Journal of Applied Microbiology

The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.

There has been a lot of controversy concerning the physiological significance of the complement system in human breast-milk. This is mainly due to the observation that human milk contains predominantly non-inflammatory and many anti-inflammatory factors, while simultaneously protecting the infant against a wide range of infectious and other diseases. The present study was carried out to assess the contribution of the complement system to the bactericidal activity of the human colostrum and early lactational milk. Using a serum-sensitive strain of Escherichia coli, different fractions of human breast-milk were assessed for their ability to kill the bacteria, with and without inactivation of their complement components, in comparison to another strain of the bacteria species. Deposition of activated C3 fragments on the killed bacteria, using an established ELISA technique, was demonstrated, further proving that the human milk complement could be activated in vitro. The bactericidal activities of human milk were almost completely abolished by complement heat inactivation at 56 degrees C or by the addition of EDTA.

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.

To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Yp/s) and productivities (Qp and qp) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 gp gS(-1), 0.104 +/- 0.012 gp l(-1) h(-1) and 0.03 gp gx(-1) h(-1), respectively when grown in a medium containing 20 gs l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0,3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2% , respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0 .1, temperature 30 +/- 0.5 degrees C, agitation 650 rev min(-1) and oxygen transfer rate 3.8 mmol O(2) l(-1) h(-1). Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/- 0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.

To purify and characterize an exo-acting chitinolytic enzyme produced from a Gram-negative bacterium Pseudomonas fluorescens JK-0412. A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK-0412. From the culture filtrates of this strain, a chito-oligosaccharides-degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS-PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK-0412 and shown to be an exo-type β-N-acetylglucosaminidase yielding GlcNAc as the final product from the natural chito-oligosaccharides, (GlcNAc)(n) , n = 2-5. As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK-0412 can be recognized as a potent producer for industry-level and cost-effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry-level production of GlcNAc. To the best of our knowledge, this is the first report on an exo-type chitinolytic enzyme of Pseudomonas species.

This work was aimed to isolate, purify and characterize an extracellular polysaccharide (EPS) produced by a freshwater dynamic sediment-attached micro-organism, Bacillus megaterium RB-05, and study its emulsifying potential in different hydrocarbon media. Bacillus megaterium RB-05 was found to produce EPSs in glucose mineral salts medium, and maximum yield (0.864 g l(-1) ) was achieved after 24-h incubation. The recovery rates of the polysaccharide material by ion-exchange and gel filtration chromatography were around 67 and 93%, respectively. As evident from HPLC and FT-IR analyses, the polysaccharide was found to be a heteropolymer-containing glucose, galactose, mannose, arabinose, fucose and N-acetyl glucosamine. Different oligosaccharide combinations namely hexose(3), hexose(4), hexose(5) deoxyhexose(1) and hexose(5) deoxyhexose(1) pentose(3) were obtained after partial hydrolysis of the polymer using MALDI-ToF-MS. The polysaccharide with an average molecular weight of 170 kDa and thermal stability up to 180°C showed pseudoplastic rheology and significant emulsifying activity in hydrocarbon media. Isolated polysaccharide was found to be of high molecular weight and thermally stable. The purified EPS fraction was composed of hexose, pentose and deoxyhexose sugar residues, which is a rare combination for bacterial polysaccharides. Emulsifying property was either better or comparable to that of other commercially available natural gums and polysaccharides. This is probably one of the few reports about characterizing an emulsifying EPS produced by a freshwater sediment-attached bacterium. The results of this study contribute to understand the influence of chemical composition and material properties of a new microbial polysaccharide on its application in industrial biotechnology. Furthermore, this work reconfirms freshwater dynamic sediment as a potential habitat for bioprospecting extracellular polymer-producing bacteria. This study will improve our knowledge on the exploitation of a nonconventional renewable resource, which also seems to be ecologically significant.

The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas-Phytophthora interaction as a biological control mechanism, and to understand the roles of these genes. Sixteen Ps. putida genes with increased expression on Phytophthora mycelial surfaces were identified using in vivo expression technology (IVET) screening. Sequence analysis of these Phytophthora mycelium-induced (pmi) genes revealed that many of them display similarity to genes known or predicted to be involved in carbohydrate catabolism, energy metabolism, amino acid/nucleotide metabolism, and membrane transport processes. Disruption of three pmi genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde-3-phosphate dehydrogenase showed significant phenotypic differences involved in the colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these pmi genes were induced by glycogen and other substances, such as organic acids and amino acids utilized by Ps. putida. The IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provide insight into the possible mechanisms of mycelial colonization by this bacterium. The IVET screening through a bacterial genome library might be a huge task. However, because the genes involved in direct interaction with Phytophthora and in bacterial adaptation can be identified, the IVET system will be a valuable tool in studying biocontrol bacteria at the molecular and ecological levels.

