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1.  Analysis of Anoxybacillus Genomes from the Aspects of Lifestyle Adaptations, Prophage Diversity, and Carbohydrate Metabolism 
PLoS ONE  2014;9(3):e90549.
Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.
PMCID: PMC3948429  PMID: 24603481
2.  Abiotic and Microbiotic Factors Controlling Biofilm Formation by Thermophilic Sporeformers 
Applied and Environmental Microbiology  2013;79(18):5652-5660.
One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.
PMCID: PMC3754176  PMID: 23851093
3.  Non-contiguous finished genome sequence of Anoxybacillus flavithermus subsp. yunnanensis type strain (E13T), a strictly thermophilic and organic solvent-tolerant bacterium 
Standards in Genomic Sciences  2014;9(3):735-743.
Anoxybacillus flavithermus subsp. yunnanensis is the only strictly thermophilic bacterium that is able to tolerate a broad range of toxic solvents at its optimal temperature of 55-60°C. The type strain E13T was isolated from water-sediment slurries collected from a hot spring. This study presents the draft genome sequence of A. flavithermus subsp. yunnanensis E13T and its annotation. The 2,838,393bp long genome (67 contigs) contains 3,035 protein-coding genes and 85 RNA genes, including 10 rRNA genes, and no plasmids. The genome information has been used to compare with the genomes from A. flavithermus subsp. flavithermus strains.
PMCID: PMC4148970  PMID: 25197458
Anoxybacillus flavithermus subsp. yunnanensis; genome; solvent tolerance; thermophile
4.  Anoxybacillus suryakundensis sp. nov, a Moderately Thermophilic, Alkalitolerant Bacterium Isolated from Hot Spring at Jharkhand, India 
PLoS ONE  2013;8(12):e85493.
Four closely related facultative anaerobe, moderately thermophilic, Gram positive rods (JS1T, JS5, JS11, and JS15) were isolated from sediment samples from a hot spring at Suryakund, Jharkhand, India. Colonies were pale yellow, rough surface with uneven edges on TSA after 72 h incubation. Heterotrophic growth was observed at 40-60°C and pH 5.5-11.5; optimum growth occurred at 55°C and pH 7.5. 16S rRNA gene sequence analysis revealed the strains belong to genus Anoxybacillus. DNA-DNA homology values among strains were above 70% and showed distinct ERIC and REP PCR profile. On the basis of morphology and biochemical characteristics, strain JS1T was studied further. Strain JS1T showed 99.30% sequence similarity with A. flavithermus subsp. yunnanensis, 99.23% with A. mongoliensis, 99.16% with A. eryuanensis, 98.74% with A. flavithermus subsp. flavithermus, 98.54% with A. tengchongensis, 98.51% with A. pushchinoensis, 97.91% with A. thermarum, 97.82% with A. kaynarcensis, 97.77% with A. ayderensis and A. kamchatkensis, 97.63% with A. salavatliensis, 97.55% with A. kestanbolensis, 97.48% with A. contaminans, 97.27% with A. gonensis and 97.17% with A. voinovskiensis. In 16S rRNA secondary structure based phylogenetic comparison, strain JS1T was clustered with Anoxybacillus eryuanensis, A. mongoliensis, and A. flavithermus subsp. yunnanensis and showed 15 species specific base substitutions with maximum variability in helix 6. Moreover, DNA-DNA relatedness between JS1T and the closely related type strains were well below 70%. The DNA G+C content was 42.1 mol%. The major fatty acids were C15:0 iso, C16:0 iso and C17:0iso. The polar lipids were a phosphatidylgylycerol, a diphosphatidylglycerol, a phosphatidylethnolamine, a phosphatidylcholine, a phosphatidyl monomethylethanolamine and four unknown lipids. Based on polyphasic approach, strain JS1T represent a novel species of the genus Anoxybacillus for which Anoxybacillus suryakundensis sp. nov. is proposed. The type strain is JS1T (= DSM 27374T = LMG 27616T =JCM19211T).
PMCID: PMC3869905  PMID: 24376881
5.  Experimental Simulation of Evaporation-Driven Silica Sinter Formation and Microbial Silicification in Hot Spring Systems 
Astrobiology  2013;13(2):163-176.
