Caracterización bioinformática y producción de L-asparaginasa de Bacillus sp. M62 aislado de las salinas de Maras, Cusco, Perú

El objetivo del estudio fue realizar la caracterización bioinformática, así como optimizar la producción de L-asparaginasa extracelular de Bacillus sp. M62 aislada de las salinas de Maras (Cusco). Para ello, se verificó la producción de L-asparaginasa mediante el viraje del medio M9 modificado con azul de bromofenol 0.0075%, pH 7.4 a 37 °C por 72 h. A la vez, se extrajo el ADN genómico para amplificar los genes ribosómicos 16S y el gen ansA3. La secuencia aminoacídica codificada por el gen ansA3 se predijo mediante análisis bioinformático. La producción de L-asparaginasa intracelular y extracelular se evaluó a diferentes niveles de glucosa, L-asparagina, NaCl y pH en el medio M9 modificado. Adicionalmente, las actividades enzimáticas de L-asparaginasa y L-glutaminasa se determinaron mediante cuantificación del amonio liberado por el método de Nessler. Así, Bacillus sp. M62 produjo el viraje del medio M9 modificado, obtuvo alta similitud y cercanía evolutiva con Bacillus licheniformis, se encontró que el gen ansA3 amplificado codificaba para 319 aa, dentro de la cual se predijo una secuencia patrón del sitio activo (GFVITHGTDTM ) y 15 sitios inmunogénicos. La producción de L-asparaginasa extracelular fue superior a la intracelular, la que se optimizó de 0.37 U/mL (0.24 U/mg) a 2.15 ± 0.39 U/mL (0.63 U/mg). Finalmente, se encontró que Bacillus sp. M62 presenta L-asparaginasa extracelular con mínima actividad de L-glutaminasa.

The aim of this study was to perform bioinformatics characterization and optimize the production of extracellular L-asparaginase from Bacillus sp. M62, isolated from the Maras salt ponds (Cusco). To achieve this, the production of L-asparaginase was verified by the change in color of modified M9 medium, containing 0.0075% bromophenol blue, at pH 7.4 and 37°C for 72 hours. Genomic DNA was extracted to amplify the 16S ribosomal genes and the ansA3 gene. The amino acid sequence encoded by the ansA3 gene was predicted using bioinformatic analysis. The production of intracellular and extracellular L-asparaginase was evaluated at different levels of glucose, L-asparagine, NaCl, and pH in modified M9 medium. Additionally, the enzymatic activities of L-asparaginase and L-glutaminase were determined by quantifying the released ammonium using the Nessler method. Bacillus sp. M62 showed the change in color of the modified M9 medium, high similarity, and evolutionary closeness to Bacillus licheniformis. The amplified ansA3 gene was found to encode for 319 amino acids, with a predicted active site pattern (GFVITHGTDTM) and 15 immunogenic sites. The production of extracellular L-asparaginase was found to be higher than intracellular L-asparaginase and was optimized from 0.37 U/mL (0.24 U/mg) to 2.15 ± 0.39 U/mL (0.63 U/mg). Finally, it was found that Bacillus sp. M62 presents extracellular L-asparaginase with minimal L-glutaminase activity.

Microbiological Assessment of Used Face Masks in Port Harcourt Secondary Schools in COVID-19 Era