To demonstrate the biodegradation of dibenzofuran (DF) and its structural analogs by a newly isolated Agrobacterium sp. PH-08. To assess the biodegradation potential of newly isolated Agrobacterium sp. PH-08, various substrates were evaluated as sole carbon sources in growth and biotransformation experiments. ESI LC-MS/MS analysis revealed that the presence of angular degrading by-products as well as lateral-dioxygenation metabolites in the upper pathway. The metabolites in the lower pathway also were detected. In addition, the cometabolically degraded daughter compounds of DF-related compounds, such as biphenyl (BP) and dibenzothiophene (DBT) in dual substrate degradation were observed. Strain PH-08 exhibited the evidence of meta-cleavage pathway as confirmed by the activity and gene expression of catechol-2,3-dioxygenase. Newly isolated bacterial strain, Agrobacterium sp. PH-08 grew well with and degraded DF via both angular and lateral dioxygenation as demonstrated by metabolites identified through ESI LC-MS/ MS and GC-MS analyses. The other heterocyclic pollutants were also cometabolically degraded. Few reports have described the complete degradation of DF by a cometabolic lateral pathway. Our study demonstrates the novel results that the newly isolated strain utilized the DF as a sole carbon source and mineralized it via multiple dioxygenation. This article is protected by copyright. All rights reserved.

Aims: To assess the effects of inoculation of Lactobacillus buchneri on the ensiling properties and aerobic stability of maize silage. Methods and Results: Chopped whole crop maize was ensiled in 0·5 litre airtight polyethylene bottles (0·4 kg per bottle) and in double-layered, thin polyethylene bags (15 kg per bag), with or without inoculation of Lact. buchneri. The silos were stored for two to four months and the chemical composition, microbial numbers and aerobic stability were determined. Inoculation lowered lactic acid and yeasts, and increased acetic acid and pH value, resulting in improved aerobic stability of the silages. Inoculated silages produced 1,2-propanediol, the content of which increased as ensiling was prolonged, and nearly 50 g kg−1 dry matter had accumulated after four months of storage. The effects of inoculation, however, were much less pronounced in silages prepared in bags. Mannitol was found in all silages; the production was lowered by Lact. buchneri treatment and appeared to be unrelated to the accumulation of 1,2-propanediol. Conclusions: Inoculation of Lact. buchneri occasionally causes accumulation of 1,2-propanediol in silages without further degradation into propionic acid and 1-propanol. Significance and Impact of the Study: Substantial amounts of 1,2-propanediol could be consumed by ruminants when fed on silages inoculated with Lact. buchneri. In addition to increasing acetic acid, attention needs to be paid to 1,2-propanediol because the two fermentation products might affect the intake and utilization of silage-based diets.

To evaluate the potential for using a novel chemiluminescence-based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2-dioxetane [(4-methoxy-4(3-β-d-glucoside-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7·3·1·0(2,7) ]tridec-2,7-ene] specific for β-d-glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R(2) > 0·776). EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance AND IMPACT OF THE STUDY: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1-7 h) of faecal pollutions in different water types.

Biphenyl-degrading bacteria are able to metabolize dibenzofuran via lateral dioxygenation and meta-cleavage of the dihydroxylated dibenzofuran produced. This degradation was considered to be incomplete because accumulation of a yellow-orange ring-cleavage product was observed. In this study, we want to characterize the 1,2-dihydroxydibenzofuran cleaving enzyme which is involved in dibenzofuran degradation in the bacterium Ralstonia sp. SBUG 290. In this strain, complete degradation of dibenzofuran was observed after cultivation on biphenyl. The enzyme shows a wide substrate utilization spectrum, including 1,2-dihydroxydibenzofuran, 2,3-dihydroxybiphenyl, 1,2-dihydroxynaphthalene, 3- and 4-methylcatechol and catechol. MALDI-TOF analysis of the protein revealed a strong homology to the bphC gene products. We therefore cloned a 3.2 kb DNA fragment containing the bphC gene of Ralstonia sp. SBUG 290. The deduced amino acid sequence of bphC is identical to that of the corresponding gene in Pseudomonas sp. KKS102. The bphC gene was expressed in Escherichia coli and the meta-fission activity was detected using either 2,3-dihydroxybiphenyl or 1,2-dihydroxydibenzofuran as substrate. These results demonstrate that complete degradation of dibenzofuran by biphenyl degraders can occur after initial oxidation steps catalysed by gene products encoded by the bph-operon. The ring fission of 1,2-dihydroxydibenzofuran is catalysed by BphC. Differences found in the metabolism of the ring fission product of dibenzofuran among biphenyl degrading bacteria are assumed to be caused by different substrate specificities of BphD. This study shows for the first time that the gene products of the bph-operon are involved in the mineralization of dibenzofuran in biphenyl degrading bacteria.

