Inducible cellulase production from an organic solvent tolerant Bacillus sp. SV1 and evolutionary divergence of endoglucanase in different species of the genus Bacillus

Abstract Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and β-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times β-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai

Abstract This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264 g/L, 2.181 g/L, 1.47 g/L, 1.742 g/L and 1.786 g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18 h and 21 h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140 °C, melting point at 170 °C and maximum thermal degradation at 287 °C. The molecular weight and poly dispersion index of the polymer were found as 199.7 kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

Comparative analysis of ampoules and vials in sterile and conventional packaging as to microbial load and sterility test / Análise comparativa de ampolas e frascos-ampolas em embalagens estéreis e convencionais quanto a carga microbiana e teste de esterilidade

ABSTRACT Objective To compare sterility and microbial (bacteria and fungi) load in the outer part of hyperbaric bupivacaine (Neocaína®) in ampoule and bupivacaine in vial, in conventional and sterile pack formulations. Methods The sterile packs were divided into two groups: G1 (n=16) with ampoules and G2 (n=16) with vials. Conventional formulations were divided into two groups, being G3 (n=16) with ampoules and G4 (n=16) with vials. The ampoules and vials were opened and had their content drawn. The empty bottles were then placed in sterile plastic bags and sent for analysis of microbial load (bacteria and fungi) and sterility testing. Data were analyzed using the χ2 test with Yates correction, and 95% confidence interval. Results G1 and G2 showed no bacterial growth when compared to conventional groups (p<0.001). The most common agent in conventional microbiological samples was Staphylococcus aureus. There was no fungal growth in both groups. Conclusion The use of (sterile pack) reduces the microbial load of bottles, and would decrease the chance of exposure to potential contamination of the anesthetic solution.

RESUMO Objetivo Comparar a esterilidade e a carga microbiana (bactérias e fungos) da parte externa dos frascos de envasamento de bupivacaína hiperbárica (Neocaína®) em ampola e bupivacaína em frasco-ampola das apresentações convencional e estéril (sterile pack). Métodos As apresentações estéreis (sterile pack) foram distribuídas em dois grupos, sendo que o G1 (n=16) continha as ampolas e o G2 (n=16), os frascos-ampola. As apresentações convencionais foram distribuídas em dois grupos, a saber G3 (n=16) com as ampolas e G4 (n=16) com os frascos-ampola. As ampolas e os frascos-ampolas eram abertos e tinham seu conteúdo aspirado. Os frascos vazios eram, então, acondicionados em sacos plásticos estéreis e enviados para análise quanto à carga microbiana (bactérias e fungos), bem como para o teste de esterilidade. Os dados foram analisados por meio do teste χ2 com correção Yates com intervalo de confiança de 95%. Resultados Os grupos G1 e G2 não apresentaram crescimento bacteriano quando comparado aos grupos convencionais (p<0,001). O microbiano mais comum nas amostras convencionais foi o Staphylococcus aureus. Não houve crescimento de fungos em nenhum dos grupos. Conclusão O uso de embalagens estéreis (sterile pack) diminui a carga microbiana dos frascos de envasamentos, o que diminuiria a chance de exposição a uma potencial contaminação da solução anestésica.

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.

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.

Evaluation of Bacillus spp. as dough starters for Adhirasam - A traditional rice based fermented food of Southern India