Aim: This study was aimed at identifying bacterial and fungal contaminations in used face masks from different secondary schools in Port Harcourt during COVID-19 EraStudy Design: The study employs statistical analysis of the data and interpretation.Place and Duration of Study: Five Secondary Schools–Three public schools: Federal Government College Rumuokoro; Rumueme and Rumuokuta Girls’ Secondary Schools; Two Private Schools: Solid Steps and Istan Secondary Schools; all located in the city of Port-Harcourt, Nigeria. Sample collection lasted for a week and the analysis lasted for six months.Methodology: The research study was facilitated through Laboratory analysis and the use of questionnaire to get the age and sex from the school children. A total of 25 used face masks samples were collected from school children between ages of 12-18years and they were examined microbiologically. Sterile swab sticks soaked in sterile nutrient broth were employed to swab the inner surface area of the used face mask of circular diameter 10 cm. The swabbed samples were dipped and shaken in 9ml of sterile saline water for 1-3 minutes to dislodge the organisms; the mixture was then diluted through a ten-fold serial dilution, after which an aliquot of 0.1ml were inoculated unto Nutrient Agar (dilution used 10-6, incubated at 370C for 24h), Mac Conkey Agar (dilution used 10-3, incubated at 44±0.20C for 24-48h) and Sabouraud Dextrose Agar (dilution used 10-3; incubated at 370C for 5-7 days). Frequency evaluation and identification of isolates were carried out using standard microbiological techniques.Results: The entire face masks sampled were found contaminated with microorganisms. The Microbial load (Log10 CFU/cm2;) and Percentage (%) occurrence of bacterial isolates from used facemask were; Bacillus spp (6.10±2.13)(30.81) > Staphylococcus auerus (3.89±3.01)(19.57%) > Proteus spp (2.25±2.45)(11.35) > Paenibacillus spp (1.55±2.52)(7.82) > Escherichia coli (0.36±0.81)(1.82) while fungal isolates were Aspergillus spp (2.20±0.55)(11.09) > Mucor spp (2.19±0.96)(11.04) > Penicillum spp (1.29±0.61)(6.51). The contaminated used face masks with microorganism were highest in school children of ages 16-18years (72%) and the lowest occurred in children of 12-14years of age (12%).Conclusion: The presence of potential pathogen such as Staphylococcus auerus, Bacillus spp etc. are of public health significance. It is therefore recommended that crowd should be controlled in such environments with high bacterial and fungal load such as schools and COVID-19 protocols duly observed.

Necrotizing placentitis in a cow caused by Bacillus cereus / Placentite necrotizante em uma vaca por Bacillus cereus

ABSTRACT: This report described a case of necrotizing placentitis caused by Bacillus cereus in a cow associated with abortion and maternal lethality. The etiological diagnosis of placentitis by B. cereus was based on histopathology of placenta, cytology and bacterial isolation from intrauterine aminiotic fluid in retained placenta and further characterization of the pathogen by the MALDI-TOF. Although, B. cereus abortions are sporadic, the bacterium has the ability to release necrotizing toxins that can lead to placentitis, fetal death and abortion.

RESUMO: Este relato descreve a placentite necrotizante causada por Bacillus cereus em uma vaca associada a aborto e mortalidade materna. O diagnóstico etiológico de placentite por B. cereus foi baseado na histopatologia da placenta, citologia e isolamento bacteriano partir do líquido aminiótico em placenta retida e identificação do patógeno pela técnica de MALDI-TOF. Embora abortos por B. cereus sejam esporádicos, a bactéria tem a capacidade de liberar toxinas necrotizantes que podem levar a placentite e aborto.

Study on biology of tea bunch caterpillar Andraca bipunctata on alternative host plant Eurya acuminata of Meghalaya

Aim: The present study was carried out to study the biology of tea caterpillar Andraca bipunctata on Eurya acuminata as an alternate host which will be useful for tea growers of Meghalaya and nearby area to monitor the incidence and timely management of this pest on alternative host itself.Methodology: Insect population was collected from Umiam, Ri-Bhoi district situated in the mid altitude hills (Khasi hills) of Meghalaya. Six locations were selected randomly within the locality for the collection of eggs, larval instars and pupae from the host plant. Host plant was identified at Agro-forestry Division, ICAR-Research Complex for NEH, Umiam. Observations on all the biological parameters were recorded under laboratory conditions and analysed accordingly. Results: In present study, an alternative host of A. bipunctata was found in mid-altitude hills of Meghalaya state during survey which was identified as Eurya acuminatea. The insect was able to easily survive and complete its whole life cycle on this host plant, E. acuminata. Interpretation: E. acuminate acted as an alternative host and safe reservoir site for all the stages of A. bipunctata and thereby this host plant helps to survive the pest during unfavorable condition in main host (tea plantation). A. bipunctata is able to multiply their population on E. acuminata and get ready to damage on the main host for successive years.

