Piper cubeba L. is the berry of a shrub that is indigenous to Java, Southern Borneo, Sumatra, and other islands in the Indian Ocean. The plant is usually used in folk traditional medicine and is an important ingredient in cooking. The purpose of this study was to isolate and purify the bioactive compounds from P. cubeba L. fractions. In addition, the isolated compounds were tested for their antibacterial and antispore activities against vegetative cells and spores of Bacilluscereus ATCC33019, B. subtilis ATCC6633, B.pumilus ATCC14884, and B.megaterium ATCC14581. The phytochemical investigation of the DCM fraction yielded two known compounds: β-asarone (1), and asaronaldehyde (2) were successfully isolated and identified from the methanol extract and its fractions of P. cubeba L. Results showed that exposing the vegetative cells of Bacillus sp. to isolated compounds resulted in an inhibition zone with a large diameter ranging between 7.21 to 9.61 mm. The range of the minimum inhibitory concentration (MIC) was between 63.0 to 125.0 µg/mL and had minimum bactericidal concentration (MBC) at 250.0 to 500.0 µg/mL against Bacillus sp. Isolated compounds at a concentration of 0.05% inactivated more than 3-Log10 (90.99%) of the spores of Bacillus sp. after an incubation period of four hours, and all the spores were killed at a concentration of 0.1%. The structures were recognizably elucidated based on 1D and 2D-NMR analyses (1H, 13C, COSY, HSQC, and HMBC) and mass spectrometry data. Compounds 1, and 2 were isolated for the first time from this plant. In conclusion, the two compounds show a promising potential of antibacterial and sporicidal activities against Bacillus sp. and thus can be developed as an anti-Bacillus agent.
Currently the development of green chemistry approach with the use of biomaterial-based activities of microbial cells in the synthesis of various nanostructures has attracted a great attention. In this study, we report on the use of bacterium, Bacillus cereus as a biotemplating agent for the formation of zinc oxide nanoparticles with raspberry- and plate-like structures through a simple thermal decomposition of zinc acetate by maintaining the original pH of the reaction mixtures. Possible mechanism on the formation of the nanostructures is proposed based on the surface chemistry and biochemistry processes involved organic-inorganic interactions between zinc oxide and the microbial cells.
Citrus canker caused by Xanthomonas citri subsp. citri is a disease affecting the yield and fruit quality of lime (Citrus aurantiifolia). This research investigated endophytic bacteria obtained from six healthy Citrus spp. to inhibit the pathogen and to control citrus canker on lime plants. Numbers of the endophytic bacteria isolated from C. aurantifolia, C. hystrix, C. maxima, C. nobilis, C. reticulata and C. sinensis were 28, 25, 29, 42, 12 and 34 isolates, respectively. The selected endophytic bacteria that were effective against X. citri subsp. citri were Bacillus amyloliquefaciens LE109, B. subtilis LE24 and B. tequilensis PO80. The optimum culture medium for an antagonistic effect on the pathogen in B. amyloliquefaciens LE109 and B. tequilensis PO80 was yeast extract peptone dextrose broth, and in B. subtilis LE24 was modified soluble starch broth. To control citrus canker in lime, young expanded leaves of lime plants were aseptically punctured and inoculated with 30 μl of bacterial suspension of the pathogen (108 CFU/ml in 0.85% NaCl) per punctured location. After the pathogenic inoculation for 24 h, the leaves were then inoculated with 30 μl of the selected endophytic bacteria (108 CFU/ml in 0.85% NaCl), and treated with 30 μl of the culture media containing bioactive compounds produced by the selected endophytic bacteria. The leaves inoculated with cell suspensions of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. However, the leaves inoculated with B. tequilensis PO80 displayed 10% disease incidence. Additionally, the leaves treated with the crude bioactive compounds of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. Notably, the leaves treated with the crude bioactive compounds of B. tequilensis PO80 displayed 5% disease incidence. The results of this study showed that the Bacillus strains play important roles in the biocontrol of citrus canker in lime.