Evaporation of silica-rich geothermal waters is one of the main abiotic drivers of the formation of silica sinters around hot springs. An important role in sinter structural development is also played by the indigenous microbial communities, which are fossilized and eventually encased in the silica matrix. The combination of these two factors results in a wide variety of sinter structures and fabrics. Despite this, no previous experimental fossilization studies have focused on evaporative-driven silica precipitation. We present here the results of several experiments aimed at simulating the formation of sinters through evaporation. Silica solutions at different concentrations were repeatedly allowed to evaporate in both the presence and absence of the cyanobacterium Synechococcus elongatus. Without microorganisms, consecutive silica additions led to the formation of well-laminated deposits. By contrast, when microorganisms were present, they acted as reactive surfaces for heterogeneous silica particle nucleation; depending on the initial silica concentration, the deposits were then either porous with a mixture of silicified and unmineralized cells, or they formed a denser structure with a complete entombment of the cells by a thick silica crust. The deposits obtained experimentally showed numerous similarities in terms of their fabric to those previously reported for natural hot springs, demonstrating the complex interplay between abiotic and biotic processes during silica sinter growth. Key Words: Silica—Cyanobacteria—Fossilization—Hot springs—Stromatolites. Astrobiology 13, 163–176.
PMCID: PMC3582282  PMID: 23384170
6.  Thermostable and Alkalistable Xylanases Produced by the Thermophilic Bacterium Anoxybacillus flavithermus TWXYL3 
ISRN Microbiology  2012;2012:517524.
With the rising cost and finite supply of fossil energy, there is an increasing economic incentive for the development of clean, efficient, and renewable domestic energy. The activities of microorganisms offer the potential conversion of lignocellulosic materials into fermentable sugars, usable for downstream fermentation processes. Strain TWXYL3, a thermophilic facultative anaerobe, was discovered in the Alvord Basin hydrothermal system in Oregon, USA. Phylogenetic analysis of strain TWXYL3 showed it to be 99% similar to the 16S rRNA gene of Anoxybacillus flavithermus WL (FJ950739). A. flavithermus TWXYL3 was shown to secrete a large multisubunit thermostable xylanase complex into the growth medium. Xylanase induction was achieved by resuspending the isolate in a selective xylan-containing medium. Extracellular xylanase activity showed a temperature optimum of 65°C and retained thermostability up to 85°C. Extracellular xylanase activity showed a bimodal pH optimum, with maxima at pH 6 and pH 8. Electrophoretic analysis of the extracellular xylanase shows 5 distinct proteins with xylanase activity. Strain TWXYL3 is the first xylanolytic isolate obtained from the Alvord Basin hydrothermal system and represents a new model system for development of processes where lignocellulosics are converted to biofuel precursors.
PMCID: PMC3671716  PMID: 23762752
7.  Influence of Cations on Growth of Thermophilic Geobacillus spp. and Anoxybacillus flavithermus in Planktonic Culture 
Free ions of Na+, K+, Ca2+, and Mg2+ influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca2+ and Mg2+ were associated with increases in optical density more so than Na+ and K+. Overall, it appeared that Ca2+ and/or Mg2+ was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.
PMCID: PMC3302610  PMID: 22287005
8.  Complete Genome Sequence of Anoxybacillus flavithermus TNO-09.006, a Thermophilic Sporeformer Associated with a Dairy-Processing Environment 
Genome Announcements  2013;1(1):e00010-13.
Spores of thermophilic spore-forming bacteria are a common cause of contamination in dairy products. We isolated the thermophilic strain Anoxybacillus flavithermus TNO-09.006 from a milk-processing plant, and we report the complete genome of this isolate consisting of a single chromosome of 2.65 Mb.
PMCID: PMC3587922  PMID: 23469333
9.  Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts 
Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.
PMCID: PMC427776  PMID: 15184171
10.  Preconditioning with Cations Increases the Attachment of Anoxybacillus flavithermus and Geobacillus Species to Stainless Steel 
Applied and Environmental Microbiology  2013;79(13):4186-4190.
Preconditioning of Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 with cations prior to attachment often significantly increased (P ≤ 0.05) the number of viable cells that attached to stainless steel (by up to 1.5 log CFU/cm2) compared with unconditioned bacteria. It is proposed that the transition of A. flavithermus and Geobacillus spp. from milk formulations to stainless steel product contact surfaces in milk powder manufacturing plants is mediated predominantly by bacterial physiological factors (e.g., surface-exposed adhesins) rather than the concentrations of cations in milk formulations surrounding bacteria.
PMCID: PMC3697576  PMID: 23645192
11.  Draft Genome Sequence of a Thermophilic Member of the Bacillaceae, Anoxybacillus flavithermus Strain Kn10, Isolated from the Kan-nawa Hot Spring in Japan 
Genome Announcements  2013;1(3):e00311-13.
