Open Science Research Excellence

Open Science Index

Commenced in January 2007 Frequency: Monthly Edition: International Paper Count: 33

Study of Adsorption Isotherm Models on Rare Earth Elements Biosorption for Separation Purposes
The development of chemical routes for the recovery and separation of rare earth elements (REE) is seen as a priority and strategic action by several countries demanding these elements. Among the possibilities of alternative routes, the biosorption process has been evaluated in our laboratory. In this theme, the present work attempts to assess and fit the solution equilibrium data in Langmuir, Freundlich and DKR isothermal models, based on the biosorption results of the lanthanum and samarium elements by Bacillus subtilis immobilized on calcium alginate gel. It was observed that the preference of adsorption of REE by the immobilized biomass followed the order Sm (III)> La (III). It can be concluded that among the studied isotherms models, the Langmuir model presented better mathematical results than the Freundlich and DKR models.
The Use of Microorganisms in the Bioleaching of Soils Polluted with Heavy Metals

This paper shows researches in order to extract Cr, Cu and Ni from the polluted soils. Research is based on preliminary studies regarding the usage of Thiobacillus ferrooxidans bacterium (9K medium) for bioleaching of soil polluted with heavy metal (Cu, Cr and Ni). The microorganisms (Thiobacillus ferooxidans) selected directly from polluted soil samples were used in this experimental work. Soil samples used in the experimental research were taken from an area polluted with heavy metals from Romania. The soil samples are subjected to the cleaning process using the 9K medium solution (20 mL and 40 mL, respectively), stirred 200 rpm for 20 hours at a controlled temperature (30 ˚C). During the experiment (0, 2, 4, 8 and 20 h), liquid samples have been extracted and analyzed using the Atomic Absorption Spectrophotometer AA-6800 (AAS) in order to determine the Cr, Cu and Ni concentration. Experiments led to the conclusion that these soils can be depolluted by bioleaching, being a biological treatment method involving the use of microorganisms to favor the extraction of Cr, Cu and Ni from polluted soils.

Bioleaching of Metals Contained in Spent Catalysts by Acidithiobacillus thiooxidans DSM 26636

Spent catalysts are considered as hazardous residues of major concern, mainly due to the simultaneous presence of several metals in elevated concentrations. Although hydrometallurgical, pyrometallurgical and chelating agent methods are available to remove and recover some metals contained in spent catalysts; these procedures generate potentially hazardous wastes and the emission of harmful gases. Thus, biotechnological treatments are currently gaining importance to avoid the negative impacts of chemical technologies. To this end, diverse microorganisms have been used to assess the removal of metals from spent catalysts, comprising bacteria, archaea and fungi, whose resistance and metal uptake capabilities differ depending on the microorganism tested. Acidophilic sulfur oxidizing bacteria have been used to investigate the biotreatment and extraction of valuable metals from spent catalysts, namely Acidithiobacillus thiooxidans and Acidithiobacillus ferroxidans, as they present the ability to produce leaching agents such as sulfuric acid and sulfur oxidation intermediates. In the present work, the ability of A. thiooxidans DSM 26636 for the bioleaching of metals contained in five different spent catalysts was assessed by growing the culture in modified Starkey mineral medium (with elemental sulfur at 1%, w/v), and 1% (w/v) pulp density of each residue for up to 21 days at 30 °C and 150 rpm. Sulfur-oxidizing activity was periodically evaluated by determining sulfate concentration in the supernatants according to the NMX-k-436-1977 method. The production of sulfuric acid was assessed in the supernatants as well, by a titration procedure using NaOH 0.5 M with bromothymol blue as acid-base indicator, and by measuring pH using a digital potentiometer. On the other hand, Inductively Coupled Plasma - Optical Emission Spectrometry was used to analyze metal removal from the five different spent catalysts by A. thiooxidans DSM 26636. Results obtained show that, as could be expected, sulfuric acid production is directly related to the diminish of pH, and also to highest metal removal efficiencies. It was observed that Al and Fe are recurrently removed from refinery spent catalysts regardless of their origin and previous usage, although these removals may vary from 9.5 ± 2.2 to 439 ± 3.9 mg/kg for Al, and from 7.13 ± 0.31 to 368.4 ± 47.8 mg/kg for Fe, depending on the spent catalyst proven. Besides, bioleaching of metals like Mg, Ni, and Si was also obtained from automotive spent catalysts, which removals were of up to 66 ± 2.2, 6.2±0.07, and 100±2.4, respectively. Hence, the data presented here exhibit the potential of A. thiooxidans DSM 26636 for the simultaneous bioleaching of metals contained in spent catalysts from diverse provenance.