This study employed Bacillus spp. with α-amylase production isolated from Malaysian hot spring for domestic kitchen food waste treatment contained grains, vegetables, chicken and tuna that mimic the food waste discharge from domestic kitchens in Malaysian household. Results showed that Bacillus licheniformis HULUB1 and Bacillus subtilis SUNGB2 possess excellent amylolytic properties. Highest α-amylase activity was obtained when both isolates were cultivated at pH 6.0 and 65 °C with concentrations of 18.15 U/mL for HULUB1 and 22.14 U/mL for SUNGB2. Stability of α-amylase with significant levels of enzyme activity were recorded at 55-85 °C and pH 5.0-9.0. The extracted mixed α-amylase of HULUB1 and SUNGB2 showed greatest reduction were achieved at day 12 with 45% ± 0.03 solid content at 65 °C. While the mixed culture of HULUB1 and SUNGB2 displayed an enhanced effect on the food waste contents reduction with 43% ± 0.02 solid content at 45 °C after day 12. The findings showed that the combination of the two Bacillus spp. isolates possessed degradation of food wastes at faster rate than α-amylase. It was also pointed out that the standard food waste (SFW) and the treatment process assimilated for this study was suitable for the growth of Bacillus spp.
Certain rhizobacteria can be applied to remove arsenic in the environment through bioremediation or phytoremediation. This study determines the minimum inhibitory concentration (MIC) of arsenic on identified rhizobacteria that were isolated from the roots of Ludwigia octovalvis (Jacq.) Raven. The arsenic biosorption capability of the was also analyzed. Among the 10 isolated rhizobacteria, five were Gram-positive (Arthrobacter globiformis, Bacillus megaterium, Bacillus cereus, Bacillus pumilus, and Staphylococcus lentus), and five were Gram-negative (Enterobacter asburiae, Sphingomonas paucimobilis, Pantoea spp., Rhizobium rhizogenes, and Rhizobium radiobacter). R. radiobacter showed the highest MIC of >1,500 mg/L of arsenic. All the rhizobacteria were capable of absorbing arsenic, and S. paucimobilis showed the highest arsenic biosorption capability (146.4 ± 23.4 mg/g dry cell weight). Kinetic rate analysis showed that B. cereus followed the pore diffusion model (R2 = 0.86), E. asburiae followed the pseudo-first-order kinetic model (R2 = 0.99), and R. rhizogenes followed the pseudo-second-order kinetic model (R2 = 0.93). The identified rhizobacteria differ in their mechanism of arsenic biosorption, arsenic biosorption capability, and kinetic models in arsenic biosorption.
Oil palm empty fruit bunch (OPEFB), contains abundant cellulose and hemicelluloses and can be used as a renewable resource for fuel and chemical production. This study, as the first attempt, aims to convert OPEFB derived sugars to polyhydroxybutyrate (PHB). OPEFB collected from a Malaysia palm oil refinery plant was chemically pretreated and enzymatically hydrolyzed by an in-house prepared cellulase cocktail. The PHB producer, Bacillus megaterium R11, was isolated in Singapore and could accumulate PHB up to 51.3% of its cell dry weight (CDW) from both glucose and xylose. Tryptone was identified as its best nitrogen source. PHB content and production reached 58.5% and 9.32 g/L, respectively, for an overall OPEFB sugar concentration of 45 g/L. These respectively reached 51.6% and 12.48 g/L for OPEFB hydrolysate containing 60 g/L sugar with a productivity of 0.260 g/L/h.