Here, we report the draft genome sequence of the Anoxybacillus flavithermus Kn10 strain (NBRC 109594), isolated from a water drain of the Kan-nawa Hot Spring in Japan. The draft genome sequence is composed of 90 contigs for 2,772,624 bp with 41.6% G+C content and contains 2,883 protein-coding genes and 80 tRNA genes.
PMCID: PMC3668014  PMID: 23723406
12.  Draft Genome Sequence of Anoxybacillus flavithermus Strain 25, Isolated from the Garga Hot Spring in the Barguzin Valley, Baikal Region, Russian Federation 
Genome Announcements  2014;2(6):e01258-14.
Anoxybacillus flavithermus strain 25 was isolated from a sediment sample from the Garga hot spring in the Barguzin Valley, Baikal Region, Russian Federation (54°19′3.72″N, 110°59′38.4″E). The sequenced and annotated genome is 2,838,680 bp and encodes 3,009 genes.
PMCID: PMC4256193  PMID: 25477412
13.  The Use of Amino Sugars by Bacillus subtilis: Presence of a Unique Operon for the Catabolism of Glucosamine 
PLoS ONE  2013;8(5):e63025.
B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source.
PMCID: PMC3648570  PMID: 23667565
14.  Stimulation of Expression of a Silica-Induced Protein (Sip) in Thermus thermophilus by Supersaturated Silicic Acid▿  
The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75°C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO2), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe3+ ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe3+-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.
PMCID: PMC2675196  PMID: 19233950
15.  Molecular phylogenetic analysis of bacterial community and characterization of Cr(VI) reducers from the sediments of Tantloi hot spring, India 
Aquatic Biosystems  2014;10:7.
A geothermal ecosystem located at Tantloi, India has been found to be an interesting habitat for microbes of diverse nature. However, the microbial diversity of this habitat is poorly explored. In this study, a detailed phylogenetic study has been carried out to understand the bacterial diversity of this habitat and to identify prospective metal reducers using culture independent approach. The bacterial diversity of the sediments, which contain undetectable levels of Cr(VI), was analysed with respect to chromium reduction and the strains highly resistant to and efficiently reducing chromium under aerobic conditions were isolated and characterized.
16S rRNA gene sequence analysis of Tantloi hot spring microbial community revealed a significant bacterial diversity represented by at least ten taxonomic divisions of Bacteria with clear predominance of Thermus. Similar sequence analysis of rRNA gene library clones derived from bacterial consortia enriched from sediments in presence of Cr(VI) revealed the abundance of the family Bacillaceae. Under aerobic conditions at 65°C, the consortia reduced 1 mM of Cr(VI) completely within 24 h and 5 mM in 6 days. A complete reduction of 1 mM Cr(VI) has been shown by five of our isolates within 36 h. 16S rRNA gene sequences of all the isolates showed high degree of similarity (97-99%) to Bacillaceae with ten of them being affiliated to Anoxybacillus. Crude extract as well as the soluble fraction from isolates TSB-1 and TSB-9 readily reduced Cr(VI); TSB-1 showed higher chromium reductase activity.
Most of the Tantloi Spring Bacterial (TSB) sequences analyzed in different taxonomic divisions could be related to representatives with known metabolic traits which indicated presence of organisms involved in redox processes of a variety of elements including iron, sulphur and chromium. Approximately 80% of the sequences obtained in this study represented novel phylotypes indicating the possibility of discovery of bacteria with biotechnologically important new biomolecules. Again, highly chromium-resistant and remarkably active Cr(VI)-reducing Anoxybacillus strains isolated in this study could serve as potential candidates for designing chromium bioremediation strategies at high temperatures and also at high chromium concentrations.
PMCID: PMC4168125  PMID: 25243065
Phylogenetic analysis; Bacterial community; Hot spring; Chromium reduction; Bioremediation
16.  New insight into silica deposition in horsetail (Equisetum arvense) 
BMC Plant Biology  2011;11:112.
The horsetails (Equisetum sp) are known biosilicifiers though the mechanism underlying silica deposition in these plants remains largely unknown. Tissue extracts from horsetails grown hydroponically and also collected from the wild were acid-digested in a microwave oven and their silica 'skeletons' visualised using the fluor, PDMPO, and fluorescence microscopy.