Effect of Bacillus subtilis Pb6 on Growth and Gut Microflora in Clostridium perfringens Challenged Broilers

The objective of current study was to investigate the effect of Bacillus subtilis PB6 (CloSTAT) as a probiotic in broilers. The corn-soybean based diet was divided into four treatment groups; T1 (basal diet with no probiotic and no Clostridium perfringens); T2 (basal diet challenged with C. perfringens without probiotic); T3 (basal diet challenged with C. perfringens having 0.05% probiotic); T4 (basal diet challenged with C. perfringens having 0.1% probiotic). Every treatment group had four replicates with 24 birds each. Body weight and feed intake were measured on weekly basis, while ileal bacterial count was recorded on day-28 following Clostridium perfringens challenge. The 0.1% probiotic treatment showed 7.2% increase in average feed intake (P=0.05) and 8% increase in body weight compared to T2. In 0.1% treatment body weight was 5% higher than T3 (P=0.02). It was also observed that 0.1% treatment had improved feed conversion ratio (1.77) on 6th week. No effect of treatment was observed on mortality and ileal bacterial count. The current study indicated that 0.1% use of probiotic had positive response in C. perfringens challenged broilers.

Bioremediation of Phenanthrene by Monocultures and Mixed Culture Bacteria Isolated from Contaminated Soil
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.
Fermentation of Germinated Native Black Rice Milk Mixture by Probiotic Lactic Acid Bacteria
This research aimed to demonstrate probiotic germinated native black rice juice fermentation by lactic acid bacteria (Lactobacillus casei TISTR 390). Germinated native black rice juice was inoculated with a 24-h old lactic culture and incubated at 30 °C for 72 hours. Changes in pH, acidity, total soluble solid, and viable cell counts during fermentation under controlled conditions at 0-h, 24-h, 48-h, and 72-h fermentations were evaluated. The study found out that the change in pH and total soluble solid of probiotic germinated black rice juice significantly (p ≤ 0.05) decreased at 72-h fermentation (5.67±0.12 to 2.86±0.04 and 7.00±0.00 to 6.40±0.00 ºbrix at 0-h and 72-h fermentations, respectively). On the other hand, the amount of titratable acidity expressed as lactic acid and the viable cell count significantly (p≤0.05) increased at 72-h fermentation (0.11±0.06 to 0.43±0.06 (% lactic acid) and 3.60 x 106 to 2.75 x 108 CFU/ml at 0-h and 72-h fermentations, respectively). Interestingly, the amount of γ-Amino Butyric Acid (GABA) had a significant difference (p≤0.05) twice as high as that of the control group (0.25±0.01 and 0.13±0.01 mg/100g, respectively). In addition, the free radical scavenging capacity assayed by DPPH method also showed that the IC50 values were significantly (p≤0.05) higher than the control (147.71±0.96 and 202.55±1.24 mg/ml, respectively). After 4 weeks of cold storage at 4 °C, the viable cell counts of lactic acid bacteria reduced to 1.37 x 106 CFU/ml. In conclusion, fermented germinated native black rice juice could be served as a healthy beverage for vegans and people who are allergic to cow milk products.
Experimental Investigation of the Impact of Biosurfactants on Residual-Oil Recovery
The increasing high price of natural gas and oil with attendant increase in energy demand on world markets in recent years has stimulated interest in recovering residual oil saturation across the globe. In order to meet the energy security, efforts have been made in developing new technologies of enhancing the recovery of oil and gas, utilizing techniques like CO2 flooding, water injection, hydraulic fracturing, surfactant flooding etc. Surfactant flooding however optimizes production but poses risk to the environment due to their toxic nature. Amongst proven records that have utilized other type of bacterial in producing biosurfactants for enhancing oil recovery, this research uses a technique to combine biosurfactants that will achieve a scale of EOR through lowering interfacial tension/contact angle. In this study, three biosurfactants were produced from three Bacillus species from freeze dried cultures using sucrose 3 % (w/v) as their carbon source. Two of these produced biosurfactants were screened with the TEMCO Pendant Drop Image Analysis for reduction in IFT and contact angle. Interfacial tension was greatly reduced from 56.95 mN.m-1 to 1.41 mN.m-1 when biosurfactants in cell-free culture (Bacillus licheniformis) were used compared to 4. 