An extractive bioconversion with Bacillus cereus cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) in aqueous two-phase system (ATPS) was investigated for the synthesis and recovery of cyclodextrins (CDs). Optimum condition for the extractive bioconversion of CDs was achieved in ATPS consisted of 7.7% (w/w) polyethylene glycol (PEG) 20,000 and 10.3% (w/w) dextran T500 with volume ratio (VR) of 4.0. Enzymatic conversion of starch occurred mainly in dextran-rich bottom phase whereas the product, CDs was transferred to top phase and a higher partition coefficient of CDs was achieved. Repetitive batch of CDs synthesis was employed by replenishment of the top phase components and addition of starch every 8h. An average total CDs concentration of 13.7 mg/mL, (4.77 mg/mLα-CD, 5.02 mg/mLβ-CD and 3.91 mg/mLγ-CD) was recovered in the top phase of PEG 20,000/dextran T500 ATPS. This study showed the effectiveness of ATPS application in extractive bioconversion of CDs synthesis with B. cereus CGTase.
There was high mortality in late larval instars of Aedes albopictus (Skuse) from laboratory and field populations in the 24 h after application of three Bactimos formulations of Bacillus thuringiensis H-14. Mortalities were higher and residual effects longer in field populations than in laboratory ones. Briquets were the most effective formulation (mortality 96-100% after five weeks; 76-92% after eight weeks). Culex quinquefasciatus Say larvae were tested only against the briquet formulation. In the laboratory, 100% mortality of late instars persisted for six weeks and dropped to 48-88% after eight weeks. In the field, late instars were reduced by 62-87% after 24 h and 69-72% after one week compared to increases in an untreated population of 160% and 176% respectively.
The pathogenicity of Bacillus sphaericus strain 1593 was tested against laboratory-reared larvae of four local species of mosquitoes of public health importance in Malaysia; Aedes aegypti, Anopheles balabacensis, Mansonia uniformis and Culex quinquefasciatus. The bacteria was shake-cultured at 28 +/- 1 degrees C for three days, using Glucose-Yeast Extract Salts medium. After which, the spores and vegetative cells were harvested and stored at 4 degrees C before use. Conditions for bioassays were mean temperature of 25 +/- 1 degrees C and relative humidity 65 +/- 5.0. Twenty third-instar larvae of each species were assayed in 90 ml of diluted spore solution. Each concentration and a control were replicated three times for each bioassay. Larval mortalities at 24 hours and 48 hours were taken and analyzed through Probit Analysis using a computer (IBM 370). LC50 values after 48 hours of exposure showed an increasing order of larval susceptibility as follows: Ae. aegypti (417.70 x 10(4)), An. balabacensis (45.84 x 10(4)), Ma. uniformis (18.23 x 10(4)) and Cx. quinquefasciatus (4.14 x 10(4) spores/ml). With the ability to kill 90% of the Cx. quinquefasciatus larvae tested with just a concentration of 10(5) spores/ml, B. sphaericus (strain 1593) has shown good potential as a biocontrol agent for this species of mosquito.
Bacillus thuringiensis israeliensis (BTI) against culicine mosquitoes was tried out in cement sullage drains in Kelang municipal area at a dosage of 0.15 ppm. and 0.6 ppm. The results of the trial showed that at 0.15 ppm. the BTI was not effective, but at 0.6 ppm. it was effective giving about 95% kill. There was no residual effect and treatment had to be repeated weekly.