Catechol 1,2-dioxygenase is a key enzyme in the degradation of monoaromatic pollutants. The detection of this gene is in focus today but recently designed degenerate primers are not always suitable. Rhodococcus species are important members of the bacterial community involved in the degradation of aromatic contaminants and their specific detection could help assess functions and activities in the contaminated environments. To reach this aim, specific PCR primer sets were designed for the detection of Rhodococcus related catechol 1,2-dioxygenase genes. Primers were tested with genetically well-characterized strains isolated in this study and community DNA samples were used as template for Rhodococcus specific PCR as well. The sequences of the catabolic gene in question were subjected to multiple alignment and a phylogenetic tree was created and compared to a 16S rRNA gene based Rhodococcus tree. A strong coherence was observed between the phylogenetic trees. The results strongly support the opinion that there was no recent lateral gene transfer among Rhodococcus species in the case of catechol 1,2-dioxygenase. In gasoline contaminated environments, aromatic hydrocarbon degrading Rhodococcus populations can be identified based upon the detection and sequence analysis of catechol 1,2-dioxygenase gene.

Aims: In this study, an attempt has been made to isolate and identify the bioactive compounds from hydroalcoholic extract of Terminalia chebula fruits effective against multidrug-resistant uropathogens and also to elucidate the influence of metal ions on the growth inhibitory activity of isolated compounds against the studied bacteria, if any. Methods and results: Bioassay-guided fractionation and extensive spectrometric analyses (FT-IR, (1) H NMR, (13) C NMR and ESI-MS) were used to isolate and characterize the bioactive compound. Growth inhibitory activities of isolated compound were studied by agar well diffusion and microbroth dilution assay methods. Checkerboard titration method was used for combination study between antibiotics and isolated compound. Influence of metal ions on growth inhibitory activity of this bioactive compound against the test isolates were also studied by INT [P-iodonitrotetrazolium violet; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride] colorimetric assay. The isolated bioactive compound 1, 2, 6-tri-O-galloyl-β-D-glucopyranose was found to be responsible for antibacterial activity against multidrug-resistant uropathogens and showed synergy with trimethoprim and gentamicin. This antibacterial activity of bioactive compound was counteracted by the supplementation of iron in the medium. Conclusion: Terminalia chebula fruit extract contains bioactive compound effective against multidrug-resistant uropathogens, and this antibacterial activity may be due to its iron-complexing property. Significance and impact of the study: To the best of our knowledge, the antibacterial activity exhibited by isolated gallotannin against multidrug-resistant uropathogens is first time reported by us. Besides, these promising findings may lead to the development of antimicrobial agents from T. chebula fruits for the treatment of urinary tract infections caused by these pathogens.

This study was conducted to understand influences of fermentation factors in NADH recycling and mechanisms of 1,3-propanediol (1,3-PDO) production in Lactobacillus panis PM1. We conducted metabolite analyses, qRT-PCR of the glycerol reductive pathway (glycerol dehydratase (DhaB) and 1,3-PDO dehydrogenase (DhaT)), and DhaT activity assays at different pH, temperature, and initial glycerol concentrations. The supplementation of 150 mmol·l(-1) glycerol caused a shift in NADH flux from ethanol to 1,3-PDO production; whereas 300 mol·l(-1) glycerol negatively affected the regeneration of NAD(+) via 1,3-PDO production. This retardation decreased transcription levels and specific activities of DhaT. The decreased DhaT activity eventually caused the shutdown of 1,3-PDO production. Temperature and pH did not significantly affect the specific activity of DhaT, whereas expression of genes for DhaB and DhaT was activated under acidic conditions. Moreover, fresh glucose addition after its depletion could not restart the glycerol reduction, but increased in ethanol production. Those environmental factors affect 1,3-PDO production in different ways through changing the expression level of enzymes and shifting the NAD(+) regeneration pathways. Our findings elucidated a key element to optimize 1,3-PDO production by L. panis PM1 which potentially improves 1,3-PDO manufacturing efficiencies. This article is protected by copyright. All rights reserved.