83mN.m-1 cell-free culture of Bacillus subtilis. As a result, cell-free culture of (Bacillus licheniformis) changes the wettability of the biosurfactant treatment for contact angle measurement to more water-wet as the angle decreased from 130.75o to 65.17o. The influence of microbial treatment on crushed rock samples was also observed by qualitative wettability experiments. Treated samples with biosurfactants remained in the aqueous phase, indicating a water-wet system. These results could prove that biosurfactants can effectively change the chemistry of the wetting conditions against diverse surfaces, providing a desirable condition for efficient oil transport in this way serving as a mechanism for EOR. The environmental friendly effect of biosurfactants applications for industrial purposes play important advantages over chemically synthesized surfactants, with various possible structures, low toxicity, eco-friendly and biodegradability.
Surface Characteristics of Bacillus megaterium and Its Adsorption Behavior onto Dolomite
Surface characteristics of Bacillus megaterium strain were investigated; zeta potential, FTIR and contact angle were measured. Surface energy components including Lifshitz-van der Waals, Hamaker constant, and acid/base components (Lewis acid/Lewis base) were calculated from the contact angle data. The results showed that the microbial cells were negatively charged over all pH regions with high values at alkaline region. A hydrophilic nature for the strain was confirmed by contact angle and free energy of adhesion between microbial cells. Adsorption affinity of the strain toward dolomite was studied at different pH values. The results showed that the cells had a high affinity to dolomite at acid pH comparing to neutral and alkaline pH. Extended DLVO theory was applied to calculate interaction energy between B. megaterium cells and dolomite particles. The adsorption results were in agreement with the results of Extended DLVO approach. Surface changes occurred on dolomite surface after the bio-treatment were monitored; contact angle decreased from 69° to 38° and the mineral’s floatability decreased from 95% to 25% after the treatment.
Biosorption of Metal Ions from Sarcheshmeh Acid Mine Drainage by Immobilized Bacillus thuringiensis in a Fixed-Bed Column

Heavy metals have a damaging impact for the environment, animals and humans due to their extreme toxicity and removing them from wastewaters is a very important and interesting task in the field of water pollution control. Biosorption is a relatively new method for treatment of wastewaters and recovery of heavy metals. In this study, a continuous fixed bed study was carried out by using Bacillus thuringiensis as a biosorbent for the removal of Cu and Mn ions from Sarcheshmeh Acid Mine Drainage (AMD). The effect of operating parameters such as flow rate and bed height on the sorption characteristics of B. thuringiensis was investigated at pH 6.0 for each metal ion. The experimental results showed that the breakthrough time decreased with increasing flow rate and decreasing bed height. The data also indicated that the equilibrium uptake of both metals increased with decreasing flow rate and increasing bed height. BDST, Thomas, and Yoon–Nelson models were applied to experimental data to predict the breakthrough curves. All models were found suitable for describing the whole dynamic behavior of the column with respect to flow rate and bed height. In order to regenerate the adsorbent, an elution step was carried out with 1 M HCl and five adsorption-desorption cycles were carried out in continuous manner.