Abstract Adhirasam is a cereal based, doughnut shaped, deep fried dessert consumed in the southern regions of India. The dough used to prepare adhirasam is fermented and contains rice flour and jaggery. The aim of the present study was to characterize the cultivable bacteria associated with this fermented dough and to identify a suitable starter culture for the production of quality adhirasam. In total, one hundred and seventy bacterial isolates were recovered from de Man Rogosa Sharp (MRS) agar, nutrient agar, lysogeny agar and tryptic soy agar media. Out of the 170 bacterial isolates, sixteen isolates were selected based on their ability to tolerate glucose and sucrose. All the bacterial isolates tolerated 15% glucose and 30% sucrose. Analyses of 16S rDNA gene sequences of the bacterial isolates showed that the dominant cultivable bacteria were members of the genus Bacillus. These strains were further used as starters and tested for their ability to ferment rice flour with jaggery to produce adhirasam dough. Organoleptic evaluation was carried out to choose the best starter strain. Adhirasam prepared from Bacillus subtilis isolates S4-P11, S2-G2-A1 and S1-G15, Bacillus tequilensis isolates S2-H16, S3-P9, S3-G10 and Bacillus siamensis isolate S2-G13 were highly acceptable to consumers. Adhirasam prepared using these starter cultures had superior product characteristics such as softness in texture, flavor and enhanced aroma and sweet taste.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1

A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseedoil as an inducer forprotease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

Agro-industrial residues and starch for growth and co-production of Polyhydroxyalkanoate copolymer and alfa-amylase by Bacillus. SP CFR67îen

Polyhydroxyalkanoates (PHA) and α-amylase (α-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1) were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH) or rice bran (RBH) individually or in combination (5-20 g L-1, based on weight of soluble substrates-SS). In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L-1 of SS) along with ammonium acetate (1.75 g L-1) and corn starch (30 g L-1) produced maximum quantity of biomass (10 g L-1) and PHA (5.9 g L-1). The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L-1) in the medium enhanced fermentative yield of α-amylase (2-40 U mL-1 min-1). The enzyme was active in a wide range of pH (4-9) and temperature (40-60ºC). This is the first report on simultaneous production of copolymer of bacterial PHA and α-amylase from unhydrolysed corn starch and agro-industrial residues as substrates.

Evaluation of Bacillus sphaericus bioinsecticide produced with white soybean meal as culture medium for the control of Culex (Culex) quinquefasciatus / Avaliação do bioinseticida de Bacillus sphaericus, produzido com meio de cultivo de farelo branco de soja no controle de Culex (Culex) quinquefasciatus

Bioinsecticides are shown to be useful in control programs to prevent several diseases, based on their specificity and efficiency against insect vectors. In the current study a bioinsecticide based on Bacillus sphaericus was produced using a white soybean culture medium and applied to larvae of Culex quinquefasciatus, the susceptible species, and Aedes aegypti, the refractory species used as the negative control. Efficacy was compared with that of the product fermented with the Luria Bertani (LB) reference medium. The experiments showed that C. quinquefasciatus was highly susceptible to the product prepared with white soybean meal, reaching 100 percent larval mortality even at 10mg/L, while A. aegypti failed to reach 70 percent mortality at a concentration of 1g/L. By comparison with the reference medium, the proposed culture medium showed high larvicidal power, reaching a LD90 of 2.26mg/L, while 4.37mg/L was needed for the LB medium to achieve the same mortality rate. Cost comparison between the formulations favored the use of the bioinsecticide produced with white soybean meal. After factoring in the LD90 value, the cost ratio favored the new raw material by nearly 1:220.

A utilização de bioinseticidas tem se mostrado útil aos programas de prevenção de diversas enfermidades devido a sua especificidade e eficiência contra insetos vetores. No presente trabalho, o bioinseticida de Bacillus sphaericus foi produzido com um meio de cultivo composto de farelo branco de soja e aplicado em larvas de Culex quinquefasciatus, espécie susce-tível, e Aedes aegypti, espécie refratária, usada como controle negativo. O desempenho foi comparado com o do produto fermentado com o meio referência Luria Bertani (LB). Os experimentos constataram que C. quinquefasciatus apresentou uma alta suscetibilidade ao produto produzido com farelo branco de soja, alcançando mortalidade de 100 por cento mesmo na diluição de 10mg/L, enquanto A. aegypti não atingiu 70 por cento na concentração de 1g/L. Quando comparado com o meio referência, a formulação proposta proporcionou um alto poder larvicida, alcançando uma DL90 de 2,26mg/L, ao passo que LB precisou de 4,37mg/L para a mesma mortalidade. A comparação do custo das formulações favoreceu o bioinseticida produzido com o meio farelo branco de soja. Considerando os valores de DL90, a relação do custo das matérias-primas ficou próxima de 1:220.