A microcosm-enrichment approach was used to investigate bacterial populations that may represent 1,3-dichloropropene (1,3-D)-degrading micro-organisms in compost-amended soil. After 8 weeks of incubation, with repeated application of 1,3-D, volatilization fluxes were much lower for compost-amended soil (CM) than with the unamended soils, indicating accelerated degradation due to addition of compost, or development of new microbial populations with enhanced degradation capacity. Denaturing gradient gel electrophoresis (DGGE) profiles of the PCR-amplified region of 16S rDNA genes were used to identify dominant bacterial populations in the fumigant-degrading soil. The DGGE results indicated that specific bacterial types had been enriched, and a more diverse fingerprint was observed in the community derived from the compost-amended soil compared with the unamended soil. Fragments from 16 different DGGE bands were cloned, sequenced and compared with published 16S rDNA sequences. Two clones, designated E1 and E4, were unique to all soils to which compost was added, and corresponded to strains of Pseudomonas and Actinomadura, respectively. The results show that the addition of compost to soil increases specific microbial populations and results in the accelerated degradation of fumigants. Application of compost manure to soil can help degrade soil fumigants at a faster rate.

Saccharomyces cerevisiae is a safe micro-organism used in fermentation industry. 1,3-Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensively chemical synthesis. The aim of this study is to engineer a S. cerevisiae strain that can produce 1,3-propanediol at low cost. By using D-glucose as a feedstock, S. cerevisiae could produce glycerol, but not 1,3-propanediol. In this study, we have cloned two genes yqhD and dhaB required for the production of 1,3-propanediol from glycerol, and integrated them into the chromosome of S. cerevisiae W303-1A by Agrobacterium tumefaciens-mediated transformation. Both genes yqhD and dhaB functioned in the engineered S. cerevisiae and led to the production of 1,3-propanediol from D-glucose. Saccharomyces cerevisiae can be engineered to produce 1,3-propanediol from low-cost feedstock D-glucose. To our knowledge, this is the first report on developing S. cerevisiae to produce 1,3-propanediol by using A. tumefaciens-mediated transformation. This study might lead to a safe and cost-efficient method for industrial production of 1,3-propanediol.

Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol.

To elucidate the roles of the β-1,3-endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. A β-1,3-endoglucanase was purified from carbon-starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene-expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. The β-1,3-endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall-degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.

Aims: To improve the β-1,3-1,4-glucanase production by Rhizomucor miehei under solid-state fermentation (SSF) for industrial application. Methods and results: The fermentation conditions for β-1,3-1,4-glucanase production by R. miehei CAU432 under SSF were optimized using a 'one-factor-at-a-time' method. Under the optimized fermentation conditions, viz. oatmeal (0·45-0·9 mm) as sole carbon source, 5% (w/w) peptone as sole nitrogen source, initial moisture of 80% (w/w), initial culture pH of 5·0, incubation temperature of 50°C and incubation time of 6 days, the highest β-1,3-1,4-glucanase activity of 20,025 U g(-1) dry substrate was achieved, which represents the highest yield for β-1,3-1,4-glucanase production ever reported. The crude enzyme was extracted and purified to homogeneity with a purification fold of 4·6 and a recovery yield of 9·0%. The addition of the purified β-1,3-1,4-glucanase in mash obviously reduced its filtration time (24·6%) and viscosity (2·61%). Conclusions: The optimal fermentation conditions for maximal β-1,3-1,4-glucanase production under SSF was obtained, and the enzyme was suitable for application in the malting process. Significance and impact of the study: The high production yield and excellent capability of the enzyme may enable it great potential in industries, especially in brewing industry.

The feasibility of the continuous production of a valuable bioplastic raw material, namely 1,3-propanediol (1,3-PDO) from biodiesel by-product glycerol, using immobilized cells was investigated. In addition, the effect of hydraulic retention time (HRT) was also analysed. Ceramic balls and ceramic rings were used for the immobilization of a locally isolated strain; Klebsiella pneumoniae (GenBank no. 27F HM063413). HRT of 1 h is the best one in terms of volumetric production rate (g 1,3-PDO l(-1) h(-1)). The results indicated that ceramic-based cell immobilization achieved a 2-fold higher production rate (10 g 1,3-PDO l(-1) h(-1)) in comparison with suspended cell system (4·9 g 1,3-PDO l(-1) h(-1)). Continuous cultures with immobilized cells revealed that 1,3-PDO production was more effective and more stable than suspended culture systems. Furthermore, cell immobilization had also obvious benefits especially for resistance of the production for extreme conditions (high organic loading rates, cell washouts). The results were important for understanding the significance of continuous immobilization process among other well-known 1,3-PDO fermentation processes. This work is a promising process for further studies, as the immobilized micro-organism was able to reach high volumetric production rates at short HRT, it has an important role in tolerating and converting glycerol during fermentation. Therefore, HRT is a very significant operational parameter (P value <0·05) directly affecting the bioreactor performance and production rate.