Effect of carbon/nitrogen ratio on polyhydroxyalkanoates production by Bacillus species under submerged fermentation

Aim: To analyze the effect of carbon/nitrogen (C/N) ratio on polyhydroxyalkanoates (PHAs) production by Bacillus species under submerged fermentation process.Methodology: Preserved polyhydroxyalkanoates producing Bacillus sp. C1 (2013) (KF626477) was revived and growth parameters were optimized by one factor at a time approach. The effect of C/N ratio and the influence of time period on polyhydroxyalkanoates production through submerged fermentation process was evaluated under optimized condition. Primary structural and morphological characterization of extracted polyhydroxyalkanoates was carried out by Fourier Transform Infra-Red Spectroscopy and Field Emission-Scanning Electron Microscopy. Results: Bacillus sp. C1 (2013) produced higher cell biomass in mineral salt medium at pH 9.0, temperature 37oC, dextrose (2%) and ammonium sulphate (1%) as carbon and nitrogen source with 15% inoculum size. Under optimized condition, higher polyhydroxyalkanoates production of 1.09 g l-1 (49.2%) was obtained at 48 hrs with 2: 0.4 C/N ratio. However, in our previous study 0.909 gl-1 of PHAs was produced by the bacteria at 6:1 carbon to nitrogen ratio. Fourier Transform Infra-Red Spectroscopic analysis showed high intense absorption bands at 1720.18 cm−1 resembled to ester carbonyl functional group of PHB, which is the most common homopolymer of PHAs. Surface morphology of poly-beta-hydroxybutyrate film was rough and fairly regular as revealed from Field Emission-Scanning Electron Microscopic imaging. Interpretation: Poly-beta-hydroxybutyrate production by the bacteria increased under higher degrees of nitrogen deficient condition. Thus, optimized C/N ratio can improve the cost affordability of poly-beta-hydroxybutyrate production, however, further research in contrast to different bacteria is highly essential in this regard.

In vitro cytotoxic activity assay of bacteria extract derived marine sponge Haliclona fascigera toward Hela, WiDr, T47D, and Vero cell line

Symbiotic association between marine sponge and microorganism was a promising chance in the discovery of leadcompound of anticancer. This association was probably concluded that symbiotic microorganism would contain thesame secondary metabolites with the host. In this continuation research, we had isolated symbiotic bacteria from amarine sponge and tested for cytotoxic activity. Twenty-six isolates of bacteria derived from marine sponge Haliclonafascigera were isolated from Setan Island, West Sumatra, Indonesia. Screening of cytotoxic activity by BSLT methodand MTT assay was conducted toward 21 ethyl acetate extracts of symbiotic bacteria with weight >50 mg. Onebacterial extract with code H2N was very toxic according to BSLT test, while 18 isolates were toxic with LC50 rangingfrom 31.17 to 283.38 ppm. All of the bacterial extracts did not show a good percentage of viability (>50%) againstHela, WiDr, T47D, dan Vero cell line in MTT assay. However, bacterial extract with code H2N have shown potentialcytotoxic compared to other extracts. As per the phytochemical study, this extract probably contained terpenoid group.Based on biochemical examination this bacterium, H2N, was identified as Bacillus sp.3.

