Search results for: Bacillus thuringiensis var israelinsis
Commenced in January 2007
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Edition: International
Paper Count: 334

Search results for: Bacillus thuringiensis var israelinsis

304 Antifungal Protein ~35kDa Produced by Bacillus cereus Inhibits the Growth of Some Molds and Yeasts

Authors: Saleh H. Salmen, Sulaiman Ali Alharbi, Hany M. Yehia, Mohammad A. Khiyami, Milton Wainwright, Naiyf S. Alharbi, Arunachalam Chinnathambi

Abstract:

An antifungal protein synthesized by Bacillus cereus has been partially purified by the use of ammonium sulfate precipitation and Sephadex-G-200 column chromatography. The protein was produced from Bacillus cereus grown in potato Dextrose Broth Medium (PDB) at 30 ºC for 3 days at 100 rpm. The protein showed antagonistic effect against some fungi and yeasts. Crude extract from medium and semi-purified protein were tested in vitro against both fungi and yeasts using the disc diffusion method in order to detect the inhibitory effect of the protein. Zones of inhibition of the following diameter were found (mm) were Alternaria alternate (28), Rhodotorula glutinis (20), Fusarium sp. (16), Rhizopus sp. (15), Penicillium digitatum (13), Mucor sp. (13) and Aspergillus niger (10). The isolated protein was found to have a molecular weight of ~35kDa by sodium deodecyl sulfate-poly acrylamide gel electrophoresis. The data showed that the protein of Bacillus cereus has antifungal activity, a fact which points to the possibility of using it as a bio-control agent against some fungi, findings which emphasize the potential role of B. cereus as an important bio-control agent.

Keywords: bacillus cereus, ~35kDa protein, molds, yeasts

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303 Bacillus cereus Bacteremia and Multi-Organ Failure With Diffuse Brain Hypoxia During Acute Lymphoblastic Leukemia Induction Therapy. A Case Report

Authors: Roni Rachel Mendelson, Caileigh Pudela

Abstract:

Bacillus cereus is a toxin-producing, facultatively anaerobic gram-positive bacterium that is widely distributed environmentally. It can quickly multiply at room temperature with an abundantly present preformed toxin. When ingested, this toxin can cause gastrointestinal illness, which is the commonly known manifestation of the disease. Bacillus cereus sepsis is a disease that is mostly concerning in the population of the immunocompromised patients. One of them is acute lymphoblastic leukemia’s patients during induction. Pediatric acute lymphoblastic leukemia is a common pediatric hematologic malignancy. It is characterized by the rapid proliferation of poorly differentiated lymphoid progenitor cells inside the bone marrow. We present here a 21-month-old boy undergoing induction chemotherapy for acute lymphoblastic leukemia who developed bacillus sepsis bacteremia and, as a result, multi organ failure leading to seizures and multiple strokes. Our case report highlights the extensive overall and neurological damage that can be caused because of bacillus cereus bacteremia, which can lead to higher mortality rate and decreased in survivorship in a highly curable disease. It is very subtle and difficult to recognize and appears to be deteriorating extremely fast. There should be a low threshold for work up and empiric coverage for neutropenic patients during acute lymphoblastic leukemia induction therapy.

Keywords: acute lymphoblastic leukemia, bacillus cereus, immunocompromised, sepsis

Procedia PDF Downloads 79
302 Entomopathogenic Bacteria as Biological Control Agents: Review Paper

Authors: Tadesse Kebede Dabsu

Abstract:

Insect pest is one the major limiting factor for sustainable food production. To overtake insect pest problem, since Second World War, producers have used excessive insecticide for insect pest management. However, in the era of 21st Century, the excessive use of insecticide caused insect resistant, insecticide bioaccumulation, insecticide hazard to environment, human health problem, and the like. Due to these problems, research efforts have been focused on the development of environmental free sustainable insect pest management method. To minimize all above mentioned risk utilizing of biological control such as entomopathogenicmicroorganism include bacteria, virus, fungus, and their productsare the best option for suppress insect population below certain density level. The objective of this review was to review the updated available studies and recent developments on the entomopathogenic bacteria (EPB) as biological control of insect pest and challenge of using them for control of insect pest. EPB’s mechanisms of insecticidal activities, type, taxonomy, and history are included in this paper body. EPB has been successfully used for the suppression of populations of insect pests. Controlling of harmful insect by entomopathogenic bacteria is an effective, low bioaccumulation in environment and food, very specific, reduce resistance risk in insect pest, economically and sustainable method of major insect pest management method. Identified and reported as potential major common type of entomopathogenic bacteria include Bacillus thuringiensis, Photorhabdus sp., Xenorhabdus spp.Walbachiaspp, Actinomycetesspp.etc. These bacteria being enter into insect body through natural opening or by vector release toxin protein inside of insect and disrupt the cell’s content cause natural mortality under natural condition. As per reported by different scientists, insect orders like Lepidoptera, Hemiptera, Hymenoptera, Coleoptera, and Dipterahave been successful controlled by entomopathogenic bacteria. As per coming across in different scientific research journals, much of the work was emphasised on Bacillus thuringiensisbsp. Therefore, for commercial production like Bacillus thuringiensi, detail research should be done on other bacteria species. The efficacy and practical application of EPB are restricted to some crops and greenhouse area, but their field application at farmers’ level very less. So still much work needs to be done to the practical application of the EPB at widely application. Their efficacy, pathogenicity, and host range test should be tested under environmental condition.

Keywords: insect pest, entomopathogenic bacteria, biological control, agent

Procedia PDF Downloads 136
301 Bioprotective Role of Soil Borne Bacillus Strains against Selected Fungal Pathogens of Agriculture Relevance

Authors: Asad Ali, Asif Jamal

Abstract:

The agriculture productivity losses due to microbial pathogens have been a serious issue in Pakistan and rest of the world. Present work was designed to isolate soil borne microorganisms having the antagonistic ability against notorious phytopathogens. From the initial collection of 23 bacterial isolates, two potent strains of Bacillus were screened on the basis of their comparative efficacy against devastating fungal pathogens. The strains AK-1 and AK-5 showed excellent inhibitory indexes against the majority of tested fungal strains. It was noted that both strains of Bacillus showed significant biocontrolling activity against Aspergillus flavus, Fusarium moniliforme, Colletotricum falcatum, Botrytis cinerea, Aspergillus niger, Fusarium oxysporum, Phythopthora capsici and Rhizopus oryzae. The strain AK-1 was efficient to suppress Aspergillus species and Rhizopus oryzae while AK-5 expressed significant antagonistic activity against Fusarium, Botrytis and Colletotricum species. On the basis of in vitro assay, it can be postulated that the Bacillus strains AK-1 and AK-5 can be used as bio-protective agent against various plant diseases. In addition, their applications as natural pesticides could be very helpful to prevent the adverse effects of chemical pesticides.