To clone and characterize the gene coding for BGN16.3, a beta-1,6-glucanase putatively implicated in mycoparasitism by Trichoderma harzianum, a biocontrol agent used against plant pathogenic fungi. Using degenerate primed PCR and cDNA library screening, we have cloned the cDNA coding BGN16.3. bgn16.3 showed a significant sequence identity (50%) to bgn16.1; however, they both have low identity to the previously cloned bgn16.2, allowing the identification of amino acid sequences putatively involved in the common catalytic activity of the three proteins. bgn16.3 is a single-copy gene and highly homologous sequences are present in all tested Trichoderma species. bgn16.3 expression pattern is analysed by Northern blot, finding that it is expressed during the interaction of T. harzianum CECT 2413 with Botrytis cinerea, supporting the implication of the enzyme in the mycoparasitic process. The cloned bgn16.3 completes the knowledge on the beta-1,6-glucanase isozyme system from T. harzianum CECT 2413. A highly homologous gene is present in all analysed Trichoderma strains. bgn16.3 is expressed under few specific conditions, including the mycoparasitic process. This study contributes to the knowledge of beta-1,6-glucanases. It implicates this group of enzymes in the mycoparasitism by some biocontrol agents such as T. harzianum.

Optimal production conditions of conjugated gamma-linolenic acid (CGLA) from gamma-linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0.03% (w/v) alpha-linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml(-1)gamma-linolenic acid as a substrate in 5 -ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml(-1) dry cells] as the catalysts produced 8.8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis-6,cis-9,trans-11-octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis-6,trans-9,trans-11-octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. The practical process of CGLA production from gamma-linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm of Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7.5 and 37 degrees C, respectively, and the Km value was 0.17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l-1 of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l-1 of CaCl2. The activity was not affected by treatment with the protein denaturants, 0.01 mol l-1 of SDS or 1 mmol l-1 of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.

The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.

Recent analyses of ribosomal RNA sequence diversity have demonstrated the extent of bacterial diversity in the human colon, and have provided new tools for monitoring changes in the composition of the gut microbial community. There is now an excellent opportunity to correlate ecological niches and metabolic activities with particular phylogenetic groups among the microbiota of the human gut. Bacteria that associate closely with particulate material and surfaces in the gut include specialized primary degraders of insoluble substrates, including resistant starch, plant structural polysaccharides and mucin. Butyrate-producing bacteria found in human faeces belong mainly to the clostridial clusters IV and XIVa. In vitro and in vivo evidence indicates that a group related to Roseburia and Eubacterium rectale plays a major role in mediating the butyrogenic effect of fermentable dietary carbohydrates. Additional cluster XIVa species can convert lactate to butyrate, while some members of the clostridial cluster IX convert lactate to propionate. The metabolic outputs of the gut microbial community depend not only on available substrate, but also on the gut environment, with pH playing a major role. Better understanding of the colonic microbial ecosystem will help to explain and predict the effects of dietary additives, including nondigestible carbohydrates, probiotics and prebiotics.

To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 10(10)R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

To develop descriptive models for the combined effect of temperature (10-40 degrees C) and water activity (0.850-0.980) on the growth of two ochratoxin A producing strains of Aspergillus carbonarius from Greek wine grapes on a synthetic grape juice medium. Fungal growth was measured as changes in colony diameter on a daily basis. The maximum specific colony growth rates (mu(max)) were determined by fitting the primary model of Baranyi describing the change in colony diameter (mm) with respect to time (days). Secondary models, relating mu(max) with temperature and a(w) were developed and comparatively evaluated based on polynomial, Parra, Miles, Davey and Rosso equations. No growth was observed at 0.850 a(w) (water activity) regardless of temperature, as well as at marginal temperature levels assayed (10 and 40 degrees C) regardless of water activity. The data set was fitted successfully in all models as indicated by the values of regression coefficients and root mean square error. Models with biological interpretable parameters were highly rated compared with the polynomial model, providing realistic cardinal values for temperature and a(w). The optimum values for growth were found in the range 0.960-0.970 a(w) and 34-35 degrees C respectively for both strains. The developed models were validated on independently derived data from the literature and presented reasonably good predictions as inferred by graphical plots and statistical indices (bias and accuracy factors). The effect of temperature and a(w) on the growth of A. carbonarius strains could be satisfactorily predicted under the current experimental conditions, and the proposed models could serve as a tool for this purpose. The results could be successfully employed as an empirical approach in the development and prediction of risk models of contamination of grapes and grape products by A. carbonarius.