Screening and identification of amylase producing strains of Bacillus

An attempt was made to isolate and screen efficient amylolytic strains of Bacillus sp. Initial screening basedon the starch hydrolysis ratio resulted in the selection of 72 amylolytic bacterial strains. Among these, 18strains were selected for further studies. Secondary screening based on amylase production in starch brothmedium led to the selection of six amylolytic strains of Bacillus sp. The selected strains were grown in fourdifferent fermentation media (FMI-FMIV) in order to screen for three most efficient amylolytic strains foroptimization and characterization. FMIV was the best basal medium as it provided required nutrients, whichstimulated highest amylase production in bacterial strains within shortest incubation time ( 24 hours). Molecularidentification based on 16S rDNA sequence revealed that three most efficient strains [BCM36 (KR1), BCM33(KR2), and BCM25 (KR3)] belonged to Bacillus sp

Rizobacterias promotoras de crecimiento en Guarianthe skinneri (Orchidaceae) / Growth-promoting rhizobacteria from Guarianthe skinneri (Orchidaceae)

Resumen La orquídea Guarianthe skinneri está incluida en la norma NOM-059-ECOL-2010 de México como una especie amenazada. Con el fin de estudiar las BPCV (bacterias promotoras del crecimiento vegetal) en esta orquídea, se recolectaron 10 raíces de diferentes plantas para aislar bacterias asociadas a las raíces, que se analizaron mediante pruebas in vitro como: producción de AIA, fijación de nitrógeno, interacción con el hongo micorrízico Thanatephorus sp. cepa RG26 y solubilización de fosfato. De los 71 aislados bacterianos se caracterizaron 10 cepas mediante secuenciación con el marcador 16s rADN y se identificaron seis cepas: Sphingomonas sp., Sinorhizobium sp., Bacillus sp., Nocardia cerradoensis, Bacillus megaterium y Burkholderia phytofirmans. Se observó que la bacteria Sinorhizobium sp. produjo mayor cantidad de AIA (69.189 µg/ml) y Bacillus sp. presentó mayor reducción de acetileno (10.251 nmol cultivo/96 h). En las interacciones de las bacterias y el hongo RG26 se presentaron cuatro categorías (sumamente positivo, positivo, antagonismo 50-50 e inhibición). En relación a la solubilización de fosfato, la bacteria Burkholderia phytofirmans presentó mayor IS a las 48 y 96 hr con IS de 3.11 y 3.48, respectivamente. Los resultados indican que Bacillus sp. pudiera tener las mejores características para promover el desarrollo de la orquídea G. skinneri mediante la inoculación de semillas y plántulas.

Abstract The Guarianthe skinneri orchid is included in NOM-059-ECOL-2010, Mexico standard as an endangered species. In order to study PGPR (promoting growth plant rhizobacteria) from this orchid, 10 roots were collected from different plants to isolate bacteria associated with the roots, which were analyzed by in vitro tests such as: production of AIA, nitrogen fixation, interaction with the mycorrhizal fungus Thanatephorus sp. strain RG26 and phosphate solubilization. We obtain 71 bacterial isolates, 10 strains of them were characterized by sequencing with the 16d rDNA marker identifying six bacteria: Sphingomonas sp. Sinorhizobium sp. Bacillus sp. Nocardia cerradoensis, Bacillus megaterium and Burkholderia phytofirmans. We observed that the bacterium Sinorhizobium sp. produced a greater amount of AIA (69.189 μg/ml) and Bacillus sp. performed greater acetylene reduction (10.251 nmol cultivo/96h). In the interactions of the bacteria and the fungus RG26, four categories were presented (extremely positive, positive, antagonism 50-50 and inhibition). In relation to the solubilization of phosphate, Burkholderia phytofirmans presented higher IS after 48 and 96 hr with an IS of 3.11 and 3.48, respectively. The results indicate that Bacillus sp. it could have the best characteristics to promote the development of the G. skinneri orchid by inoculating seeds and seedlings. Rev. Biol. Trop. 66(3): 953-968. Epub 2018 September 01.