Colonization of Bacillus spp. on seeds and in plant rhizoplane.

Seed coating, dipping and Scanning Electron Microscopy (SEM) were employed to study bacterial and fungal colonization of the seeds and rhizoplane of maize (Zea mays L.) during the early stages of growth. Isolation of Bacillus spp. entailed screening soil bacteria with potential growth stimulation and plant pathogen suppressive abilities isolated from the rhizospheres and rhizoplanes of vegetable crops. The bacterial colonization of the spermosphere was 90%. When the coated seeds were fully germinated, bacteria moved to the emerging radicle. Virtually no bacteria occurred on the root tip both for the treated and untreated. However, colonization was 20% in the basal portion of the roots close to the seed-root junction. SEM observations showed that the bacterial cells were arranged linearly and laterally on the growing root axis. This phenomenon was more noticeable in the seedlings dipped in the bacterial culture on the 3rd day after germination. The results indicate that attachment to the seed coat and the rhizoplane by the plant growth-promoting rhizobacterium (PGPR) is an important factor in the successful colonization of the rhizoplane. The significance of the work is to ascertain that the inoculated Bacillus spp. adhered to and established in the rhizoplane of maize. It can therefore be used as a PGPR and as a biocontrol agent.

Microbial counts of dark red latosol samples stored at different temperatures

The number of colony forming units (CFU) of different groups of bacteria and fungi in samples stored at temperatures of 5 to -12§C for 0-32 weeks was evaluated. The number of CFU obtained after the different periods of storage of red latossol soil was compared with the number of colonies obtained immediately after removal of soil samples (time zero). The number of total bacteria and actinomycetes in the samples remained pratically unchanged throughout the storage period. The number of Gram-negative bacteria decreased by as much as 69 per cent compared to control, while the number of Bacillus spp and of fungi increased 1.9 to 4.9 times starting from the 12th week in sample stored at 5§C. Except for the variations observed in fungal counts, the remaining groups of bacteria pratically showed the same variation in number of colonies in soil samples stored at 5§C and -12§.

Production of alkaline protease bacillus macerans cells entrapped in different gel matrices

The extracellular alkaline protease activity of 8 bacterial isolates was investigated in shake and static cultures A 72 hold. Bacillus macerans showed the highest alkaline protease activity under shaking conditions [71.3 U/ml]. Entrapment of B. macerans cells in Ca-alginate, K-carrageenan or agar, increased the alkaline protease activity in the cultures by 1.60 and 1.37-fold, respectively. Cell leakage was relatively lower from alginate beads. Cell entrapment decreased the protein content in the culture supernatants by 20 to 30% that of the free cultures. Some properties of the crude enzyme were studied. Optimum enzyme and substrate concentrations were 1.22 mg protein/ml and 30 mg casein/ml, respectively. Optimum temperature for the enzyme activity was 45°C and optimum pH was 10. The enzyme showed its maximal stability at pH 10 retaining 66% of its activity after exposure to 55°C for 30 min, while it retained only 25% of activity at pH 11

Purification and characterization of chitinase produced by bacillus amyloliquefaciens