To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin. Carbon and nitrogen sources and growth factors, such as amino acids and vitamins, were screened initially in a mineral medium for the biomass and antagonistic compound of Pseudomonas MCCB 103. The selected ingredients were further optimized using a full-factorial central composite design of the response surface methodology. The medium optimized as per the model for biomass contained mannitol (20 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5 g l(-1)), urea (3.3 g l(-1)) and mineral salts solution (20 ml l(-1)), and the one optimized for the antagonistic compound contained mannitol (2 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5.1 g l(-1)), urea (3.6 g l(-1)) and mineral salts solution (20 ml l(-1)). Subsequently, the model was validated experimentally with a biomass increase by 19% and fivefold increase of the antagonistic compound. Significant increase in the biomass and antagonistic compound production could be obtained in the new media. Media formulation and optimization are the primary steps involved in bioprocess technology, an attempt not made so far in the production of aquaculture probiotics.

To develop a suite of group-specific, rRNA-targeted oligonucleotide scissor probes for the quantitative detection of the predominant bacterial groups within the ruminal microbial community with the rRNA cleavage reaction-mediated microbial quantification method. Oligonucleotides that complement the conserved sites of the 16S rRNA of phylogenetically defined groups of bacteria that significantly contribute to the anaerobic fermentation of carbohydrates in ruminal ecosystems were selected from among published probes or were newly designed. For each probe, target-specific rRNA cleavage was achieved by optimizing the formamide concentration in the reaction mixture. The set of scissor probes was then used to analyse the bacterial community in the rumen fluids of four healthy dairy cows. In the rumen fluid samples, the genera Bacteroides/Prevotella and Fibrobacter and the Clostridium coccoides-Eubacterium rectale group were detected in abundance, accounting for 44-48%, 2.9-10%, and 9.1-10% of the total 16S rRNA, respectively. The coverage with the probe set was 71-78% of the total bacterial 16S rRNA. The probe set coupled with the sequence-specific small-subunit rRNA cleavage method can be used to analyse the structure of a ruminal bacterial community. The probe set developed in this study provides a tool for comprehensive rRNA-based monitoring of the community members that dominate ruminal ecosystems. As the ruminal microbial community can be perturbed, it is important to track its dynamics by analysing microbiological profiles under specific conditions. The method described here will provide a convenient approach for such tracking.

To use ELISA and immunoblotting assays to examine the serum antibody response of cattle infected with Salmonella Typhimurium DT104 and following vaccination with Bovivac S. Three hundred and twenty-nine cattle, including 16 shedding multiresistant Salmonella Typhimurium DT104, were screened for serum antibodies binding to O=1, 4, 5, 12 lipopolysaccharide (LPS) antigens before and after vaccination with Bovivac S. Sera with an ELISA reading of 0.9A405 or above were shown to contain antibodies, of the IgG-class only, to the LPS of Salmonella Typhimurium using immunoblotting. Prior to vaccination, only 11 cattle had serum IgG-class antibodies to the O=4, 5 LPS antigens, and of these one also had antibodies to outer membrane proteins and H=i flagellar antigens. Following vaccination, 87 out of 315 cattle developed serum antibodies to the LPS of Salmonella Typhimurium. Evidence of infection of cattle with Salmonella Typhimurium was readily obtained with an LPS-based ELISA in association with an immunoblotting procedure, supplementing existing bacteriological procedures. This enabled the detection of an increase in the number of cattle with serum antibodies to Salmonella Typhimurium LPS following vaccination with Bovivac S. The immunoassays described provided evidence of infection with Salmonella Typhimurium and served as a valuable adjunct to established bacteriology.

Most studies involving prebiotic oligosaccharides have been carried out using inulin and its fructo-oligosaccharide (FOS) derivatives, together with various forms of galacto-oligosaccharides (GOS). Although many intestinal bacteria are able to grow on these carbohydrates, most investigations have demonstrated that the growth of bifidobacteria, and to a lesser degree lactobacilli, is particularly favoured. Because of their safety, stability, organoleptic properties, resistance to digestion in the upper bowel and fermentability in the colon, as well as their abilities to promote the growth of beneficial bacteria in the gut, these prebiotics are being increasingly incorporated into the Western diet. Inulin-derived oligosaccharides and GOS are mildly laxative, but can result in flatulence and osmotic diarrhoea if taken in large amounts. However, their effects on large bowel habit are relatively minor. Although the literature dealing with the health significance of prebiotics is not as extensive as that concerning probiotics, considerable evidence has accrued showing that consumption of GOS and FOS can have significant health benefits, particularly in relation to their putative anti-cancer properties, influence on mineral absorption, lipid metabolism, and anti-inflammatory and other immune effects such as atopic disease. In many instances, prebiotics seem to be more effective when used as part of a synbiotic combination.