Keywords: bacillus species, biocontrol agent, biopesticides, phytopathogens

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300 Investigation into the Effectiveness of Bacillus Mucilaginosus in Liberation of Platinum Group Metals Locked in Silicates

Authors: Nokubonga G. Zulu, Bongephiwe M. Thethwayo, Mapilane S. Madiba, Peter A. Olubambi

Abstract:

In South Africa, PGMs’ metallurgy industry is now leaned on the Upper Group 2 (UG2) reef for the beneficiation of 4PGEs (Platinum, Palladium, Rhodium, and Ruthenium). The current effective beneficiation method is direct froth flotation which uses the hydrophobicity of liberated valuables minerals to carefully float them while hydrophilic gangue minerals report to the residue. PGMs are known to be associated with base metal sulphides which are hydrophobic; however, approximately 25% of PGMs from UG2 are associated with hydrophilic silicates, which results in high PGMs grade in the flotation residue. Further, the smallest size in which UG2 PGMs occur is approximately 9 microns which demands high grinding for liberation, imposing energy and cost implications. The use of Bacillus mucilaginosus to liberate PGMs using Bio-leaching of PGMs bearing Silicates is a promising cost-effective, energy-saving, and green solution to liberate PGMs locked in silicates. This is due to the ability of Bacillus mucilaginosus to generate extracellular polysaccharides (EPS) that are responsible for the leaching of silicate minerals. The bioleaching is done at a laboratory beaker using a cultivated Bacillus mucilaginosus as a lixiviant. The bioleaching residue is expected to have a reduced particle size due to silicate consumption, which reduces the need and cost associated with a secondary milling circuit. Moreover, the grade of the bioleaching product is increased since the silicates (gangue minerals) are consumed by Bacillus mucilaginosus; this serves as a pre-concentration step. This paper discusses an alternative liberation and pre-concentrating technique of PGMs that are associated with silicates using Bacillus mucilaginosus leaching to dissolve silicates.

Keywords: Bacillus mucilaginosus, bio-leaching of PGMs bearing silicates, liberation of PGMs, pre-concentration of PGMs

Procedia PDF Downloads 131
299 Bioprotective Role of Soil Borne Bacillus Strain against Selected Fungal Pathogens of Agriculture Relevance

Authors: Asif Jamal, Asad Ali, Muhammad Ishtiaq Ali

Abstract:

The agriculture productivity losses due to microbial pathogens have been a serious issue in Pakistan and rest of the world. Present work was designed to isolate soil borne microorganisms having the antagonistic ability against notorious phytopathogens. From the initial collection of 23 bacterial isolates, two potent strains of Bacillus were screened on the basis of their comparative efficacy against devastating fungal pathogens. The strains AK-1 and AK-5 showed excellent inhibitory indexes against the majority of tested fungal strains. It was noted that both strains of Bacillus showed significant biocontrolling activity against Aspergillus flavus, Fusarium moniliforme, Colletotricum falcatum, Botrytis cinerea, Aspergillus niger, Fusarium oxysporum, Phythopthora capsici and Rhizopus oryzae. The strain AK-1 was efficient to suppress Aspergillus species and Rhizopus oryzae while AK-5 expressed significant antagonistic activity against Fusarium, Botrytis, and Colletotricum species. On the basis of in vitro assay, it can be postulated that the Bacillus strains AK-1 and AK-5 can be used as a bio-protective agent against various plant diseases. In addition, their applications as natural pesticides could be very helpful to prevent the adverse effects of chemical pesticides.

Keywords: biological control, Bacillus spp, fungal pathogens, agriculture

Procedia PDF Downloads 270
298 Effect of Bacillus Pumilus Strains on Heavy Metal Accumulation in Lettuce Grown on Contaminated Soil

Authors: Sabeen Alam, Mehboob Alam

Abstract:

The research work entitled “Effect of Bacillus pumilus strains on heavy metal accumulation in lettuce grown on contaminated soil” focused on functional role of Bacillus pumilus strains inoculated with lettuce seed in mitigating heavy metal in chromite mining soil. In this experiment, factor A was three Bacillus pumilus strains (sequence C-2PMW-8, C-1 SSK-8 and C-1 PWK-7) while soil used for this experiment was collected from Prang Ghar mining site and lettuce seeds were grown in three levels of chromite mining soil (2.27, 4.65 and 7.14 %). For mining soil minimum days to germinate noted in lettuce grown on garden soil inoculated with sequence. Maximum germination percentage noted was for C-1 SSK-8 grown on garden soil, maximum lettuce height for sequence C-2 PWM-8, fresh leaf weight for C-1 PWK-7 inoculated lettuce, dry weight of lettuce leaf for lettuce inoculated with C-1 SSK-8 and C-1 PWK-7 strains, number of leaves per plant for lettuce inoculated with C-1 SSK-8, leaf area for C-2 PMW-8 inoculated lettuce, survival percentage for C-1 SSK-8 treated lettuce and chlorophyll content for C-2 PMW-8. Results related to heavy metals accumulation showed that minimum chromium was in lettuce and in soil for all three sequences, cadmium (Cd) in lettuce and in soil for all three sequences, manganese (Mn) in lettuce and in soil for three sequences, lead (Pb) in lettuce and in soil for three sequences. It can be concluded that chromite mining soil significantly reduced the growth and survival of lettuce, but when lettuce was inoculated with Bacillus.pumilus strains, it enhances growth and survival. Similarly, minimum heavy metal accumulation in plant and soil, regardless of type of Bacillus pumilus used, all three sequences has same mitigating effect on heavy metal in both soil and lettuce. All the three Bacillus pumilus strains ensured reduction in heavy metals content (Mn, Cd, Cr) in lettuce, below the maximum permissible limits of WHO 2011.

Keywords: bacillus pumilus, heavy metals, permissible limits, lettuce, chromite mining soil, mitigating effect

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297 Metagenomic Assessment of the Effects of Genetically Modified Crops on Microbial Ecology and Physicochemical Properties of Soil

Authors: Falana Yetunde Olaitan, Ijah U. J. J, Solebo Shakirat O.