Isolation, molecular identification and screening of halophilic and thermophiliclipase producing bacterial strains from extreme environmental conditions

Aims@#The aim of this study was to isolate and screen lipolytic-thermophilic and halophilic strains from extreme environments and at the same time studying the influence of physiological conditions such as temperature and sodium chloride concentrations on growth of the strains.@*Methodology and results@# A total of 45 halophilic bacterial strains and 4 thermophilic bacterial strains were isolated from various saline environments and hot spring respectively, and were screened for lipolytic activity on tributyrincontaining agar. The strains that showed lipolytic potential on the solid agar were further subjected to secondary screening by submerged fermentation using the same substrate (tributyrin). One halophilic (SWJ2) and thermophilic (HS2) strain each with highest extracellular lipase production ability was outsourced for molecular identification. The result from 16S rRNA partial gene sequencing and amplification of the selected isolates designated the halophilic strain SWJ2 as Marinobacter sp. SWJ2 and the thermophilic strain HS2 as Bacillus sp. HS2. Upon screening under submerged fermentation, Bacillus sp. HS2 gave 0.17 U/mL lipase production while Marinobacter sp. SWJ2 had 0.57 U/mL lipase production.@*Conclusion, significance and impact of study@#It could be inferred that the selected strains possess high lipase producing potential with interesting thermal and halophilic properties. Continuous screening of extremophilic environment for strains producing lipases with novel properties will improve their potential industrial and biotechnological applications especially for those processes conducted in harsh reaction conditions.

Phosphate solubilization by endophytic bacteria isolated from banana trees

ABSTRACT Forty isolates of endophytic bacteria isolated from banana tree roots were assessed as to their capacity to solubilize phosphate in a solid culture medium supplemented with different inorganic and one organic source of phosphorus. The amount of phosphorus (P) in each liquid medium was quantified, and an indirect assessment of acid phosphatase activity was performed. All assays had a fully randomized design, with three repetitions. Approximately 67.5% of the 40 isolates assessed in solid medium solubilized phosphorus from tricalcium phosphate and 7.5% of the isolates solubilized phosphorus from soy lecithin; no isolates exhibited P solubilization capacity in medium supplemented with iron phosphate. Acid phosphatase activity was detected in 65% of the isolates; Aneurinibacillus sp. and Lysinibacillus sp. isolates presented with the best solubilization indexes. All of the assessed isolates exhibited a capacity to reduce the potential of hydrogen in liquid medium supplemented with tricalcium phosphate. Isolate EB. 78 (Bacillus sp.) exhibited P solubilization capacity in solid media when Ca3(PO4)2 and soy lecithin were used as P sources; this isolate significantly reduced the pH of the liquid medium and exhibited acid phosphatase activity. The results of the present study highlight isolates that exhibit variations in their capacity to solubilize P. These isolates should be used in future tests to assess their field performance.

Consortium of heterotrophic nitrification bacteria Bacillus sp. and its application on urea fertilizer industrial wastewater treatment

Aims: Industrial wastewater can be processed by physical, chemical and biological treatment. Most biological treatment have no negative impact on environment and can minimize more cost. The aim of this study was to reduce ammonia in urea fertilizer industrial wastewater by Bacillus bacterial consortium. This strategy tried to develop biological processing of ammonia in wastewater with nitrification process. Methodology and results: Bacterial consortium consisted of three isolates: isolate W1.6 identified as Bacillus sp. A16ZZ, isolate W2.5 identified as Bacillus sp. BNPK-13, and isolate S2.6 identified as B. cereus strain CP1. Consortium culture was made based on shortest generation time on each isolate in order to be in exponential phase when it was used. Bacterial consortium was able to decrease ammonia concentration in wastewater seven days after incubation. Conclusion, significance and impact of study: Consortium of heterotrophic nitrification works optimally with concentration of 350 mg/L ammonia and decrease 96.28%. This consortium has potency to be developed as alternative biological agent in reducing ammonia compounds in high-concentrated urea fertilizer industrial wastewater.

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.