To understand the diversity, taxonomy and antagonistic potential of rice-associated bacteria, and to discover new bacteria for biocontrol of rice foliar pathogens. Amplified ribosomal DNA restriction analysis (ARDRA), BOX-PCR and 16S rRNA gene sequence analysis were used to identify the diversity of 203 rice-associated antagonistic bacteria. Eleven potential biocontrol bacteria were used to test their biological control of rice blast in a natural field experiment. Eleven different genera were encountered in five divisions, including Bacilli, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deinococci. The most prominent genus in all microenvironments was Bacillus (68 x 5%). The efficacy of rice leaf blast biocontrol was 64 x 35% for strain 1Pe2, 57 x 86% for strain 2R37 and 56 x 44% for strain 1Re14. Biocontrol data from the field experiments demonstrated no positive correlation between antagonism, physiological characteristics and biocontrol efficacy. There was significant diversity among the rice-associated bacteria isolated from different microenvironments. The most prominent genus of all microenvironments was Bacillus. Brevibacillus brevis strain 1Pe2 and Deinococcus aquaticus strain 1Re14 have good potential for field application and commercial use. This is the first attempt to study the diversity and identification of rice-associated antagonistic bacteria from different microenvironments, and endophytic bacteria Deinococcus aquaticus strain 1Re14, Acidovorax sp. isolate 3Re21 and Brevibacillus brevis strain 1Pe2 are first reported as rice-associated bacteria.

Salmonella Typhimurium DT 104 is generally assumed to be spread by contact between live animals, e.g. by trading. The aim of the present study was to assess the importance of other routes of transmission in the dissemination of this bacterium. An outbreak among 14 cattle and pig herds located in a geographically narrow area in Denmark was investigated. Epidemiological information and disease history of the herds was obtained through interviews. Based on this, the hypothesis for horizontal spread was proposed, and these were confirmed by comparison of the pulsed field gel electrophoresis (PFGE) and the plasmid profiles of isolates obtained by continuous sampling over a period of almost 3 years. The study indicated that other routes might play an important role, than the trading of live animals, in the spread of S. Typhimurium DT 104 among livestock. Salmonella Typhimurium DT 104 infected herd might pose a significant risk to herds located within the same geographic area. In advising on how to avoid the spread of this bacterium, factors like person contacts, sharing of equipment and contaminated slurry should be focussed on in addition to infected animals.

The aim of this work was to observe bacteria associated with the spores of Gigaspora margarita, an arbuscular mycorrhizal fungus (AMF). First, a direct analysis of DNA from sterilized spores indicated the bacteria belonging to the genus Janthinobacterium. In the second assay, two bacterial strains were isolated by osmosis from protoplasts, which were derived from spores by using two particular enzymes: lysing enzymes and yatalase. After isolation, cultivation and identification by their DNA as performed in the first experiment, the species with the closest relation were Janthinobacterium lividum (KCIGM01) and Paenibacillus polymyxa (KCIGM04) isolated with lysing enzymes and yatalase respectively. Morphologically, J. lividum was Gram negative and oval, while P. polymyxa was also oval, but Gram positive. Both strains had antagonistic effects to the pathogenic fungi Rosellimia necatrix, Pythium ultimum, Fusarium oxysporum and Rhizoctonia solani. In particular, J. lividum was much stronger in this role. However, in phosphorus (P) solubilization P. polymyxa functioned better than J. lividum. This experiment had revealed two new bacteria species (P. polymyxa and J. lividum), associated with AMF spores, which functioned to suppress diseases and to solubilize P. AMF spores could be a useful source for bacterial antagonists to soil-borne diseases and P solubilization.

The phyllosphere represents the habitat provided by the aboveground parts of plants, and on a global scale supports a large and complex microbial community. Microbial interactions in the phyllosphere can affect the fitness of plants in natural communities, the productivity of agricultural crops, and the safety of horticultural produce for human consumption. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonists, which is influenced by numerous environmental factors in addition to leaf physico-chemical properties. The recent use of culture-independent techniques has demonstrated considerable previously unrecognized diversity in phyllosphere bacterial communities. Furthermore, there is significant recent evidence that plant genotype can play a major role in determining the structure of phyllosphere microbial communities. The main aims of this review are: (i) to discuss the diversity of phyllosphere microbial populations; (ii) to consider the processes by which microbes colonize the phyllosphere; (iii) to address the leaf characteristics and environmental factors that determine the survival and growth of colonists; (iv) to discuss microbial adaptations that allow establishment in the phyllosphere habitat and (v) to evaluate evidence for plant genotypic control of phyllosphere communities. Finally, we suggest approaches and priority areas for future research on phyllosphere microbiology.

