Characterization and emulsifying property of a novel bioemulsifier by Aeribacillus pallidus YM-1

Aims: Biosurfactants and bioemulsifiers commonly have the advantages of biodegradability, low toxicity, selectivity and biocompatibility over chemically synthesized surfactants. The goal of the study is to present a novel bioemulsifier with great application potential. Methods and Results: Aeribacillus pallidus YM‐1, isolated from crude oil contaminated soil, was found to produce a novel high molecular bioemulsifier with an emulsification index of 60 ± 1% without remarkable surface tension reduction (45·7 ± 0·1 mN m^?1). The number‐average molecular weight was determined as 526 369 Da by gel permeation chromatography analysis. Bioemulsifier was subjected to FT‐IR and a complex of carbohydrates (41·1%), lipids (47·6%) and proteins (11·3%) was determined. Conclusions: The bioemulsifier of A. pallidus YM‐1 was isolated from the glucose‐based culture medium and characterized with the help of chemical analytical techniques. The bioemulsifier exhibited a promising emulsifying property for biotechnology application potential in bioremediation and microbial enhanced oil recovery. Significance and Impact of the Study: This is the first report of the bioemulsifier production by A. pallidus. The potential emulsifying activity of the bioemulsifier in the present study may be explored in various biotechnological and industrial applications.     

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35?kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60?°C. It was determined that the enzyme had remained stable at the range of pH 7.0–10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20–80?°C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K_M and V_max values were calculated as 0.197?mg/mL and 7.29?μmol.mL^?1.min^?1, respectively.     

Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders

AIMS: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder. METHODS AND RESULTS: The BactiFlow flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0.1% peptone were treated with 0.8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55 degrees C for 2-3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results. CONCLUSIONS: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product.     

Anoxybacillus and Geobacillus biofilms in the dairy industry: effects of surface material,incubation temperature and milk type

Abstract

Anoxybacillus (A. flavithermus, A. kamchatkensis subsp. asachharedens, A. caldiproteolyticus and A. tepidamans) and Geobacillus (two strains of G. thermodenitrificans, G. thermoglucosidans and G. vulcanii) isolates and reference strains in whole milk were evaluated for their biofilm production on six different abiotic surfaces. G. thermodenitrificans DSM 465^T had the highest cell counts (>4 log₁₀ CFU cm^?2) on glass and stainless steel (SS) at 55 and 65?°C, respectively. G. thermodenitrificans D195 had the highest counts on SS at 55?°C (>5 log₁₀ CFU cm^?2) and polyvinyl chloride (PVC) at 65?°C (>4 log₁₀ CFU cm^?2), indicating the existence of strain variation. The ideal surfaces for all strains were SS and glass at 55?°C, but their preferences were polystyrene and SS at 65?°C. Moreover, Anoxybacillus members were more prone to form biofilms in skim milk than in semi-skim and whole milk, whereas the results were the opposite for Geobacillus. Both the attachment and sporulation of Geobacillus in whole milk was higher than in semi-skim or skim milk. This study proposes that the surface material, temperature and milk type had a cumulative effect on biofilm formation.     

Short communication: Protoplast formation from the thermophilic fungus Malbranchea sulfurea,using the thermostable chitinase and laminarinase of Paecilomyces varioti

Two thermostable enzymes produced by the thermophilic fungus Paecilomyces varioti, a chitinase and laminarinase, were used to isolate protoplasts of a thermophilic fungus, Malbranchea sulfurea. The frequency of protoplast regeneration observed (35%) was considerably higher than that obtained using commercial lytic enzymes.     

Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel

AIMS: To determine the mechanism for both the removal and inactivation of 18-h biofilms of a thermophilic Bacillus species that optimally grows at 55 degrees C on stainless steel. METHODS AND RESULTS: The cleaning strategies tested were based on biofilm biochemistry and physiology, and focused on the chemistry of the cleaners, the duration and temperature of the cleaning process and a combination of various cleaners. The success of the cleaning regimes was determined based on the removal of cells and organic debris and the elimination of viable cells. The results confirmed that a caustic (75 degrees C for 30 min) and acid (75 degrees C for 30 min) wash, relied upon heavily in most food processing industries for cleaning-in-place systems, was successful in removing these biofilms. However, any changes in the concentrations of these cleaners or the temperature of cleaning drastically affected the overall outcome. Alternative cleaning agents based on enzymatic or nonenzymatic breakdown of cellular proteins or polysaccharides, surfactant action, use of oxidative attack and free radicals varied in degrees of their success. Combining proteolytic action with surfactants increased wetability and therefore enhanced the cleaning efficiency. CONCLUSIONS: Several procedures, including caustic/acid and enzyme based cleaners, will be satisfactory, provided that the correct process parameters are observed i.e. concentration, time, temperature and kinetic energy (flow). Confirmation of these results should be carried out in a pilot plant through several use/clean cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: Confidence in standard and alternative cleaning procedures for food manufacturing plant to prevent contamination with thermophilic bacilli that threaten product quality.     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Influence of microbial interactions and EPS/polysaccharide composition on nutrient removal activity in biofilms formed by strains found in wastewater treatment systems

The study of biofilm function, structure and microbial interactions might help to improve our understanding of biofilm wastewater treatment processes. However, few reports specifically address the influence of interactions within multispecies biofilms on microbial activity and biofilm composition. Thus, the relationship between biofilm formation, denitrification activity, phosphorus removal and the composition of extracellular polymeric substances (EPS), exopolysaccharides and the bacterial community was investigated using biofilms of denitrifying and phosphorus removing strains Comamonas denitrificans 110, Brachymonas denitrificans B79, Aeromonas hydrophila L6 and Acinetobacter calcoaceticus ATCC23055. Denitrification activity within the biofilms generally increased with the amount of biofilm while phosphorus removal depended on bacterial growth rate. Synergistic effects of co-growth on denitrification (B. denitrificans B79 and A. hydrophila L6) and phosphorus removal (C. denitrificans 110 with either A. calcoaceticus or A. hydrophila L6) were observed. B. denitrificans B79 was highly affected by interspecies interactions with respect to biofilm formation, denitrification activity and EPS composition, while C. denitrificans 110 remained largely unaffected. In some of the dual and quadruple strain biofilms new exopolysaccharide monomers were detected which were not present in the pure strain samples.     

Design of a rotating disk reactor to assess the colonization of biofilms by free-living amoebae under high shear rates

The present study was aimed at designing and optimizing a rotating disk reactor simulating high hydrodynamic shear rates (γ), which are representative of cooling circuits. The characteristics of the hydrodynamic conditions in the reactor and the complex approach used to engineer it are described. A 60 l tank was filled with freshwater containing free-living amoebae (FLA) and bacteria. Adhesion of the bacteria and formation of a biofilm on the stainless steel coupons were observed. FLA were able to establish in these biofilms under γ as high as 85,000 s^?1. Several physical mechanisms (convection, diffusion, sedimentation) could explain the accumulation of amoeboid cells on surfaces, but further research is required to fully understand and model the fine mechanisms governing such transport under γ similar to those encountered in the industrial environment. This technological advance may enable research into these topics.     

A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms

A rapid method for screening the metabolic susceptibility of biofilms to toxic compounds was developed by combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology. The method was developed using Pseudomonas alcaliphila 34, a Cr(VI)-hyper-resistant bacterium, as the test organism. P. alcaliphila produced a robust biofilm after incubation for 16 h, reaching the maximum value after incubation for 24 h (9.4 × 10⁶ ± 3.3 × 10⁶ CFU peg^?1). In order to detect the metabolic activity of cells in the biofilm, dye E (5×) and menadione sodium bisulphate (100 μM) were selected for redox detection chemistry, because they produced a high colorimetric yield in response to bacterial metabolism (340.4 ± 6.9 Omnilog Arbitrary Units). This combined approach, which avoids the limitations of traditional plate counts, was validated by testing the susceptibility of P. alcaliphila biofilm to 22 toxic compounds. For each compound the concentration level that significantly lowered the metabolic activity of the biofilm was identified. Chemical sensitivity analysis of the planktonic culture was also performed, allowing comparison of the metabolic susceptibility patterns of biofilm and planktonic cultures.     

Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates

Heterotrophic CaCO₃-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO₃ precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO₃ was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO₃ precipitation by bacteria.     

Osteomyelitis and the role of biofilms in chronic infection

Understanding the mechanisms implicated in the initial attachment, development, and maturation of a biofilm phenotype are of tremendous importance for their effect on the medical, industrial, and public health arenas. This review explores the current understanding of the nature of biofilms and the impact that molecular interactions between the bacteria themselves, as well as between bacteria and the host, may have on biofilm development and phenotype using the nonmotile Gram-positive coccus, Staphylococcus aureus, as an example.     

Microstructural characteristics of thin biofilms through optical and scanning electron microscopy

The combination of a conventional optical microscope with a specially designed glass flow cell was used to visualize in situ biofilms formed on opaque thin biomaterials through a simple non-invasive way (optical microscopy of thin biofilms, OMTB). Comparisons of OMTB with scanning electron microscopy (SEM) images were made. Thin metallic dental biomaterials were used as substrata. They were immersed in a synthetic saliva and in a modified Mitis–Salivarius medium inoculated with a consortium of oral microorganisms. To study the effect of bacterial motility, Pseudomonas fluorescens cultures were also used. The processes which give rise to the formation of the biofilm were monitored through OMTB. Biofilm microstructures like pores, water channels, streamers and chains of Streptococci, attached to the surface or floating in the viscous interfacial environment, could be distinguished. Thickness and roughness of the biofilms formed on thin substrata could also be evaluated. Distortions introduced by pretreatments carried out to prepare biological materials for SEM observations could be detected by comparing OMTB and SEM images. SEM images (obtained at high magnification but ex situ, not in real time and with pretreatment of the samples) and OMTB images (obtained in situ, without pretreatments, in real time but at low magnification) in combination provided complementary information to study biofilm processes on thin substrata.     

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPS_C putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPS_C in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPS_C led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (eps^T) in EPS_C containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of eps^T resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPS_C that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.     

Sulfolobus solfataticus P2嗜热嗜酸α-淀粉酶在大肠杆菌和毕赤酵母中的表达

通过PCR方法从Sulfolobus solfataticus P2中扩增到2．6kb的α-淀粉酶基因（SS01172）,将其分别克隆到表达载体pET32a（＋）和pPICZaA,并在E．coliRosetta和Pichia pastoris GS115中进行表达。结果表明α-淀粉酶基因在Rosetta中得到了高效表达,酶活为143．466U／mL;而在GS115中表达量稍低,发酵液酶活力为98．102U／mL。     

Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus

AIMS: The development of a rapid method for the selective detection and enumeration of the total and viable vegetative cell and spore content of thermophilic bacilli in milk powder by PCR. METHODS AND RESULTS: Quantitative PCR and microscopy indicate the presence of up to 2.9 log units more cells in milk powder than accounted for by plate counting due to the majority of cells being killed during milk processing. Two approaches for viable and dead cell differentiation of thermophilic bacilli by quantitative PCR were evaluated, these being the nucleic binding dye ethidium monoazide (EMA) and DNase I digestion. The former agent exposed to a viable culture of Anoxybacillus flavithermus caused considerable cell inactivation. In contrast, DNase I treatment had no effect on cell viability and was utilized to develop DNA extraction methods for the differential enumeration of total, viable vegetative cells and spores in milk powder. Moreover, the methods were further applied and evaluated to 41 factory powder samples taken throughout eight process runs to assess changes in numbers of vegetative cells and spores with time. DNase I treatment reduced vegetative cell numbers enumerated with PCR by up to 2.6 log units. The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log units more spores than determined by plate counting. CONCLUSIONS: The method presented in this study provides the ability to selectively enumerate the total and viable cell and spore content of reconstituted milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides a tool to monitor the extent of thermophilic contamination during milk powder manufacturing 60-90 min after sampling.     

Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.