Adulticidal and larvicidal performances of a water-based pyrethroid microemulsion Pesguard PS 102 (AI d-allethrin and d-phenothrin, both at 5.0% w/w) and Vectobac 12AS, an aqua-suspension Bacillus thuringiensis israelensis (B.t.i.) formulation (AI 1,200 ITU/mg) were assessed against mosquitoes Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using a Leco ULV Fog Generator Model 1600 and a Scorpion 20 ULV AirBlast Sprayer. Laboratory-cultured mosquito adults and larvae were used for efficacy assessment. For trials using Leco, both pyrethroid and bacterial formulations were dispersed both singly and in combination with Pesguard PS 102 at a dosage of 0.2 liters/ha and B.t.i. at a dosage of 1.0 liter/ha. Similar trials with the Scorpion were also conducted with Pesguard PS 102 at a dosage of 0.2 liters/ha and a higher dosage of B.t.i. (1.5 liters/ha). Experiments were conducted in a football field (200 x 100 m) where five check points at 10, 25, 50, 75, and 100 m downwind from the spray nozzle were chosen for efficacy assessments. Knockdown and mortality were scored at 1 and 24 h postspraying. Results from both trials showed that mortality values varied with distance from spray nozzle. For trials with Leco, fogging with the combination of Pesguard PS 102 and B.t.i. provided larvicidal mortality of > 80% for both Aedes species and of > 60% for Cx. quinquefasciatus larvae at several check points, depending on wind conditions. Complete mortality of adult Aedes mosquitoes at 24 h posttreatment was also achieved, while mortality values for Culex adults reached > 90% under strong wind conditions. As for trials with the Scorpion 20, high adult and larval mortalities were also achieved, with > 90% mortality at some check points. The above study demonstrated the possibility of achieving both larvicidal and adulticidal effects when using a combination of B.t.i. and Pesguard PS 102 in ULV space spray.
Evaluation of the effectiveness of Bacillus thuringiensis ssp. israelensis (B.t.i.) against mosquito larvae dispersed by ultralow volume (ULV) spraying was conducted in simulated field trials. Effectiveness was measured using 3 different indicators: larval mortality, colony-forming unit enumeration, and droplet analysis. B.t.i. was dispersed with a ULV generator using 2 different flow rates: 0.3 and 0.5 liter/min on 2 different days. Based on the results of this study, it can be concluded that an output of 0.3 liter/min is effective for controlling Aedes aegypti. although a dosage of 0.5 liter/min can be used when high residual activity is desired. For Culex quinquefasciatus control, both dosages were effective but with low residual activity. For Anopheles maculatus control, only a discharge rate of 0.5 liter/min was effective with low residual activity. B.t.i. application at both dosages penetrated tires well, indicating that B.t.i. ULV application is an effective method for controlling container-inhabiting mosquitoes. Good coverage of target area and penetration were attributed to satisfactory droplet profiles.
Susceptibility levels of a few laboratory-cultured and dengue-endemic area field-collected strains of Aedes aegypti and Aedes albopictus to Bacillus thuringiensis israelensis (Bti) at different storage ages were studied. The susceptibility of laboratory-cultured World Health Organization (WHO) Bora Bora reference, Vector Control Research Unit (VCRU), and Fumakilla Malaysia Berhad (FMB) strains of Ae. aegypti to Bti was examined. The sensitivity to Bti decreased with storage age. The median lethal concentration (LC50) for Bti increased by 2-3 times after 2 years compared to a fresh sample (3-6 months of storage). However, after the 2-year storage period, Bti still provided very good efficacy against all laboratory-cultured susceptible strains of Ae. aegypti and Ae. albopictus. The observed 95% lethal concentration values were about 20 times lower than the recommended concentration (6,000 international toxic units (ITU)/liter). Results obtained from the study against the dengue-endemic area field-collected strains of Ae. aegypti and Ae. albopictus confirmed the effectiveness of the Bti after storage for 2 years (18-24 months). For Ae. aegypti, the Ujung Batu strain was the most susceptible to Bti, whereas the Sungai Nibong strain showed the most tolerance. Susceptibility of laboratory-cultured strains varied; the Air Itam strain of Ae. albopictus was the most susceptible to Bti, whereas the Kampung Serani strain was the most tolerant among the field strains. However, the laboratory strain of Ae. albopictus was more susceptible than all the field strains.