Potential of γ-Polyglutamic Acid for Cadmium Toxicity Alleviation in Rice
Cadmium (II) (Cd) is one of the major toxic elemental pollutants, which is hazardous for humans, animals and plants. γ- Polyglutamic acid (γ-PGA) is an extracellular biopolymer produced by several species of Bacillus which has been reported to be an effective biosorbent for metal ions. The effect of γ-PGA on growth of rice grown under laboratory conditions was investigated. Rice seeds were germinated and then grown at 30±1°C on filter paper soaked with Cd solution and γ-PGA for 7 days. The result showed that Cd significantly inhibited the growth of roots, shoots by reducing root, and shoot lengths. Fresh and dry weights also decreased compared with control; however, the addition of 500 mg·L-1 γ-PGA alleviated rice seedlings from the adverse effects of Cd. The analysis of physiological traits revealed that Cd caused a decrease in the total chlorophyll and soluble protein contents and amylase activities in all treatments. The Cd content in seedling tissues increased for the Cd 250 μM treatment (P
The Construction of a Probiotic Lactic Acid Bacterium Expressing Acid-Resistant Phytase Enzyme
The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.
Vaccinated Susceptible Infected and Recovered (VSIR) Mathematical Model to Study the Effect of Bacillus Calmette-Guerin (BCG) Vaccine and the Disease Stability Analysis
Tuberculosis (TB) remains a leading cause of infectious mortality. It is primarily transmitted by the respiratory route, individuals with active disease may infect others through airborne particles which releases when they cough, talk, or sing and subsequently inhale by others. In order to study the effect of the Bacilli Calmette-Guerin (BCG) vaccine after vaccination of TB patient, a Vaccinated Susceptible Infected and Recovered (VSIR) mathematical model is being developed to achieve the desired objectives. The mathematical model, so developed, shall be used to quantify the effect of BCG Vaccine to protect the immigrant young adult person. Moreover, equations are to be established for the disease endemic and free equilibrium states and subsequently utilized in disease stability analysis. The stability analysis will give a complete picture of disease annihilation from the total population if the total removal rate from the infectious group should be greater than total number of dormant infections produced throughout infectious period.
Application of Statistical Approach for Optimizing CMCase Production by Bacillus tequilensis S28 Strain via Submerged Fermentation Using Wheat Bran as Carbon Source

Biofuels production has come forth as a future technology to combat the problem of depleting fossil fuels. Bio-based ethanol production from enzymatic lignocellulosic biomass degradation serves an efficient method and catching the eye of scientific community. High cost of the enzyme is the major obstacle in preventing the commercialization of this process. Thus main objective of the present study was to optimize composition of medium components for enhancing cellulase production by newly isolated strain of Bacillus tequilensis. Nineteen factors were taken into account using statistical Plackett-Burman Design. The significant variables influencing the cellulose production were further employed in statistical Response Surface Methodology using Central Composite Design for maximizing cellulase production. The optimum medium composition for cellulase production was: peptone (4.94 g/L), ammonium chloride (4.99 g/L), yeast extract (2.00 g/L), Tween-20 (0.53 g/L), calcium chloride (0.20 g/L) and cobalt chloride (0.60 g/L) with pH 7, agitation speed 150 rpm and 72 h incubation at 37oC. Analysis of variance (ANOVA) revealed high coefficient of determination (R2) of 0.99. Maximum cellulase productivity of 11.5 IU/ml was observed against the model predicted value of 13 IU/ml. This was found to be optimally active at 60oC and pH 5.5.

Extracellular Protein Secreted by Bacillus subtilis ATCC21332 in the Presence of Streptomycin Sulfate

The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.