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Genetically modified crops are already phenomenally successful and are grown worldwide in more than eighteen countries on more than 67 million hectares. Nigeria, in October 2018, approved Bacillus thuringiensis (Bt) cotton and maize; therefore, the need to carry out environmental risk assessment studies. A total of 15 4L octagonal ceramic pots were filled with 4kg of soil and placed on the bench in 2 rows of 10 pots each and the 3rd row of 5 pots, 1st-row pots were used to plant GM cotton seeds, while the 2nd-row pots were used for non-GM cotton seeds and the 3rd row of 5 pots served as control, all in the screen house. Soil samples for metagenomic DNA extraction were collected at random and at the monthly interval after planting at a distance of 2mm from the plant’s root and at a depth of 10cm using a sterile spatula. Soil samples for physicochemical analysis were collected before planting and after harvesting the GM and non-GM crops as well as from the control soil. The DNA was extracted, quantified and sequenced; Sample 1A (DNA from GM cotton Soil at 1st interval) gave the lowest sequence read with 0.853M while sample 2B (DNA from GM cotton Soil at 2nd interval) gave the highest with 5.785M, others gave between 1.8M and 4.7M. The samples treatment were grouped into four, Group 1 (GM cotton soil from 1 to 3 intervals) had between 800,000 and 5,700,000 strains of microbes (SOM), Group 2 (non GM cotton soil from 1 to 3 intervals) had between 1,400,600 and 4,200,000 SOM, Group 3 (control soil) had between 900,000 and 3,600,000 SOM and Group 4 (initial soil) had between 3,700,000 and 4,000,000 SOM. The microbes observed were predominantly bacteria (including archaea), fungi, dark matter alongside protists and phages. The predominant bacterial groups were the Terrabacteria (Bacillus funiculus, Bacillus sp.), the Proteobacteria (Microvirga massiliensis, sphingomonas sp.) and the Archaea (Nitrososphaera sp.), while the fungi were Aspergillus fischeri and Fusarium falciforme. The comparative analysis between groups was done using JACCARD PERMANOVA beta diversity analysis at P-value not more than 0.76 and there was no significant pair found. The pH for initial, GM cotton, non-GM cotton and control soil were 6.28, 6.26, 7.25, 8.26 and the percentage moisture was 0.63, 0.78, 0.89 and 0.82, respectively, while the percentage Nitrogen was observed to be 17.79, 1.14, 1.10 and 0.56 respectively. Other parameters include, varying concentrations of Potassium (0.46, 1,284.47, 1,785.48, 1,252.83 mg/kg) and Phosphorus (18.76, 17.76, 16.87, 15.23 mg/kg) were recorded for the four treatments respectively. The soil consisted mainly of silt (32.09 to 34.66%) and clay (58.89 to 60.23%), reflecting the soil texture as silty – clay. The results were then tested with ANOVA at less than 0.05 P-value and no pair was found to be significant as well. The results suggest that the GM crops have no significant effect on microbial ecology and physicochemical properties of the soil and, in turn, no direct or indirect effects on human health.

Keywords: genetically modified crop, microbial ecology, physicochemical properties, metagenomics, DNA, soil

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296 Production of Lignocellulosic Enzymes by Bacillus safensis LCX Using Agro-Food Wastes in Solid State Fermentation

Authors: Abeer A. Q. Ahmed, Tracey McKay

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The increasing demand for renewable fuels and chemicals is pressuring manufacturing industry toward finding more sustainable cost-effective resources. Lignocellulose, such as agro-food wastes, is a suitable equivalent to petroleum for fine chemicals and fuels production. The complex structure of lignocellulose, however, requires a variety of enzymes in order to degrade its components into their respective building blocks that can be used further for the production of various value added products. This study aimed to isolate bacterial strain with the ability to produce a variety of lignocellulosic enzymes. One bacterial isolate was identified by 16S rRNA gene sequencing and phylogenetic analysis as Bacillus safensis LCX found to have CMCase, xylanase, manganese peroxidase, lignin peroxidase, and laccase activities. The enzymes production was induced by growing Bacillus safensis LCX in solid state fermentation using wheat straw, wheat bran, and corn stover. The activities of enzymes were determined by specific colorimetric assays. This study presents Bacillus safensis LCX as a promising source for lignocellulosic enzymes. These findings can extend the knowledge on agro-food wastes valorization strategies toward a sustainable production of fuels and chemicals.

Keywords: Bacillus safensis LCX, high valued chemicals, lignocellulosic enzymes, solid state fermentation

Procedia PDF Downloads 294
295 Sensory and Microbial Properties of Fresh and Canned Calocybe indica

Authors: Apotiola Z. O., Anyakorah C. I., Kuforiji O. O.

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Sensory and microbial properties of fresh and canned Calocybe indica (milky mushroom) were evaluated. The mushroom was grown under a controlled environment with hardwood (Cola nitida) and rice bran substrate (4:1) canned in a brine solution of salt and citric acid. Analysis was carried out using standard methods. The overall acceptability ranged between 5.62 and 6.50, with sample S30 adjudged the best. In all, significant differences p<0.01 exist in the panelist judgment. Thus, the incorporation of salt and citric acid at 3.5g and 1.5g, respectively, improved sensory attributes such as texture, aroma, color, and overall acceptability. There was no coliform and fungi growth on the samples throughout the storage period. The bacterial count, on the other hand, was observed only in the fifth and sixth week of the storage period which varied between 0.2 to 0.9 x 103 cfu/g. The highest value was observed in sample S20 of the sixth week of storage, while the lowest value was recorded in sample S30 of the sixth week of storage. Based on 16S rRNA gene sequencing, bacterial species were taxonomically confirmed as Bacillus thuringiensis. The percentile compositions and Sequence ID of the bacterial species in the mushroom was 90%.