Avaliação in vitro da adsorção de aflatoxina B1 por produtos comerciais utilizados na alimentação animal / In vitro evaluation of aflatoxin B1 adsorption by commercial products used in animal feed

Objetivou-se avaliar a capacidade de adsorção in vitro de aflatoxina B1 (AFB1) por produtos comerciais utilizados na alimentação animal. Muitas pesquisas estão sendo realizadas para a descontaminação de AFB1 em alimentos. Os produtos comerciais utilizados frequentemente na alimentação de peixes, disponíveis na forma de probióticos, são formados por cepas de bactérias e leveduras utilizadas na maioria dos ensaios de adsorção de micotoxinas. Foram utilizados três produtos comerciais: A, composto por Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium e Lactobacillus acidophilus; B, por leveduras secas de Saccharomyces cerevisiae provenientes de cervejaria; e C, por Bacillus subtilis, Bacillus licheniformis e Bacillus pumilus. Cinco suspensões da dose máxima recomendada pelo fabricante de cada produto (0; 25; 50; 75 e 100%) foram testadas contra AFB1 (1000 ng.mL-1) em microtubos para determinação da capacidade de adsorção. Para simular o pH do estômago e do intestino de tilápias do Nilo (Oreochromis niloticus) foram formuladas soluções tampão fosfato salino (PBS), com pH 1,5 e 7,5; respectivamente. Os microtubos foram introduzidos em uma centrífuga com agitação mecânica, a 37ºC por 1 h e depois centrifugados por 10 min a 14.000 rpm; os sobrenadantes foram quantificados por cromatografia líquida de alta eficiência. Os produtos comerciais, nas concentrações máximas, foram capazes de adsorver AFB1 em quantidades de 45,01 a 129,59; 123,90 a 215,59 e 209,98 a 370,73 ng.mL-1, respectivamente. Concluiu-se que todos os produtos comerciais analisados adsorvem AFB1 em condições simuladas de pH gastrointestinal e são candidatos potenciais para adsorção de AFB1 para futuros ensaios in vivo.(AU)

The aim of this study was to evaluate the aflatoxin B1 (AFB1) adsorption capacity, in vitro, by commercial products used in animal feed. Many studies are being conducted for the decontamination of aflatoxins in feed. The commercial products destined to fish feed that are available as probiotics and are formed by strains of bacteria and yeasts used in most mycotoxins adsorption assays. Three commercial products were studied: A, consisting of Bacillus subtilis, Bifidobacterium bifidum, Enterococcus faecium and Lactobacillus acidophilus; B, consisting of dry yeast of Saccharomyces cerevisiae from brewery; and C, consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Five suspensions of the maximum dose recommended by the manufacturer of each product (0; 25; 50; 75 and 100%) were tested against AFB1 (1000 ng.mL-1) in microtubes to determine the adsorption capacity. To simulate the pH of the stomach and intestine of the Nile tilapia (Oreochromis niloticus), phosphate buffered saline solutions (PBS) at pH 1.5 and 7.5, respectively, were formulated. Microtubes were introduced into a centrifuge with mechanical agitation at 37ºC for 1 h and then centrifuged for 10 min at 14.000 rpm; the supernatants were quantified by high-performance liquid chromatography. The commercial products in the maximum concentration were capable of adsorbing AFB1 in amounts from 45.01 to 129.59; from 123.90 to 215.59; and from 209.98 to 370.73 (ng.mL-1), respectively. It was concluded that all commercial products, which are added to animal feed, adsorbed AFB1 under simulated gastrointestinal pH conditions and are potential candidates for AFB1 adsorption for future in vivo studies.(AU)

Production and characterization of polyhydroxybutyrate (PHB) produced by Bacillus sp. isolated from Egypt.