Effect of Capsule Storage on Viability of Lactobacillus bulgaricus and Streptococcus thermophilus in Yogurt Powder
Yogurt capsule was made by mixing 14% w/v of reconstitution of skim milk with 2% FOS. The mixture was fermented by commercial yogurt starter comprising Lactobacillus bulgaricus and Streptococcus thermophilus. These yogurts were made as yogurt powder by freeze-dried. Yogurt powder was put into capsule then stored for 28 days at 4oc. 8ml of commercial yogurt was found to be the most suitable inoculum size in yogurt production. After freeze-dried, the viability of L. bulgaricus and S. thermophilus reduced from 109 to 107 cfu/g. The precence of sucrose cannot help to protect cell from ice crystal formation in freeze-dried process, high (20%) sucrose reduced L. bulgaricus and S. thermophilus growth during fermentation of yogurt. The addition of FOS had reduced slowly the viability of both L. bulgaricus and S. thermophilus similar to control (without FOS) during 28 days of capsule storage. The viable cell exhibited satisfactory viability level in capsule storage (6.7x106cfu/g) during 21 days at 4oC.
Laboratory Evaluation of Bacillus subtilis Bioactivity on Musca domestica (Linn) (Diptera: Muscidae) Larvae from Poultry Farms in South Western Nigeria

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.

Enzyme Involvement in the Biosynthesis of Selenium Nanoparticles by Geobacillus wiegelii Strain GWE1 Isolated from a Drying Oven

The biosynthesis of nanoparticles by microorganisms, on the contrary to chemical synthesis, is an environmentally-friendly process which has low energy requirements. In this investigation, we used the microorganism Geobacillus wiegelii, strain GWE1, an aerobic thermophile belonging to genus Geobacillus, isolated from a drying oven. This microorganism has the ability to reduce selenite evidenced by the change of color from colorless to red in the culture. Elemental analysis and composition of the particles were verified using transmission electron microscopy and energy-dispersive X-ray analysis. The nanoparticles have a defined spherical shape and a selenium elemental state. Previous experiments showed that the presence of the whole microorganism for the reduction of selenite was not necessary. The results strongly suggested that an intracellular NADPH/NADH-dependent reductase mediates selenium nanoparticles synthesis under aerobic conditions. The enzyme was purified and identified by mass spectroscopy MALDI-TOF TOF technique. The enzyme is a 1-pyrroline-5-carboxylate dehydrogenase. Histograms of nanoparticles sizes were obtained. Size distribution ranged from 40-160 nm, where 70% of nanoparticles have less than 100 nm in size. Spectroscopic analysis showed that the nanoparticles are composed of elemental selenium. To analyse the effect of pH in size and morphology of nanoparticles, the synthesis of them was carried out at different pHs (4.0, 5.0, 6.0, 7.0, 8.0). For thermostability studies samples were incubated at different temperatures (60, 80 and 100 ºC) for 1 h and 3 h. The size of all nanoparticles was less than 100 nm at pH 4.0; over 50% of nanoparticles have less than 100 nm at pH 5.0; at pH 6.0 and 8.0 over 90% of nanoparticles have less than 100 nm in size. At neutral pH (7.0) nanoparticles reach a size around 120 nm and only 20% of them were less than 100 nm. When looking at temperature effect, nanoparticles did not show a significant difference in size when they were incubated between 0 and 3 h at 60 ºC. Meanwhile at 80 °C the nanoparticles suspension lost its homogeneity. A change in size was observed from 0 h of incubation at 80ºC, observing a size range between 40-160 nm, with 20% of them over 100 nm. Meanwhile after 3 h of incubation at size range changed to 60-180 nm with 50% of them over 100 nm. At 100 °C the nanoparticles aggregate forming nanorod structures. In conclusion, these results indicate that is possible to modulate size and shape of biologically synthesized nanoparticles by modulating pH and temperature.

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

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.

Control of Staphylococcus aureus in Meat System by in situ and ex situ Bacteriocins from Lactobacillus sakei and Pediococcus spp.