Keywords: bacterial count, microbial property, sensory, sawdust, texture

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294 Effect of Marine Stress Starvation Conditions on Survival and Retention of the Properties of Potential Probiotic Bacillus Strains

Authors: Abdelkarim Mahdhi, Fdhila Kais, Faouzi Lamari, Zeineb Hmila, Fathi Kamoun, Maria Ángeles Esteban, Amina Bakhrouf

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Pathogenic bacteria are considered to be responsible for several infectious diseases in aquaculture. To overcome diseases in fish culture, the use of antimicrobial drugs as strategy, have been adopted. The use of probiotic was a promising approach to avoid the risk associated to pathogenic bacteria. To find a biological control treatment against pathogens, we undertook this investigation to study the maintain of the probiotic properties of Bacillus sp., such as viability, adhesive ability to abiotic surface, antibacterial activity and pathogenicity/toxicity, under marine starvation conditions. Our data revealed that the tested strains maintained their capacity to inhibit pathogens in vivo and in vitro conditions. These strains maintain their adhesive capacity to polystyrene and do not demonstrate the pathogenic or toxic effect to the host. The obtained results give insight about the effect of starvation conditions on the physiological responses of these Bacillus strains that can be considered as a potential candidate’s probiotic.

Keywords: bacillus, probiotic, cell viability, starvation conditions

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293 Influence of Bio-Based Admixture on Compressive Strength of Concrete for Columns

Authors: K. Raza, S. Gul, M. Ali

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Concrete is a fundamental building material, extensively utilized by the construction industry. Problems related to the strength of concrete is an immense issue for the sustainability of concrete structures. Concrete mostly loses its strength due to the cracks produced in it by shrinkage or hydration process. This study aims to enhance the strength and service life of the concrete structures by incorporating bio-based admixture in the concrete. By the injection of bio-based admixture (BBA) in concrete, it will self-heal the cracks by producing calcium carbonate. Minimization of cracks will compact the microstructure of the concrete, due to which strength will increase. For this study, Bacillus subtilis will be used as a bio-based admixture (BBA) in concrete. Calcium lactate up to 1.5% will be used as the food source for the Bacillus subtilis in concrete. Two formulations containing 0 and 5% of Bacillus subtilis by weight of cement, will be used for the casting of concrete specimens. Direct mixing method will be adopted for the usage of bio-based admixture in concrete. Compressive strength test will be carried out after 28 days of curing. Scanning electron microscopy (SEM) and X-ray diffraction analysis (XRD) will be performed for the examination of micro-structure of concrete. Results will be drawn by comparing the test results of 0 and 5% the formulations. It will be recommended to use to bio-based admixture (BBA) in concrete for columns because of the satisfactory increase in the compressive strength of concrete.

Keywords: bio-based admixture, Bacillus subtilis, calcium lactate, compressive strength

Procedia PDF Downloads 222
292 Biological Control of Sclerotium rolfsii, Damping-off Disease on Centella asiatica

Authors: K. Sunitra, T. Srisuda

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Centella asiatica, asiatic pennywort is a medicinal herb plant used widely which held in herbal health care group. The problem of asiatic pennywort production is the outbreak of Sclerotium rolfsii causing a damp-off disease which caused plant stem turn yellowish, finally they begin to die and result in extremely damaging to growers. Therefore, the studies were caried out to control damping off with Trichoderma sp., Bacillus subtilis and fermented banana as compared to the control to suppress with bi-culture under the laboratory condition. It was found that Trichoderma harzianum showed the highest percentage of inbihition, 69.44%. The pot experiments in greenhouse condition showed that chemical had minimum of damping-off (31.54%) and highest yield (1.20 tons/rai) and following by Trichoderma harzianum and Bacillus subtilis treatment. Due to the chemical usage leaving toxic residues on plants and affect the human bodies. Trichoderma harzianum and Bacillus subtilis should be considered as alternatives which have percent of damp-off disease and yields as follows: 45.50 and 43.75%, and 1.12 and 1.09 tons/rai, respectively. These two products are known that they have no health risk for growers and consumers in the future.

Keywords: Centella asiatica, Sclerotium rolfsii, Trichoderma harzianum, Bacillus subtilis

Procedia PDF Downloads 299
291 In Vitro Assessment of Anti-microbial Properties of Murraya Koenigii Extract

Authors: Kinza Khan, Dad Muhmmad, Asif Saleem, Nadia Mukhtar, Tahir Yaqub

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Ethomedicines are more commonly used in underdeveloped and developing countries. These medicines are sometimes more potent in controlling microbial infections than conventional medicines. Medicinal plants have been common practice to cure many diseases for centuries. Murraya koenigii is one of these plants and is commonly used in South Asian countries as a flavoring agent in food. To evaluate its anti-microbial activity, six different bacterial strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Bacillus cereus and Klebsiella pneumonia were used. N-hexane extract of Murraya koenigii leaves shows maximum activity against Bacillus cereus. Acetone extract of Murraya koenigii shoots showed more efficient activity against Pseudomonas aeruginosa Dichloromethane extracts showed maximum activity against Bacillus cereus. Ethanol extract exhibited maximum activity against Pseudomonas aeruginosa and Klebsiella pneumoniae. The methanol extract of Murraya koenigii shoots displayed maximum antibacterial activity against Bacillus cereus. Antifungal activity Ethanol extract was more effective against Candida albicans.

Keywords: ethnomedicines, bacteria, fungi, murraya koenigii, antimicrobial activity

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290 Effect of Nitrogen Source on Production of CMCase by Bacillus megaterium 1295S Isolated from Sewage Treatment Plants

Authors: Adel A. S. Al-Gheethi, M. O. Abdul-Monem

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Cellulase-producing bacteria were isolated from wastewater and sludge, and identified as Bacillus megaterium 1295S, Sporosarcina pasteurii 586S, Bacillus subtilis 117S, Burkholderia cepacia 120S and Staphylococcus xylosus 222W. Among bacteria, B. megaterium 1295S was the best cellulase producer under the catabolic repression and was therefore selected to study the factors affecting cellulase production. The optimum conditions for cellulase production were observed in CMC-Yeast Extract (CYE) agar medium (pH 6.5) inoculated with 0.4 mL of bacterial culture and incubated at 45˚C for 72 h. Twenty amino acids were introduced into the production medium as nitrogen source to investigate the production of cellulase in presence of amino acids in comparison to peptone (as an organic source) and sodium nitrate (as an inorganic source). The results found that the maximum production of cellulase was recorded at 50 ppm when L-hydroxy proline, L-arginine, glycine, L-histidine, L-leucine, DL-isoleucine, DL-β-phenylalanine were used as sole nitrogen sources and at 100 ppm when DL-threonine, L-ornithine 12.29, L-proline were used as sole nitrogen sources. The highest biomass yield was found when glycine 5 ppm and DL-serine 100 ppm used as a nitrogen source.