Five formulations of Bacillus sphaericus (strain 2362) including aqueous suspension BSP 1, BSP 2, technical powder ABG 6184, corncob granules ABG 6185 (potencies 2 x 10(10), 2 x 10(7), 9.5 x 10(10), 5 x 10(10), 5 x 10(10) spore/g, respectively) and wettable powder ABG 6232 (1,000 BS ITU/mg) were tested against laboratory-cultured late third/early fourth instar larvae of Mansonia uniformis in floating screened cages in small plots at swampy ditches on Penang Island, Malaysia. Mean dosage/response values at 90% mortality levels were 6.93, 95.32, 1.45, 11.92 and 2.86 liters or kg per ha, respectively, for the formulations tested. There were practically no residual effects for the formulations tested with larvae introduced at 48, 96, and 168 h post-treatment. In trials of BSP 1, ABG 6184 and ABG 6185 (1 liter or 1 kg per ha) against immature Mansonia spp. in impounded paddy field ditches, improved efficacy and residual effects were obtained with mean reductions of 93.1, 91.9 and 80.4% at days 3, 7 and 14 posttreatment, respectively.
We report on the cloning of the lipase gene from Bacillus licheniformis IBRLCHS2
and the expression of the recombinant lipase. DNA sequencing analysis of the
cloned lipase gene showed that it shares 99% identity with the lipase gene from
B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino
acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then
cloned into the pET-15b(+) expression vector and the construct was transformed into
E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDSPAGE
where the lipase was found to have a molecular weight of about 23 kDa.
Bacillus thuringiensis is an anaerobic, spore forming bacterium that produces various toxic proteins both during its vegetative stage and sporulative stage. During its sporulative stage, it produces parasporal proteins that have long been used in the agriculture fields as insecticides. Although anticancer effect of Bacillus thuringiensis parasporal proteins can be dated back to the 1970s, research in this area went through a giant leap in the late 1990s, with much of the work being done in Japan. It has been found that some strains of non-insecticidal Bacillus thuringiensis produce parasporal proteins that exhibit anticancer activity. Due to their selectivity against human cancer cells but not normal cells, some of these proteins have been extensively studied for their anticancer effect and the mechanism of action by which these proteins kill cancer cells have also been widely explored in Japan and Malaysia with sporadic reports from other parts of the world. The abundance of these bacilli in nature and their selectivity have made them potential candidates for cancer treatment. However, literature on the in vivo effect of these proteins is scarce. Since different Bacillus thuringiensis strains produce different cytotoxic proteins with wide variations in their anticancer effect and mechanism of action, further investigations are necessary and their effect in vivo must be well established before they can be used in human subjects.
Matched MeSH terms: Bacillus; Bacillus thuringiensis; Lactobacillus casei
The lead (Pb2+) bioaccumulation capacities and mechanisms of three different physiological structures (vegetative cells, decay cells and spores) of B. coagulans R11 isolated from a lead mine were examined in this study. The results showed that the total Pb2+ removal capacity of vegetative cells (17.53 mg/g) was at its optimal and higher than those of the spores and decay cells at the initial lead concentration of 50 mg/L. However, when the initial lead concentration surpassed 50 mg/L, Pb2+ removal capacity of decay cells was more efficient. Zeta potential, Fourier transform infrared (FTIR) and functional group modification analyses demonstrated that the electrostatic attraction and chelating activity of the functional groups were the primary pathways involved in the extracellular accumulation of Pb2+ by the vegetative cells and spores. However, the primary Pb2+ binding pathway in the decay cells was hypothesized to be due to physical adsorption, which easily led to Pb2+ desorption. Based on these results, we conclude that the vegetative cell is the ideal lead sorbent. Therefore, it is important to inhibit the transformation of the vegetative cells into decay cells and spores, which can be achieved by culturing the bacteria under anaerobic conditions to prevent spore formation. Heat stimulation can effectively enhance spore germination to generate vegetative cells.