The present study consisted of an applied test in meat system to assess the effectiveness of three bio agents bacteriocinproducing strains: Lm24: Lactobacillus sakei, Lm14and Lm25: Pediococcus spp. Two tests were carried out: The ex situ test was intended for three batches added with crude bacteriocin solutions at 12.48 AU/ml for Lm25 and 8.4 AU/ml for Lm14 and Lm24. However, the in situ one consisted of four batches; three of them inoculated with one bacteriocinogenic Lm25, Lm14, Lm24, respectively. The fourth one was used in mixture: Lm14+m24 at approximately of 107 CFU/ml. The two used tests were done in the presence of the pathogen St. aureus ATCC 6538, as a test strain at 103 CFU/ml. Another batch served as a positive or a negative control was used too. The incubation was performed at 7°C. Total viable counts, staphylococci and lactic acid bacteria, at the beginning and at selected times with interval of three days were enumerated. Physico-chemical determinations (except for in situ test): pH, dry mater, sugars, fat and total protein, at the beginning and at end of the experiment, were done, according to the international norms. Our results confirmed the ex situ effectiveness. Furthermore, the batches affected negatively the total microbial load over the incubation days, and showed a significant regression in staphylococcal load at day seven, for Lm14, Lm24, and Lm25 of 0.73, 2.11, and 2.4 log units. It should be noticed that, at the last day of culture, staphylococcal load was nil for the three batches. In the in situ test, the cultures displayed less inhibitory attitude and recorded a decrease in staphylococcal load, for Lm14, Lm24, Lm25, Lm14+m24 of 0.73, 0.20, 0.86, 0.032 log units. Therefore, physicochemical analysis for Lm14, Lm24, Lm25, Lm14+m24 showed an increase in pH from 5.50 to 5.77, 6.18, 5.96, 7.22, a decrease in dry mater from 7.30% to 7.05%, 6.87%, 6.32%, 6.00%.This result reflects the decrease in fat ranging from 1.53% to 1.49%, 1.07%, 0.99%, 0.87%; and total protein from 6.18% to 5.25%, 5.56%, 5.37%, 5.5%. This study suggests that the use of selected strains as Lm25 could lead to the best results and would help in preserving and extending the shelf life of lamb meat.

Evaluation of Attribute II Bt Sweet Corn Resistance and Reduced-Risk Insecticide Applications for Control of Corn Earworm

The corn earworm, Helicoverpa zea Boddie, is a serious pest of corn. Larval feeding in ear tips destroys kernels and allows growth of fungi and production of mycotoxins. Infested sweet corn is not marketable. Development of improved transgenic hybrids expressing insecticidal toxins from Bacillus thuringiensis (Bt) may limit or prevent crop losses. The effectiveness of Attribute® II Bt resistance and applications of Voliam Xpress insecticide were evaluated for effectiveness in controlling corn earworm in plots near Urbana, IL, USA, in 2013. Where no insecticides were applied, ear infestations and kernel damage in Attribute® II ‘Protector’ plots were consistently lower (near zero) than in plots of the non-Bt isoline ‘Garrison.’ Multiple applications of Voliam Xpress significantly reduced the number of corn earworm larvae and kernel damage in the Garrison plots, but infestations and damage in these plots were greater than in Protectorplots that did not receive insecticide applications. Our results indicate that Attribute® II Bt resistance is more effective than multiple applications of an insecticide for preventing losses caused by corn earworm in sweet corn.

Biochemical and Multiplex PCR Analysis of Toxic Crystal Proteins to Determine Genes in Bacillus thuringiensis Mutants

The Egyptian Bacillus thuringiensis isolate (M5) produce crystal proteins that is toxic against insects was irradiated with UV light to induce mutants. Upon testing 10 of the resulting mutants for their toxicity against cotton leafworm larvae, the three mutants 62, 64 and 85 proved to be the most toxic ones. Upon testing these mutants along with their parental isolate by SDS-PAGE analysis of spores-crystals proteins as well as vegetative cells proteins, new induced bands appeared in the three mutants by UV radiation and also they showed disappearance of some other bands as compared with the wild type isolate. Multiplex PCR technique, with five sets of specific primers, was used to detect the three types of cryI genes cryIAa, cryIAb and cryIAc. Results showed that these three genes exist, as distinctive bands, in the wild type isolate (M5) as well as in mutants 62 and 85, while the mutant 64 had two distinctive bands of cryIAb and cryIAc genes, and a faint band of cryI Aa gene. Finally, these results revealed that mutant 62 is considered as the promising mutant since it is UV resistant, highly toxic against Spodoptera littoralis and active against a wide range of Lepidopteran insects.