Keywords: CMCase, Bacillus megaterium 1295S, factors, amino acids

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289 Bioremediation of Phenanthrene by Monocultures and Mixed Culture Bacteria Isolated from Contaminated Soil

Authors: A. Fazilah, I. Darah, I. Noraznawati

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Three different bacteria capable of degrading phenanthrene were isolated from hydrocarbon contaminated site. In this study, the phenanthrene-degrading activity by defined monoculture was determined and mixed culture was identified as Acinetobacter sp. P3d, Bacillus sp. P4a and Pseudomonas sp. P6. All bacteria were able to grow in a minimal salt medium saturated with phenanthrene as the sole source of carbon and energy. Phenanthrene degradation efficiencies by different combinations (consortia) of these bacteria were investigated and their phenanthrene degradation was evaluated by gas chromatography. Among the monocultures, Pseudomonas sp. P6 exhibited 58.71% activity compared to Acinetobacter sp. P3d and Bacillus sp. P4a which were 56.97% and 53.05%, respectively after 28 days of cultivation. All consortia showed high phenanthrene elimination which were 95.64, 79.37, 87.19, 79.21% for Consortia A, B, C and D, respectively. The results indicate that all of the bacteria isolated may effectively degrade target chemical and have a promising application in bioremediation of hydrocarbon contaminated soil purposes.

Keywords: phenanthrene, consortia, acinetobacter sp. P3d, bacillus sp. P4a, pseudomonas sp. P6

Procedia PDF Downloads 294
288 Protective Effect of hsa-miR-124 against to Bacillus anthracis Toxins on Human Macrophage Cells

Authors: Ali Oztuna, Meral Sarper, Deniz Torun, Fatma Bayrakdar, Selcuk Kilic, Mehmet Baysallar

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Bacillus anthracis is one of the biological agents most likely to be used in case of bioterrorist attack as well as being the cause of anthrax. The bacterium's major virulence factors are the anthrax toxins and an antiphagocytic polyglutamic capsule. TEM8 (ANTXR1) and CMG2 (ANTXR2) are ubiquitously expressed type I transmembrane proteins, and ANTXR2 is the major receptor for anthrax toxins. MicroRNAs are 21-24 bp small noncoding RNAs that regulate gene expression by base pairing with the 3' UTR (untranslated regions) of their target mRNAs resulting in mRNA degradation and/or translational repression. MicroRNAs contribute to regulation of most biological processes and influence numerous pathological states like infectious disease. In this study, post-exposure (toxins) protective effect of the hsa-miR-124-3p against Bacillus anthracis was examined. In this context, i) THP-1 and U937 cells were differentiated to MΦ macrophage, ii) miRNA transfection efficiencies were evaluated by flow cytometry and qPCR, iii) protection against Bacillus anthracis toxins were investigated by XTT, cAMP ELISA and MEK2 cleavage assays. Acknowledgements: This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under Grant SBAG-218S467.

Keywords: ANTXR2, hsa-miR-124-3p, MΦ macrophage, THP-1, U937

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287 Current Status of Mosquitoes Vector Research and Control in Iran

Authors: Seyed Hassan Moosa-kazemi, Hassan Vatandoost

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Malaria, Dirofilaria immitis (dog heart worm), and D. repens (dirofilariasis), which are transmitted by mosquitoes, have been reported in Iran. The Iranian mosquito fauna includes seven genera, 65 species, and three subspecies. Aedes albopictus has been reported since. West Nile, Sindbis, Dengue, Japanese encephalitis viruses, and the nematode Setaria (setariasis) has been reported in the country but there are no information about their vectors in Iran. Iran is malaria elimination phase. Insecticides residual spraying (IRS), distributed of insecticides long lasting treated nets (ITNs), fogging, release of larvivours fishes and Bacillus thuringiensis, chemical larviciding, as well as case finding and manipulation and modification of breeding places carried out thought the IVM program in the country. Prolonged exposure to insecticides over several generations of the vectors, develop resistance, a capacity to survive contact with insecticides. However, use of insecticides in agriculture has often been implicated as contributing to resistance in mosquito’s vectors. Resistance of mosquitoes to some insecticides has been documented just within a few years after the insecticides were introduced. Some enzymes such as monooxygenases, esterases and glutathione S-transferases have been considered as a reason for resistance to pyrethroid insecticides. In conclusion, regarding to documented resistance and tolerance of mosquitoes vectors to some insecticides, resistance management is suggested by using new insecticide with novel mode of action.

Keywords: control, Iran, resistance, vector

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286 Impact of Bacillus subtilis Exotoxins on Fecundity, Sex Hormones and Release of Schistosoma mansoni cercariae in Biomphalaria alexandrina Snails

Authors: Alaa A. Youssef, Mohamed A. El-Emam, Momeana B. Mahmoud, Mona Ragheb

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Schistosomiasis, also known as bilharzia, is a disease caused by a parasitic trematode worm called Schistosoma. Biological control of the snail intermediate hosts of Schistosoma is one of the promising methods for eliminating this disease in Egypt. The molluscicidal activity of exotoxins secreted from Bacillus subtilis bacteria was studied. The effect of these exotoxins was studied on the fecundity of Biomphalaria alexandrina snails the intermediate host of Schistosoma mansoni; the fecundity includes the reproductive rate (R0) of B. alexandrina snails and levels of sex hormones (progesterone, testosterone, and estradiol). Moreover, the cercarial production of S. mansoni was determined. The results showed a significant reduction in the egg-laying capacity of the treated snails after exposure to sublethal concentrations ( LC10 and LC25) of B. Subtilis exotoxins; this reduction reached 70% at LC25. Moreover, B. Subtilis exotoxins' significantly suppressed the cercarial production of B. alexandrina snails. It is concluded that the exotoxins of Bacillus subtilis bacteria play an important role in the interference of the Schistosomiasis transmission, hence should be applied in the strategy of schistosomiasis control.