The compatibility of the commercial aqueous Bacillus thuringiensis serovar israelensis (B.t.i.) formulation, Vectobac 12AS, with the chemical insecticides Actellic 50EC, Aqua Resigen, Resigen, and Fendona SC, for the simultaneous control of Aedes larvae and adults was studied by dispersing nine different formulations using a portable mist blower, in single story half-brick houses. The effectiveness of the treatment was evaluated by measuring the larval mortality, adult mortality, and droplet analysis at varying distances from the sprayer. Persistence of the larvicidal activity of the chemical insecticides and B.t.i was also determined by measuring the larval mortality in the test samples 7 days posttreatment. The sprayed particles in all the trials were 50-60 microns in size, indicating that the particles were those of mist spray. Test samples placed within 3 m from the sprayer gave the maximum larval and adult mortality. Chemical insecticides exhibited maximum larval mortality in the 1 h posttreatment test samples and it was comparable to the larvicidal activity of B.t.i. The larvicidal toxins of B.t.i were more stable and were able to affect sufficient larval mortality for 7 days posttreatment. The larvicidal activity of the mixtures, i.e., chemical insecticides with B.t.i, in the 1 h posttreatment test samples was not significantly different from the larvicidal activity of the chemical insecticides and it was comparable to the larvicidal activity of B.t.i alone. However, the larvicidal activity of the mixtures was significantly more than the chemical insecticides alone in the 7 days posttreatment test samples except for the Actellic 50EC and Vectobac 12AS mixture. In all the trials, with or without B.t.i, there was no significant difference in adult mortality, indicating that this B.t.i formulation, Vectobac 12AS, was not antagonistic to the adulticidal activity of the chemical insecticides. From this study, it can be concluded that chemical insecticides can be used effectively for both adult and larval control, but the chemical insecticides do not exhibit residual larvicidal activity. Hence, for an effective control of both Aedes larvae and adults, it is advisable to add B.t.i. to the chemical insecticides, as B.t.i is specifically larvicidal and is also able to effect extended residual larvicidal activity.
Increased scientific interest has led to the rise in biotechnological uses of halophilic and halotolerant microbes for hypersaline wastewater bioremediation. Hence, this study performed molecular docking, molecular dynamic (MD) simulations, and validation by Molecular Mechanic Poisson-Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis H2. We aimed to identify the interactions of DehH2 with substrates haloacids, haloacetates, and chlorpyrifos under extreme salinity (35% NaCl). MD simulations revealed that DehH2 preferentially degraded haloacids and haloacetates (-6.3 to -4.7 kcal/mol) by forming three or four hydrogen bonds to the catalytic triad, Asp125, Arg201, and Lys202. Conversely, chlorpyrifos was the least preferred substrate in both MD simulations and MM-PBSA calculations. MD simulation results ranked the DehH2-L-2CP complex (RMSD □0.125-0.23 nm) as the most stable while the least was the DehH2-chlorpyrifos complex (RMSD 0.32 nm; RMSF 0.0 - 0.29). The order of stability was as follows: DehH2-L-2CP > DehH2-MCA > DehH2-D-2CP > DehH2-3CP > DehH2-2,2-DCP > DehH2-2,3-DCP > DehH2-TCA > DehH2-chlorpyrifos. The MM-PBSA calculations further affirmed the DehH2-L-2CP complex's highest stability with the lowest binding energy of -45.14 kcal/mol, followed closely by DehH2-MCA (-41.21 kcal/mol), DehH2-D-2CP (-31.59 kcal/mol), DehH2-3CP (-30.75 kcal/mol), DehH2-2,2- DCP (-29.72 kcal/mol), DehH2-2,3-DCP (-22.20 kcal/mol) and DehH2-TCA (-18.46 kcal/mol). The positive binding energy of the DehH2-chlorpyrifos complex (+180.57 kcal/mol) proved the enzyme's non-preference for the substrate. The results ultimately illustrated the unique specificity of the DehH2 to degrade the above-said pollutants under a hypersaline condition.Communicated by Ramaswamy H. Sarma.