New Effective Strains of Bacteria Bacillus thuringiensis ssp. israelensis for Bloodsucking Mosquito Control
Five original strains of entomopathogenic bacteria with insecticidal activity against mosquito larvae of the genera Aedes, Culex and Anopheles have been isolated from natural conditions in Armenia and characterized. According to morphological, physiological and biochemical parameters, all isolates were identified as Bacillus thuringiensis spp. israelensis (Bti). High larvicidal activity has been showed by three strains Bti. These strains can be recommended for industrial production of bacterial preparations.
Effect of Crude Extract from Bacillus Subtilis LB5 Cultivated Broth on Conidial Germination of Colletotrichum Gloeosporioides
Bacillus subtilis strain LB5 produced lipopeptide antibiotic iturin A-2 in liquid medium. Crude extract from cell-free supernatant of B. subtilis cultivated broth extracted with n-butanol showed antifungal activity to conidial germination of Colletotrichum gloeosporioides. The germination of conidia was completely inhibited by crude extract. The ultrastructure of conidia after treated with crude extract was found an accumulation of vesiclelike material between cell wall and plasma membrane while this accumulation was not observed in untreated and germinated conidia. Besides, the cell wall was not affected by crude extract.
Inhibition on Conidial Germination of Colletotrichum gloeosporiodes and Pestalotiopsis eugeniae by Bacillus subtilis LB5
The effect of antifungal compound from Bacillus subtilis strain LB5 was tested against conidial germination of Colletotrichum gloeosporioides and Pestalotiopsis eugeniae, causal agent of anthracnose and fruit rot of wax apple, respectively. Observation under scanning electron microscope and light compound microscope revealed that conidial germination was completely inhibited when treated with culture broth, culture filtrate, or crude extract from strain LB5. Identification of purified antifungal compound produced by strain LB5 in cell-free supernatant by nuclear magnetic resonance and fast atom bombardment showed that the active compound was iturin A-2.
Optimization of Lipase Production Using Bacillus subtilis by Response Surface Methodology

A total of 6 isolates of Bacillus subtilis were isolated from oil mill waste collected in Namakkal district, Tamilnadu, India. The isolated bacteria were screened using lipase screening medium containing Tween 80. BS-3 isolate exhibited a greater clear zone than the others, indicating higher lipase activity. Therefore, this isolate was selected for media optimization studies. Ten process variables were screened using Plackett–Burman design and were further optimized by central composite design of response surface methodology for lipase production in submerged fermentation. Maximum lipase production of 16.627 U/min/ml were predicted in medium containing yeast extract (9.3636g), CaCl2 (0.8986g) and incubation periods (1.813 days). A mean value of 16.98 ± 0.2286 U/min/ml of lipase was acquired from real experiments.

