria were also isolated from the oil-bearing sandstone core samples. One lipase-esterase producing isolate, Bacillus licheniformis L3-2, produced the enzyme with highest activity at 80 °C. The enzyme retained up to 50% of its activity after incubation at 60 °C for 4 h. The results suggest the possibility of applying an in situ EOR approach using a combination of Enzyme Enhanced Oil Recovery (EEOR) and Microbial Enhanced Oil Recovery (MEOR) methods, with this lipase-esterase producing Bacillus licheniformis isolate, which is indigenous to one of the wells in this oil field.

The Potential Source of B. licheniformis Contamination During Whey Protein Concentrate 80 Manufacture.

The objective of this study was to determine the possible source of predominant Bacillus licheniformis contamination in a whey protein concentrate (WPC) 80 manufacturing plant. Traditionally, microbial contaminants of WPC were believed to grow on the membrane surfaces of the ultrafiltration plant as this represents the largest surface area in the plant. Changes from hot to cold ultrafiltration have reduced the growth potential for bacteria on the membrane surfaces. Our recent studies of WPCs have shown the predominant microflora B. licheniformis would not grow in the membrane plant because of the low temperature (10 °C) and must be growing elsewhere. Contamination of dairy products is mostly due to bacteria being released from biofilm in the processing plant rather from the farm itself. Three different reconstituted WPC media at 1%, 5%, and 20% were used for biofilm growth and our results showed that B. licheniformis formed the best biofilm at 1% (low solids). Further investigations were done using 3 different media; tryptic soy broth, 1% reconstituted WPC80, and 1% reconstituted WPC80 enriched with lactose and minerals to examine biofilm growth of B. licheniformis on stainless steel. Thirty-three B. licheniformis isolates varied in their ability to form biofilm on stainless steel with stronger biofilm in the presence of minerals. The source of biofilms of thermo-resistant bacteria such as B. licheniformis is believed to be before the ultrafiltration zone represented by the 1% WPC with lactose and minerals where the whey protein concentration is about 0.6%.

English

Head rot of cabbage caused by Sclerotinia sclerotiorum leads to rotting of fully matured cabbage heads in the field. In the present study the antagonistic effects of twenty Bacillus isolates was tested against S. sclerotiorum in vitro. Eight effective Bacillus isolates obtained from studies in vitro, commercial formulations of Trichoderma viride isolate TV-1 and Pseudomonas fluoroscens isolate Pf-1along with a fungicide check (Nativo-Tebuconazole+Trifloxystrobin) were carried further for field studies. Results of field studies indicated that fungicide check of Nativo (1.5 g/L) was highly effective with least disease incidence of 10.36% indicating 74.50% reduction over control. Among the biocontrol agents commercial formulation of Trichoderma viride isolate TV-1 was the most effective showing disease incidence of 11.38% indicating 72.00% reduction over control followed by Bacillus amyloliquefaciens isolate B15 and Pseudomonas fluoroscens isolate Pf-1 showing disease incidence of 13.24 and 13.31% indicating 67.41 and 67.24% reduction over control respectively and both treatments were on par. B. licheniformis isolate B16 was found to be least effective with 20.41 percent disease incidence indicating 49.76% reduction over control. &nbsp; Key words: Bacillus, commercial formulation, fungicide check, Pseudomonas fluoroscens,&nbsp; Trichoderma viride, Sclerotinia sclerotiorum.

English

In the aim of selecting starter cultures, thirteen species of Bacillus spp. including six Bacillus subtilis ssp. subtilis, four Bacillus licheniformis and three Bacillus amyloliquefaciens ssp. plantarum isolated from traditional Bikalga were investigated. The study included, for all isolates, genes, determination of minimal inhibitory concentration (MIC) for 24 antimicrobials and detection of resistance by PCR using specific primers. The isolates were also examined for their resistance to pH 2.5 and their tolerance to 0.3% bile over 4 h. Results showed that most studied isolates, in particular B. subtilis ssp. subtilis G2, H4, C6, I7 and B. amyloliquefaciens ssp. plantarum A4, I8, G3 were susceptible to most antimicrobials tested while all B. licheniformis isolates showed high resistance level. The resistance observed towards the antimicrobials (chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin and trimethoprim) in this study may be intrinsic as no positive amplicon was observed for the most prevalent resistance genes investigated (catpIP501, erm(A), erm(B), erm(C), aph(3”)-I, aph(3”)-III, ant(2”)-I, blaZ, aadA, aadE, StrA, StrB, dfr(A)). Furthermore, based on their good survival in pH 2.5 and in 0.3% bile all the tested isolates may be able to resist passage through the gastro-intestinal tract conditions. Regarding these results, isolates G2, C6, I7, H4, A4, I8 and G3 may be useful as starter cultures to optimize Hibiscus sabdariffa seeds fermentation into Bikalga. Key words: Bikalga, Bacillus, antimicrobial resistance, acid resistance, bile tolerance, starter cultures.

Investigation of the Antimicrobial Activity of Bacillus licheniformis Strains Isolated from Retail Powdered Infant Milk Formulae

This study investigated the potential antimicrobial activity of ten Bacillus licheniformis strains isolated from retail infant milk formulae against a range of indicator (Lactococcus lactis, Lactobacillus bulgaricus and Listeria innocua) and clinically relevant (Listeria monocytogenes, Staphylococcus aureus, Streptococcus agalactiae, Salmonella Typhimurium and Escherichia coli) microorganisms. Deferred antagonism assays confirmed that all B. licheniformis isolates show antimicrobial activity against the Gram-positive target organisms. PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses indicated that four of the B. licheniformis isolates produce the bacteriocin lichenicidin. The remaining six isolates demonstrated a higher antimicrobial potency than lichenicidin-producing strains. Further analyses identified a peptide of ~1,422 Da as the most likely bioactive responsible for the antibacterial activity of these six isolates. N-terminal sequencing of the ~1,422 Da peptide from one strain identified it as ILPEITXIFHD. This peptide shows a high homology to the non-ribosomal peptides bacitracin and subpeptin, known to be produced by Bacillus spp. Subsequent PCR analyses demonstrated that the six B. licheniformis isolates may harbor the genetic machinery needed for the synthesis of a non-ribosomal peptide synthetase similar to those involved in production of subpeptin and bacitracin, which suggests that the ~1,422 Da peptide might be a variant of subpeptin and bacitracin.

Skin-associated Bacillus, staphylococcal and micrococcal species from the house dust mite, Dermatophagoides pteronyssinus and bacteriolytic enzymes

Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents' and children's mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14K lytic enzyme is derived from a microbial source.