Silica deposits were observed in all plant regions from the rhizome through to the stem, leaf and spores. Numerous structures were silicified including cell walls, cell plates, plasmodesmata, and guard cells and stomata at varying stages of differentiation. All of the major sites of silica deposition in horsetail mimicked sites and structures where the hemicellulose, callose is known to be found and these serendipitous observations of the coincidence of silica and callose raised the possibility that callose might be templating silica deposition in horsetail. Hydroponic culture of horsetail in the absence of silicic acid resulted in normal healthy plants which, following acid digestion, showed no deposition of silica anywhere in their tissues. To test the hypothesis that callose might be templating silica deposition in horsetail commercially available callose was mixed with undersaturated and saturated solutions of silicic acid and the formation of silica was demonstrated by fluorimetry and fluorescence microscopy.
The initiation of silica formation by callose is the first example whereby any biomolecule has been shown to induce, as compared to catalyse, the formation of silica in an undersaturated solution of silicic acid. This novel discovery allowed us to speculate that callose and its associated biochemical machinery could be a missing link in our understanding of biosilicification.
PMCID: PMC3160890  PMID: 21801378
Biosilicification; biogenic silica; silicic acid; horsetails; callose; PDMPO; fluorescence; acid digestion.
17.  A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization 
An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.
PMCID: PMC3709733  PMID: 23759984
Anoxybacillus; amylase; Bacillus; Geobacillus; glycoside hydrolase 13; pullulan; pullulanase; starch; thermostable enzyme
18.  Effect of Processing and Sintering on Controlled Release Wax Matrix Tablets of Ketorolac Tromethamine 
The objective of present study was to evaluate the effect of processing methods and sintering condition on matrix formation and subsequent drug release from wax matrix tablets for controlled release. Ketorolac tromethamine and compritol were processed with appropriate diluent using either dry blending, spray drying, partial melt granulation or melt granulation.The tablets were then sintered at 80°. The sintered tablets were characterized by their physical parameters and in vitro dissolution tests. The micro-morphology and wettability of the tablets was also investigated. It was evident that different processing methods for identical formulation significant impact the release profile of drug. Sintering further retarded drug release and its effect was related to the manufacturing processes. Scanning electron microscopy showed that heat treatment redistributed the wax and formed a film-like structure covering drug and excipient particle. The contact angle of tablets made by dry blending, spray drying and partial melt granulation methods increased after sintering, while that of tablets made by melt granulation remained constant. Drug release from the wax tablets with or without heat treatment was best described by the Higuchi equation. Different processing methods produced different matrix structures that resulted in different drug release rates. Sintering retarded drug release mainly by decreasing the porosity of the matrix. Contact angle measurement and SEM analysis indicated that heat treatment caused the wax to melt, redistribute, coat the drug and diluents and form a network structure. Differential scanning calorimetry studies ruled out the occurrence of solid solution of the drug during sintering condition.
PMCID: PMC2866346  PMID: 20502573
Controlled release; scanning electron microscopy (SEM); sintering; wax
19.  Multiparametric Analyses Reveal the pH-Dependence of Silicon Biomineralization in Diatoms 
PLoS ONE  2012;7(10):e46722.
Diatoms, the major contributors of the global biogenic silica cycle in modern oceans, account for about 40% of global marine primary productivity. They are an important component of the biological pump in the ocean, and their assemblage can be used as useful climate proxies; it is therefore critical to better understand the changes induced by environmental pH on their physiology, silicification capability and morphology. Here, we show that external pH influences cell growth of the ubiquitous diatom Thalassiosira weissflogii, and modifies intracellular silicic acid and biogenic silica contents per cell. Measurements at the single-cell level reveal that extracellular pH modifications lead to intracellular acidosis. To further understand how variations of the acid-base balance affect silicon metabolism and theca formation, we developed novel imaging techniques to measure the dynamics of valve formation. We demonstrate that the kinetics of valve morphogenesis, at least in the early stages, depends on pH. Analytical modeling results suggest that acidic conditions alter the dynamics of the expansion of the vesicles within which silica polymerization occurs, and probably its internal pH. Morphological analysis of valve patterns reveals that acidification also reduces the dimension of the nanometric pores present on the valves, and concurrently overall valve porosity. Variations in the valve silica network seem to be more correlated to the dynamics and the regulation of the morphogenesis process than the silicon incorporation rate. These multiparametric analyses from single-cell to cell-population levels demonstrate that several higher-level processes are sensitive to the acid-base balance in diatoms, and its regulation is a key factor for the control of pattern formation and silicon metabolism.