Keywords: schistosomiasis, Biomphalaria alexandrina snails, Bacillus subtilis bacteria, fecundity, sex hormones

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285 Evaluation of Paper Effluent with Two Bacterial Strain and Their Consortia

Authors: Priya Tomar, Pallavi Mittal

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As industrialization is inevitable and progress with rapid acceleration, the need for innovative ways to get rid of waste has increased. Recent advancement in bioresource technology paves novel ideas for recycling of factory waste that has been polluting the agro-industry, soil and water bodies. Paper industries in India are in a considerable number, where molasses and impure alcohol are still being used as raw materials for manufacturing of paper. Paper mills based on nonconventional agro residues are being encouraged due to increased demand of paper and acute shortage of forest-based raw materials. The colouring body present in the wastewater from pulp and paper mill is organic in nature and is comprised of wood extractives, tannin, resins, synthetic dyes, lignin and its degradation products formed by the action of chlorine on lignin which imparts an offensive colour to the water. These mills use different chemical process for paper manufacturing due to which lignified chemicals are released into the environment. Therefore, the chemical oxygen demand (COD) of the emanating stream is quite high. This paper presents some new techniques that were developed for the efficiency of bioremediation on paper industry. A short introduction to paper industry and a variety of presently available methods of bioremediation on paper industry and different strategies are also discussed here. For solving the above problem, two bacterial strains (Pseudomonas aeruginosa and Bacillus subtilis) and their consortia (Pseudomonas aeruginosa and Bacillus subtilis) were utilized for the pulp and paper mill effluent. Pseudomonas aeruginosa and Bacillus subtilis named as T–1, T–2, T–3, T–4, T–5, T–6, for the decolourisation of paper industry effluent. The results indicated that a maximum colour reduction is (60.5%) achieved by Pseudomonas aeruginosa and COD reduction is (88.8%) achieved by Bacillus subtilis, maximum pH changes is (4.23) achieved by Pseudomonas aeruginosa, TSS reduction is (2.09 %) achieved by Bacillus subtilis, and TDS reduction is (0.95 %) achieved by Bacillus subtilis. When the wastewater was supplemented with carbon (glucose) and nitrogen (yeast extract) source and data revealed the efficiency of Bacillus subtilis, having more with glucose than Pseudomonas aeruginosa.

Keywords: bioremediation, paper and pulp mill effluent, treated effluent, lignin

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284 The Genus Bacillus, Effect on Commercial Crops of Colombia

Authors: L. C. Sánchez, L. C. Corrales, A. G. Lancheros, E. Castañeda, Y. Ariza, L. S. Fuentes, L. Sierra, J. L. Cuervo

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The importance of environment friendly alternatives in agricultural processes is the reason why the research group Ceparium, the Colegio Mayor de Cundinamarca University, Colombia, investigated the genus Bacillus and its applicability for improving crops of economic importance in Colombia. In this investigation, we presented a study in which the genus Bacillus plays a leading role as beneficial microorganism. The objective was to identify the biochemical potential of three indigenous species of Bacillus, which were able to carry out actions for biological control against pathogens and pests or promoted growth to improve productivity of crops in Colombia. The procedures were performed in three phases: first, the production of biomass of an indigenous strain and a reference strain starting from culture media for production of spores and toxins were made. Spore count was done in a Neubauer chamber, concentrations of spores of Bacillus sphaericus were prepared and a bioassay was done at the Laboratory of Entomology at the University Jorge Tadeo Lozano of Plutella xylostella larvae, insect pest of crucifers in several Colombian regions. The second phase included the extraction in the liquid state fermentation, a secondary metabolite that has antibiosis action against fungi, call iturin B, and was obtained from strains of Bacillus subtilis. The molecule was identified using High Resolution Chromatography (HPLC) and its biocontrol effect on Fusarium sp fungus causes vascular wilt in economically important plant varieties, was confirmed using testing of antagonism in Petri dish. In the third phase, an initial procedure in that let recover and identify microorganisms of the genus Bacillus from the rhizosphere in two aromatic herbs, Rosmarinus officinalis and Thymus vulgaris L. was used. Subsequently, testing of antagonism against Fusarium sp were made and an assay was done under greenhouse conditions to observe biocontrol and growth promoting action by comparing growth in length and dry weight. In the first experiment, native Bacillus sphaericus was lethal to 92% Plutella xylostella larvae in 10 DDA. In the second experiment, iturin B was identified and biological control of Fusarium sp was demonstrated. In the third study, all strains demonstrated biological control and the B14 strain identified as Bacillus megaterium increased root length and productivity of the two plants in terms of weight. It was concluded that the native microorganisms of the genus Bacillus has a great biochemical potential that provides a beneficial interactions with plants, improve their growth and development and therefore a greater impact on production.

Keywords: genus bacillus, biological control, PGPRs, biochemical potential

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283 Cellolytic Activity of Bacteria of the Bacillus Genus Isolated from the Soil of Zailiskiy Alatau Slopes

Authors: I. Savitskaya, A. Kistaubayeva, A. Zhubanova, I. Blavachinskaiya, D. Ibrayeva, M. Abdulzhanova, A. Otarbay, A.Isabekova

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This study was conducted for the investigation of number of cellulolytic bacteria and their ability in decomposition. Seven samples surface soil were collected on cellulose Zailiskii Alatau slopes. Cellulolitic activity of new strains of Bacillus, isolated from soil is determined. Isolated cellulose degrading bacteria were screened for determination of the highest cellulose activity by quantitative assay using Congo red, gravimetric assay and colorimetric DNS method trough of the determination of the parameters of sugar reduction. Strains are assigned to: B.subtilis, B.licheniformis, B. cereus and, В. megaterium. Bacillus strains consisting of several different types of cellulases have broad substrate specificity of cellulase complexes formed by them. Cellulolitic bacteria were recorded to have highest cellulase activity and selected for optimization of cellulase enzyme production.