Synthetic polymers are non-degradable and accumulated in the environment so, the efforts of scientists were forwarded to provide us with alternative environmentally biopolymers. Polyhydroxyalkanoates (PHAs) including polyhydroxybutyrate (PHB) are Group of the interesting biopolymers which have several medical applications such as drug delivery, suture, scaffold and heart valves. PHAs are biological macromolecules, thermoplastics, biodegradable and biocompatible. In this study, new bacterial isolates from Egypt were screened for their ability to produce PHB using Nile red dye. Out of 44 isolates, 19 bacterial isolates were selected according to strong of their fluorescence on mineral salt medium (MSM) agar plates supplemented with Nile red. The most potent strain was identified using biochemical tests as Bacillus sp. N-2. Production of PHB was carried out in limitation of nitrogen source using a minimal salt medium (MSM) supplemented with an excess of glucose as sole carbon source. PHB was accumulated in relation to cell dry weight about 20% (PHB/CDW). The obtained biopolymer was purified and analyzed using NMR, FT-IR, TGA and DSC thus; it was highly pure and identified as PHB. Optimization of PHB production from cheap sources appears to be a realistic goal in the future for reducing the costs and obtaining high yield.

Bacterial treatment of alkaline cement kiln dust using Bacillus halodurans strain KG1

Abstract This study was conducted to isolate an acid-producing, alkaliphilic bacterium to reduce the alkalinity of cement industry waste (cement kiln dust). Gram-positive isolate KG1 grew well at pH values of 6–12, temperatures of 28–50 °C, and NaCl concentrations of 0–16% and thus was further screened for its potential to reduce the pH of an alkaline medium. Phenotypic characteristics of the KG1 isolate were consistent with those of the genus Bacillus, and the highest level of 16S rRNA gene sequence similarity was found with Bacillus halodurans strain DSM 497 (94.7%). On the basis of its phenotypic characteristics and genotypic distinctiveness from other phylogenetic neighbors belonging to alkaliphilic Bacillus species, the isolated strain was designated B. halodurans strain KG1, with GenBank accession number JQ307184 (= NCIM 5439). Isolate KG1 reduced the alkalinity (by 83.64%) and the chloride content (by 86.96%) of cement kiln dust and showed a potential to be used in the cement industry for a variety of applications.

Protease with collagenolytic activity produced by Bacillus sp. DPUA 1728 from Amazonian soil

Qualitative analyses were carried out on solid medium with insoluble collagen 0.25% (w/v) to detect proteases with collagenolytic activity produced by Bacillus sp. In cultures incubated for 24 h, a 23 full factorial design with four repetitions at the center point was developed to analyze the effects and interactions between initial pH, temperature and the concentration of gelatin. Based on the results of the first 23 full factorial design, a successive 23 full factorial design was performed. The most favorable production conditions were found to be 1.5% (w/v) gelatin, pH 9.0 and 37 °C with enzymatic activity of 86.27 U/mL. The enzyme showed optimal activity at 50 °C and pH 9.0, and it was stable over wide pH (7.2-10.0) and temperature (45 °C-60 °C) ranges. These results indicate that Bacillus sp DPUA 1728 is a potential source for producing collagenolytic protease with possible biotechnological applications, such as in the food, cosmetics and leather industries.

Application of bacterial and yeast biosurfactants for enhanced removal and biodegradation of motor oil from contaminated sand

Background This study investigated the potential application of two biosurfactants for enhanced removal capability and biodegradation of motor oil contaminated sand under laboratory conditions. The biosurfactants were produced by the yeast Candida sphaerica and by the bacterium Bacillus sp. cultivated in low-cost substrates. The ability of removing motor oil from soil by the two biosurfactants was identified and compared with that of the synthetic surfactants Tween 80 and Triton X-100. Results Both crude and isolated biosurfactants showed excellent effectiveness on motor oil removal from contaminated sand under kinetic conditions (70-90%), while the synthetic surfactants removed between 55 and 80% of the oil. A contact time of 5-10 min under agitation seemed to be enough for oil removal with the biosurfactants and synthetic surfactants tested. The crude and the isolated biosurfactant from C. sphaerica were able to remove high percentages of motor oil from packed columns (around 90%) when compared to the biosurfactant from Bacillus sp. (40%). For the degradation experiments conducted in motor oil contaminated sand enriched with sugar cane molasses, however, oil degradation reached almost 100% after 90 d in the presence of Bacillus sp. cells, while the percentage of oil degradation did not exceed 50% in the presence of C. sphaerica. The presence of the biosurfactants increased the degradation rate in 10-20%, especially during the first 45 d, indicating that biosurfactants acted as efficient enhancers for hydrocarbon biodegradation. Conclusions The results indicated the biosurfactants enhancing capability on both removal and rate of motor oil biodegradation in soil systems.