PMCID: PMC3483172  PMID: 23144697
20.  Multiparticle Sintering Dynamics: From Fractal-Like Aggregates to Compact Structures 
Multiparticle sintering is encountered in almost all high temperature processes for material synthesis (titania, silica, and nickel) and energy generation (e.g., fly ash formation) resulting in aggregates of primary particles (hard- or sinter-bonded agglomerates). This mechanism of particle growth is investigated quantitatively by mass and energy balances during viscous sintering of amorphous aerosol materials (e.g., SiO2 and polymers) that typically have a distribution of sizes and complex morphology. This model is validated at limited cases of sintering between two (equally or unequally sized) particles, and chains of particles. The evolution of morphology, surface area and radii of gyration of multiparticle aggregates are elucidated for various sizes and initial fractal dimension. For each of these structures that had been generated by diffusion limited (DLA), cluster–cluster (DLCA), and ballistic particle–cluster agglomeration (BPCA) the surface area evolution is monitored and found to scale differently than that of the radius of gyration (moment of inertia). Expressions are proposed for the evolution of fractal dimension and the surface area of aggregates undergoing viscous sintering. These expressions are important in design of aerosol processes with population balance equations (PBE) and/or fluid dynamic simulations for material synthesis or minimization and even suppression of particle formation.
PMCID: PMC3569621  PMID: 21488641
21.  Bio-inspired Silicification of Silica-binding Peptide-Silk Protein Chimeras: Comparison of Chemically and Genetically Produced Proteins 
Biomacromolecules  2012;13(3):683-690.
Novel protein chimeras constituted of ‘silk’ and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]n) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 25 equivalents of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial compatible conditions the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared to the genetically derived variants. In all cases, the structure of the protein / chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source) and approach to synthesis (genetic or chemical) can be used to ‘tune’ the properties of the composite materials formed and is a general approach which can be used to prepare a range of materials for biomedical and sensor based applications.
PMCID: PMC3304446  PMID: 22229696
fusion protein; silk; silica; peptide; chemical chimera; genetic chimera
22.  Chemoselective silicification of synthetic peptides and polyamines 
Biosilicification sets the standard for the localized in vitro precipitation of silica at low orthosilicate concentrations in aqueous environment under ambient conditions. Numerous parameters must be controlled for the development of new technologies in designing inventive nanosilica structures, which are able to challenge the biological templates. A long neglected requirement that came into focus in the recent years are the cellular techniques of preventing unintentional lithification of cellular structures since numerous cellular components such as membranes, DNA, and proteins are known to precipitate nanosilica. The diatom metabolism makes use of techniques that restrict silicification to an armor of silica around the cell wall while avoiding the petrifying gaze of Medusa, which turns the whole cell into stone. Step by step, biochemistry unveils the hierarchical interplay of an arsenal of low-molecular weight molecules, proteins, and the cytoskeletal architecture and it becomes clearer why the organisms invest much metabolic effort for an obviously simple chemical reaction like the precipitation of amorphous silica. The discrimination between different soluble components in the silicification process (chemoselective silicification) is not only vitally important for the diatom but poses an interesting challenge for in vitro experiments. Until now, silica precipitation studies were mainly focused on the amount, the morphology, and composition of the precipitate while disregarding a quantitative analysis of the remaining soluble components. Here, we turn the tables and quantify the soluble components by 1H NMR in the progress of precipitation and present experiments which quantify the additivity, and potential cooperativity of long chain polyamines (LCPAs) and cationic peptides in the silicification process.
PMCID: PMC4311759
biomineralisation; biosilicification; NMR spectroscopy; polyamines; silaffin
23.  Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia 
BMC Microbiology  2007;7:77.
Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5–99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.
Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70°C and was also stable up to 60°C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T) and Geobacillus kaustophilus (DSM 7263T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T).
Strain T1T was able to secrete extracellular thermostable lipase into culture medium. The strain T1T was identified as Geobacillus zalihae T1T as it differs from its type strains Geobacillus kaustophilus (DSM 7263T) and Geobacillus thermoleovorans (DSM 5366T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile.
PMCID: PMC2000885  PMID: 17692114
24.  The genetic diversity of genus Bacillus and the related genera revealed by 16s rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey 
Brazilian Journal of Microbiology  2012;43(1):309-324.
Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4–100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.
PMCID: PMC3768990  PMID: 24031834
isolation; temperature requirement; endospore-forming bacilli; 16S rRNA gene; ARDRA
25.  High-Aluminum-Affinity Silica Is a Nanoparticle That Seeds Secondary Aluminosilicate Formation 
PLoS ONE  2013;8(12):e84397.
Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m2 g-1 and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates.
PMCID: PMC3862809  PMID: 24349573

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