Keywords: cellulose-degrading bacteria, cellulase complex, foothills soil, screening

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282 Improving the Quality and Nutrient Content of Palm Kernel Cake through Fermentation with Bacillus subtilis

Authors: Mirnawati, Gita Ciptaan, Ferawati

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Background and Objective: Palm kernel cake (PKC) is a waste of the palm oil industry. Indonesia, as the largest palm oil producer in the world, produced 45-46% palm kernel cake. Palm kernel cake can potentially be used as animal ration but its utilization for poultry is limited. Thus, fermentation process was done in order to increase the utilization PKC in poultry ration. An experiment was conducted to study the effect between Inoculum Doses with Bacillus subtilis and fermentation time to improve the quality and nutrient content of fermented Palm Kernel Cake. Material and Methods: 1) Palm kernel cake derived from Palm Kernel Processing Manufacture of Andalas Agro Industry in Pasaman, West Sumatra. 2) Bacillus subtilis obtained from The Research Center of Applied Chemistry LIPI, Bogor. 3) Preparations nutrient agar medium (NA) produced by Difoo - Becton Dickinson. 4) Rice bran 5) Aquades and mineral standard. The experiment used completely randomize design (CRD) with 3 x 3 factorial and 3 replications. The first factors were three doses of inoculum Bacillus subtilis: (3%), (5%), and (7%). The second factor was fermentation time: (1) 2 day, (2) 4 day, and (3) 6 day. The parameters were crude protein, crude fiber, nitrogen retention, and crude fiber digestibility of fermented palm kernel cake (FPKC). Results: The result of the study showed that there was significant interaction (P<0.01) between factor A and factor B and each factor A and B also showed significant effect (P<0.01) on crude protein, crude fiber, nitrogen retention, and crude fiber digestibility. Conclusion: From this study, it can be concluded that fermented PKC with 7% doses of Bacillus subtilis and 6 days fermentation time provides the best result as seen from 24.65% crude protein, 17.35% crude fiber, 68.47% nitrogen retention, 53.25% crude fiber digestibility of fermented palm kernel cake (FPKC).

Keywords: fermentation, Bacillus Subtilis, inoculum, palm kernel cake, quality, nutrient

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281 Laboratory Evaluation of Bacillus subtilis Bioactivity on Musca domestica (Linn) (Diptera: Muscidae) Larvae from Poultry Farms in South Western Nigeria

Authors: Funmilola O. Omoya

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Muscid flies are known to be vectors of disease agents and species that annoy humans and domesticated animals. An example of these flies is Musca domestica (house fly) whose adult and immature stages occur in a variety of filthy organic substances including household garbage and animal manures. They contribute to microbial contamination of foods. It is therefore imperative to control these flies as a result of their role in Public health. The second and third instars of Musca domestica (Linn) were infected with varying cell loads of Bacillus subtilis in vitro for a period of 48 hours to evaluate its larvicidal activities. Mortality of the larvae increased with incubation period after treatment with the varying cell loads. Investigation revealed that the second instars larvae were more susceptible to treatment than the third instars treatments. Values obtained from the third instar group were significantly different (P0.05) from those obtained from the second instars group in all the treatments. Lethal concentration (LC50) at 24 hours for 2nd instars was 2.35 while LC50 at 48 hours was 4.31.This study revealed that Bacillus subtilis possess good larvicidal potential for use in the control of Musca domestica in poultry farms.

Keywords: Bacillus subtilis, Musca domestica, larvicidal activities, poultry farms

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280 Effect of Food Supplies Holstein Calves Supplemented with Bacillus Subtilis PB6 in Morbidity and Mortality

Authors: Banca Patricia Pena Revuelta, Ramiro Gonzalez Avalos, Juan Leonardo Rocha Valdez, Jose Gonzalez Avalos, Karla Rodriguez Hernandez

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Probiotics are a promising alternative to improve productivity and animals' health. In addition, they can be part of the composition of different types of products, including foods (functional foods), medicines, and dietary supplements. The objective of the present work was to evaluate the effect of the feeding of Holstein calves supplemented with bacillus subtilis PB6 in morbidity and mortality. 60 newborn animals were used, randomly included in 1 of 3 treatments. The treatments were as follows: T1 = control, T2 = 10 g / calf / day. The first takes within 20 min after birth, T3 = 10 g / calf/day. The first takes between 12 and 24 h after birth. In all the treatments, 432 L of pasteurized whole milk divided into two doses/day 07:00 and 15:00, respectively, were given for 60 days. The addition of bacillus subtilis PB6 was carried out in the milk tub at the time of feeding them. The first colostrum intake (2 L • intake) was given within 2 h after birth, after which they were given a second 6 h after the first one. The diseases registered to monitor the morbidity and mortality of the calves were: diarrhea and pneumonia. The registry was carried out from birth to 60 days of life. The parameter evaluated was food consumption. The variable statistical analysis was performed using analysis of variance, and comparison of means was performed using the Tukey test. The value of P < 0.05 was used to consider the statistical difference. The results of the present study in relation to the consumption of food show no statistical difference P < 0.05 between treatments (14,762, 11,698, and 12,403 kg of food average, respectively). Calves group to which they were not provided Bacillus subtilis PB6 obtained higher feed intake. The addition of Bacillus subtilis PB6 in feeding calves does not increase feed intake.

Keywords: feeding, development, milk, probiotic

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279 Isolation, Characterization and Optimization of Alkalophilic and Thermotolerant Lipase from Bacillus subtilis Strain

Authors: Indu Bhushan Sharma, Rashmi Saraswat

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The thermotolerant, solvent stable and alkalophilic lipase producing bacterial strain was isolated from the water sample of the foothills of Trikuta Mountain in Kakryal (Reasi district) in Jammu and Kashmir, India. The lipase-producing microorganisms were screened using tributyrin agar plates. The selected microbe was optimized for maximum lipase production by subjecting to various carbon and nitrogen sources, incubation period and inoculum size. The selected strain was identified as Bacillus subtilis strain kakrayal_1 (BSK_1) using 16S rRNA sequence analysis. Effect of pH, temperature, metal ions, detergents and organic solvents were studied on lipase activity. Lipase was found to be stable over a pH range of 6.0 to 9.0 and exhibited maximum activity at pH 8. Lipolytic activity was highest at 37°C and the enzyme activity remained at 60°C for 24hrs, hence, established as thermo-tolerant. Production of lipase was significantly induced by vegetable oil and the best nitrogen source was found to be peptone. The isolated Bacillus lipase was stimulated by pre-treatment with Mn2+, Ca2+, K+, Zn2+, and Fe2+. Lipase was stable in detergents such as triton X 100, tween 20 and Tween 80. The 100% ethyl acetate enhanced lipase activity whereas, lipase activity were found to be stable in Hexane. The optimization resulted in 4 fold increase in lipase production. Bacillus lipases are ‘generally recognized as safe’ (GRAS) and are industrially interesting. The inducible alkaline, thermo-tolerant lipase exhibited the ability to be stable in detergents and organic solvents. This could be further researched as a potential biocatalyst for industrial applications such as biotransformation, detergent formulation, bioremediation and organic synthesis.