The crude chitinase preparation obtained from Bacillus amloliquefaciens cultures was partially purified by fractional precipitation with ethanol, acetone or ammonium sulphate. The 65% ammonium sulphate fraction was the most active and was further purified by gel filtration on Sephadex G-200 followed by ion exchange chromatography on Ecteola ET-11 cellulose yielding 4 chitinolytic enzyme components. Two enzyme components, CHII and CHIII, showed high activity and protein recovery. The purity of both enzymes was checked by polyacrylamide gel electrophoresis. CHII enzyme was more specifid for chitin as substrate and showed a higher thermostability than CHIII. Both enzymes showed a temperature optimum of 45°C and pH optimum of 5.5 to 5.9. They did not show specific cationic requirements and were partially inhibited by zinc, cupric, silver and cobalt ions. Their amino acid compositions were different. SDS electrophoresis revealed that each enzyme was formed of 2 subunits of different molecular weights, CHII subunit had a M.W. of 26.5 and 23.5 KD while CHIII was 31.6 and 27.5 KD

Degradation of shrimp-shell waste and production of chitinase by bacillus amyloliquefaciens under some cultural conditions

The study of chitinase production by Bacillus amyloliquefaciens grown on shrimp-shell waste as the sole carbon source showed that chitinase production with maximum enzyme activity was reached when 2% inoculum of 24 h age seed culture was used. The inoculated medium contained [g/I] shrimpshell waste, 80; NH[4]CI, 1.2: KH[2]PO [4]; K[2]HPO[4], 1.6; MgSO[4].7H[2]O, 0.1: ZnSO[4]. 7H[2]O, 0.1; ZnSO[4] .7H[2]O. FeCI[3]. 6H[2]O, 0.01; CaCI[2]2]H[2]O, 0.02 at pH 7 and the culture shaken at 30°C for 36 hours

Some factors influencing chitinase production by bacillus amyloliquefaciens

The effect of several analogues and breakdown products of chitin on the iodsetioa of the chitinase of B. amyloliquefaciens was examined. Glucose. N -acetyl-glucosamine and chitobiose inhibited the enzyme productivity. The effect of the addition of some natural extracts was also investigated and the maximum enzyme activity was produced with 2% sand extract. A simplified medium composed of 8% shrimp-shell waste and 80% sand extract yielded an enzyme of highest activity. Reaction conditions supporting best activity sum achieved using 5-7-5mg swollen chitin in 0.03 M phosphate buffer at pH 5.916.98; enzyme concentration of 11.35mg protein/reaction mixture and incubating the mixture at 45°C for 60 min

Production of alpha-amylase from bacillus lentus

A number of nutritional factors affecting growth and alpha-amylase by amylase by Bacillus lentus were investigated. The production of enzyme increased with increase of temperature reaching its maximum between 40°C - 45°C after 60h incubation when cultivated in a medium with 30 g/1 starch as carbon source. Supplementation of the medium with different carbon sources did not enhance either enzyme activity or growth. Of the nitrogen sources examined a mixture of peptone, yeast and beef extracts stimulated growth and yielded the highest enzyme production

On the production of alpha-amylase by free and immobilized cultures of bacillus lentus

The effect of natural additive, amino acids, vitamins, surfactants on the production of alpha-amylase by Bacillus lentus was studied. Also the production of alpha-amylase was followed with cells of B. lentus immobillized by entrapment in Ca-alginate [2%] in static, shaked and insemi-continuous cultures, and by adsorption on granular clay

Production & formulation of Bacillus thuringiensis var. israelensis & B. sphaericus 1593.

Three fermentation media each for bulk growth of B. thuringiensis var. israelensis and B. sphaericus 1593 were formulated using defatted groundnut cake (Arachis hypogea) as the first nitrogen source and gram flour (Cicer arientinum), soy bean (Glycine max) and defatted milk powder as the second nitrogen source. Medium containing gram flour showed highest toxicity (14.45 micrograms/l) in case of B. thuringiensis var. israelensis whereas medium containing milk powder was found to be highly toxic with B. sphaericus 1593 (51.39 micrograms/l). Sustained release floating pellet formulations of B. thuringiensis var. israelensis and B. sphaericus 1593 exhibited toxicity of 77 per cent and above for 42 days at a dose of 500 micrograms/l for 4th instar larvae of Culex pipiens quinquefasciatus Say.