Phytase-Producing Bacteria from Extreme Regions in Indonesia

ABSTRACTIn this study, 154 isolates capable of producing extracellular phytate-degrading activity were isolated from four soil samples from volcanic areas in Central Java, Indonesia. Six strains with high phytate-degrading activity were selected for strain identification and characterization of the corresponding phytate-degrading enzyme. Blast analysis of 16S rRNA gene sequences revealed high similarities for all the six isolates to reference sequences belonging to the genusBacillus. Isolates MS5, MC6, D10 and D16 showed 99% sequence identity toB. cereus, while isolate MC8 exhibited 99% sequence identity toB. aryabhatti and D6 99% sequence identity toB. psychrotolerans. The crude extracellular phytase preparations from the isolates showed following optimal conditions for phytate dephosphorylation: pH 4.0 and 50°C (isolate D10), pH 5.0 and 60°C (isolate MC6, and isolate MS5), pH 6.0 and 50°C (isolate D16) and pH 6.0 and 60°C (isolate D6) and pH 6.0 and 40°C (isolate MC8). Zn2+ and Fe3+ strongly inhibited phytate dephosphorylation with all phytase preparations studied. In the presence of Ca2+, an increase in phytase activity of 10-15% was obtained.

Aislamiento de bacterias degradadoras de pesticidas organofosforados encontrados en suelos y en leche bovina / Isolation of organophosphorus pesticides degrading bacteria found in soil and bovine milk

The increasing use of organophosphorus pesticides in agricultural practices over the past few years has generated a number of environmental problems; these compounds tend to bioaccumulate through food chains showing high levels of toxicity triggering potential health risk for species that are exposed to these substances. In this research were used the Soxhlet and solid phase micro extraction in headspace (HS-SPME) methods for the extraction of organophosphorus pesticides in agricultural cattle soils and bovine milk, respectively. The presence of demeton-S-methylsulfon was determined at concentrations between 272.9 and 1793.3 ppm in cropland and 12.9 ppm in cow milk. Native soil bacteria were isolated showing degrading capacity of these pesticides, Bacillus sp and Pantoea agglomerans which gave results of degradation of 73.5% and 68.6 %, respectively, in the concentration of chlorpyrifos, showing that these microorganisms are a possible solution for improving soils contaminated by this class of pesticides.

El incremento en el uso de pesticidas organofosforados en las prácticas agrícolas a través de los últimos años, ha generado una serie de problemas ambientales. Estos compuestos tienden a bioacumularse a través de las cadenas tróficas presentando altos niveles de toxicidad desencadenando potenciales riesgos para la salud de las especies que son expuestas a este tipo de sustancias. En esta investigación se usaron los métodos de extracción soxhlet y micro extracción en fase sólida en espacio de cabeza (HS-SPME) para la extracción de pesticidas organofosforados en suelos de cultivo y leche de ganado bovino, respectivamente. Se determinó la presencia de demetón-S-metilsulfón en concentraciones entre 272.9 y 1793.3 ppm en los suelos de cultivo y 12.9 ppm en leche de vaca. Se aislaron bacterias nativas de suelo con capacidad degradadora de estos pesticidas, Bacillus sp y Pantoea agglomerans, obteniéndose resultados de degradación del compuesto organofosforado, clorpirifos de 73.5% y 68.67%, respectivamente, evidenciando que estos microorganismos son una posible solución para el mejoramiento de suelos contaminados por esta clase de pesticidas.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Background Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. Results A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0-20%), showing maximal growth and enzyme production at 0-5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). Conclusions A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.