Keywords: bacillus, lipase, thermotolerant, alkalophilic

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278 Bacillus licheniformis sp. nov. PS-6, an Arsenic Tolerance Bacterium with Biotransforming Potential Isolated from Sediments of Pichavaram Mangroves of South India

Authors: Padmanabhan D, Kavitha S

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The purpose of the study is to investigate arsenic resistance ability of indigenous microflora and its ability to utilize arsenic species form containing water source. PS-6 potential arsenic tolerance bacterium was screened from thirty isolates from Pichavaram Mangroves of India having tolerance to grow up to 1000 mg/l of As (V) and 800 mg/l of As (III) and arsenic utilization ability of 98 % of As (V) and 97% of As (III) with initial concentration of 3-5 mg/l within 48 hrs. Optimum pH and temperature was found to be ~7-7.4 and 37°C. Active growth of PS-6 in minimal salt media (MSB) helps in cost effective biomass production. Dry weight analysis of PS-6 has shown significant difference in biomass when exposed to As (III) and As (V). Protein level study of PS-6 after exposing to As (V) and As (III) shown modification in total protein concentration and variation in SDS-PAGE pattern. PS-6 was identified as Bacillus licheniformis based on partially sequenced of 16S rRNA using NCBI Blast. Further investigation will help in using this potential bacterium as a well-grounded source for urgency.

Keywords: arsenite, arsenate, Bacillus licheniformis, utilization

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277 Screening of Minimal Salt Media for Biosurfactant Production by Bacillus spp.

Authors: Y. M. Al-Wahaibi, S. N. Al-Bahry, A. E. Elshafie, A. S. Al-Bemani, S. J. Joshi, A. K. Al-Bahri

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Crude oil is a major source of global energy. The major problem is its widespread use and demand resulted is in increasing environmental pollution. One associated pollution problem is ‘oil spills’. Oil spills can be remediated with the use of chemical dispersants, microbial biodegradation and microbial metabolites such as biosurfactants. Four different minimal salt media for biosurfactant production by Bacillus isolated from oil contaminated sites from Oman were screened. These minimal salt media were supplemented with either glucose or sucrose as a carbon source. Among the isolates, W16 and B30 produced the most active biosurfactants. Isolate W16 produced better biosurfactant than the rest, and reduced surface tension (ST) and interfacial tension (IFT) to 25.26mN/m and 2.29mN/m respectively within 48h which are characteristics for removal of oil in contaminated sites. Biosurfactant was produced in bulk and extracted using acid precipitation method. Thin Layer Chromatography (TLC) of acid precipitate biosurfactant revealed two concentrated bands. Further studies of W16 biosurfactant in bioremediation of oil spills are recommended.

Keywords: oil contamination, remediation, Bacillus spp, biosurfactant, surface tension, interfacial tension

Procedia PDF Downloads 276
276 Isolation of a Bacterial Community with High Removal Efficiencies of the Insecticide Bendiocarb

Authors: Eusebio A. Jiménez-Arévalo, Deifilia Ahuatzi-Chacón, Juvencio Galíndez-Mayer, Cleotilde Juárez-Ramírez, Nora Ruiz-Ordaz

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Bendiocarb is a known toxic xenobiotic that presents acute and chronic risks for freshwater invertebrates and estuarine and marine biota; thus, the treatment of water contaminated with the insecticide is of concern. In this paper, a bacterial community with the capacity to grow in bendiocarb as its sole carbon and nitrogen source was isolated by enrichment techniques in batch culture, from samples of a composting plant located in the northeast of Mexico City. Eight cultivable bacteria were isolated from the microbial community, by PCR amplification of 16 rDNA; Pseudoxanthomonas spadix (NC_016147.2, 98%), Ochrobacterium anthropi (NC_009668.1, 97%), Staphylococcus capitis (NZ_CP007601.1, 99%), Bosea thiooxidans. (NZ_LMAR01000067.1, 99%), Pseudomonas denitrificans. (NC_020829.1, 99%), Agromyces sp. (NZ_LMKQ01000001.1, 98%), Bacillus thuringiensis. (NC_022873.1, 97%), Pseudomonas alkylphenolia (NZ_CP009048.1, 98%). NCBI accession numbers and percentage of similarity are indicated in parentheses. These bacteria were regarded as the isolated species for having the best similarity matches. The ability to degrade bendiocarb by the immobilized bacterial community in a packed bed biofilm reactor, using as support volcanic stone fragments (tezontle), was evaluated. The reactor system was operated in batch using mineral salts medium and 30 mg/L of bendiocarb as carbon and nitrogen source. With this system, an overall removal efficiency (ηbend) rounding 90%, was reached.

Keywords: bendiocarb, biodegradation, biofilm reactor, carbamate insecticide

Procedia PDF Downloads 278
275 Survival and Retention of the Probiotic Properties of Bacillus sp. Strains under Marine Stress Starvation Conditions and Their Potential Use as a Probiotic for Aquaculture Objectives

Authors: Abdelkarim Mahdhi, Fdhila Kais, Faouzi Lamari, Zeineb Hmila, Fathi Kamoun, Maria Ángeles Esteban, Amina Bakhrouf

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Aquaculture is the world’s fastest growing food-production sector. However, one of the most serious problems regarding the culture of marine fishes is the mortality associated with pathogenic bacteria that occurs in the critical phases of larval development. Conventional approaches, such as the use of antimicrobial drugs to control diseases, have had limited success in the prevention or cure of aquatic diseases. Promising alternatives to antibiotics are probiotics, which are food supplements consisting of live microorganisms that benefit the host organism. In the search for more effective and environmentally friendly treatments with probionts against pathogenic species in shrimp larval culture, the probiotic properties of Bacillus strains isolated from Artemia culture such as antibacterial activity, adhesion, pathogenicity, toxicity and the effect of marine stress on viability and survival were investigated, as well as the changes occurring in their properties. Analyses showed that these bacteria corresponded to the genus Bacillus sp. Antagonism and adherence assays revealed that these strains have an inhibitory effect against pathogenic bacteria in vitro and in vivo conditions and are fairly adherent. Challenge tests performed with Artemia larvae provided evidence that the tested Bacillus strains were neither pathogenic nor toxic to the host. The tested strains maintained their viability and their probiotic properties during the period of study. The results suggest that the tested strains have suffered changes allowing them to survive in seawater in the absence of nutrients and outside their natural host, identifying them as potential probiotic candidates for Artemia culture.

Keywords: bacillus, probiotic, cell viability, stress response

Procedia PDF Downloads 384