Proteolytic Dedradation of Anchovy (Spolephorus spp.) Proteins by Halophilic Proteinase from Halobacillus sp. SR5-3
The halophilic proteinase showed a maximal activity at 50°C and pH 9~10, in 20% NaCl and was highly stabilized by NaCl. It was able to hydrolyse natural actomyosin (NAM), collagen and anchovy protein. For NAM hydrolysis, the myosin heavy chain was completely digested by halophilic proteinase as evidenced by the lowest band intensity remaining, but partially hydrolysed actin. The SR5-3 proteinase was also capable hydrolyzing two major components of collagen, β- and α-compounds, effectively. The degree of hydrolysis (DH) of the halophilic proteinase and commercial proteinases (Novozyme, Neutrase, chymotrypsin and Flavourzyme) on the anchovy protein, were compared, and it was found that the proteinase showed a greater degree of hydrolysis towards anchovy protein than that from commercial proteinases. DH of halophilic proteinase was sharply enhanced according to the increase in the concentration of enzyme from 0.035 U to 0.105 U. The results warranting that the acceleration of the production of fish sauce with higher quality, may be achieved by adding of the halophilic proteinase from this bacterium.
Spent Caustic Bioregeneration by using Thiobacillus denitrificans Bacteria
Spent Sulfidic Caustic was biologically treated and regenerated for reusing by Thiobacillus denitrificans bacteria, sulfide content oxidized and RSNa reduced dramatically.PH in this test was 11.8 and no neutralization has been done on spent caustic, so spent caustic as the most difficult of industrial wastes to dispose could be regenerate and reuse instead of disposing to sea or deep wells
Protein Production by Bacillus Subtilis Atcc 21332 in the Presence of Cymbopogon Essential Oils
Proteins levels produced by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antimicrobial agents or antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics or natural compounds in nature as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was focusing on the effect of essential oils from Cymbopogon flexuosus and C. nardus in regulating proteins production by Bacillus subtilis ATCC 21332. The Minimum Inhibition Concentrations (MICs) of both essential oils on B. subtilis were determined by using microdilution assay, resulting 0.2% and 1.56% for each C. flexuosus and C. nardus subsequently. The bacteria were further exposed to each essential oils at concentration of 0.01XMIC for 2 days. The proteins were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein profile showed that a band with approximate size of 250 kD was appeared for the treated bacteria with essential oils. Thus, Bacillus subtilis ATCC 21332 in stressful condition with the presence of essential oils at low concentration could induce the protein production.
Anti-microbial Activity of Aristolochic Acid from Root of Aristolochia bracteata Retz
The present research was designed to investigate the anti-microbial activity of aristolochic acid from the root of Aristolochia bracteata. From the methanolic & ethyl extract extracts of Aristolochia bracteata aristolochic acid I was isolated and conformed through IR, NMR & MS. The percentage purity of aristolochic acid I was determined by UV & HPLC method. Antibacterial activity of extracts of Aristolochia bracteata and the isolated compound was determined by disc diffusion method. The results reveled that the isolated aristolochic acid from methanolic extract was more pure than the compound from ethyl acetate extract. The various extracts (500μg/disc) of Aristolochia bracteata showed moderate antibacterial activity with the average zone of inhibition of 7-18 mm by disc diffusion method. Among the extracts, ethyl acetate & methanol extracts were shown good anti-microbial activity and the growth of E.coli (18 mm) was strongly inhibited. Microbial assay of isolated compound (Aristolochic acid I) from ethyl acetate & methanol extracts were shown good antimicrobial activity and the zone of inhibition of both at higher concentration 50 μg/ml was similar with the standard aristolochic acid. It may be concluded that the isolated compound of aristolochic acid I has good anti-bacterial activity.
Bactericidal Properties of Carbohydrate-Stabilized Platinum Oxide Nanoparticles
Platinum oxide nanoparticles were prepared by a simple hydrothermal route and chemical reduction using carbohydrates (Fructose and sucrose) as the reducing and stabilizing agents. The crystallite size of these nanoparticles was evaluated from X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) and was found to be 10 nm as shown in figure 1, which is the demonstration of EM bright field and transmission electron microscopy. The effect of carbohydrates on the morphology of the nanoparticles was studied using TEM (Figure 1). The nanoparticles (100 μg/ml) were administered to the Pseudomonas Stutzeri and Lactobacillus cultures and the incubation was done at 35 oC for 24 hours. The nanocomposites exhibited interesting inhibitory as well as bactericidal activity against P. Stutzeri and and Lactobacillus species. Incorporation of nanoparticles also increased the thermal stability of the carbohydrates.

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