To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Bio-process development for isomer selective and efficient production of cis-9,trans-11-octadecadienoic acid (CLA) from trans-vaccenic acid (t-VA, trans-11-octadecenoic acid) through microbial fatty acid Delta9-desaturation reaction. A total of 550 strains of fungi and yeasts were screened for CLA production from t-VA through Delta9 desaturation. Delacroixia coronata IFO 8586 was selected as a potent producer of CLA from t-VA. Efficient CLA production was observed during cultivation in medium supplemented with the methyl ester of t-VA (t-VAME). Under the optimal conditions with 33.3 mg ml(-1) of t-VAME as substrate, 10.5 mg ml(-1) CLA was produced by D. coronata IFO 8586 after 7 days of cultivation in the medium containing dextrin (5.0%), tryptone (2.0%) and thiourea (0.83 micromol ml(-1)). The strain produced the cis-9,trans-11 isomer of CLA selectively (98% of total CLA), with a small amount of the trans-9,trans-11 isomer (2% of total CLA), mainly in the form of triacylglycerols (69% of total CLA). A practical bio-process for selective production of cis-9,trans-11 isomer of CLA using filamentous fungus D. coronata IFO 8586 was successfully established. Isomer selective bio-process for the practical production of cis-9,trans-11-CLA was first established. The process is benefitable for expanding the application of CLA for medicinal and nutraceutical purposes.

To isolate and characterize an oxalate-degrading Pandoraea sp. OXJ-11. A new bacterium Pandoraea sp. OXJ-11 was isolated from soil samples, which can grow in the medium with oxalate as the sole carbon and energy source. The isolate OXJ-11 is Gram-negative straight rod. It occurs singly and is motile by means of a double polar flagellum. Catalase is positive and nitrate is not reduced. It grows aerobically and the optimum growth temperature and the optimum pH are at 30 degrees C and pH 6.0, respectively. The polyphasic taxonomic data along with 16S rRNA sequence comparison demonstrate that the isolate OXJ-11 should belong to the genus Pandoraea and represent a new member in this family. Oxalate could be degraded and the oxalate-degrading enzyme activity was detected when the isolate OXJ-11 grew in the medium with oxalate as carbon source. Oxalate-degrading Pandoraea sp. OXJ-11 would be beneficial to the potential application in the control of sclerotinia stem rot in economically important plants caused by fungus Sclerotinia sclerotiorum, and in making plants resistant to the white mold disease by oxalate-degrading enzyme transgene.

Lanzhou reach of the Yellow River is contaminated by cadmium (Cd(II)).The aim of this study was to screen bacterial strains that is able to resist and absorb cadmium from soil sediment and elucidate the molecular mechanism. A strain named LZ-11 which can resist 1 mmol l(-1) and absorb 0.3 mmol l(-1) cadmium was isolated from a petrochemical wastewater discharge site. 16S rRNA gene sequencing data and Vitek phenotype results revealed that it was closely related to Enterococcus faecalis. Transmission Electron Microscopy images and Energy Dispersive X-Ray Analysis results showed that Cd(II) was absorbed both intracellularly and extracellularly. Blast results showed that Enterococcus faecalis genome owns cadA, ppx and dsbA which are proven to be involved in Cd(II) resistance and absorption. Quantitative real-time PCR data demonstrated that thesethree genes were upregulated 2-3 folds in LZ-11 under Cd(II) treatment. We've isolated a strain named LZ-11 from Lanzhou reach of the Yellow River which can resist and absorb Cd(II). LZ-11 was closely related to Enterococcus faecalis. Genes encoding CadA, Ppx and DsbA were up-regulated under Cd(II) treatment. These genes might confer Cd(II) resistance and absorption in Enterococcus faecalis strain LZ-11. Lanzhou reach of the Yellow River is contaminated by heavy metals. Microbial research and remediation is still scarce. LZ-11 is the first strain that is able to resist and absorb Cd(II) isolated from this area and might be a good candidate for future cadmium bioremediation. This article is protected by copyright. All rights reserved.

The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation. The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor. The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour. The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%). The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose. Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%). Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides. The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides.

Aims: The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition. Methods and results: A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO-075, BMO-111 and BMO-147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36-h-old culture of BMO-111 (1 × 10(8) colony-forming units ml(-1) ) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO-111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO-111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses. Conclusions: It could be concluded that the biocontrol agent O. anthropi BMO-111 was effective against blister blight disease of tea. Significance and impact of the study: Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.

The present study was aimed to evaluate the integration of Ochrobactrum anthropi BMO-111 and chemical fungicides (copper oxychloride and hexaconazole) against blister blight disease of tea. Application of the liquid culture of O. anthropi BMO-111 (36-h-old culture broth) was found to be effective in combined sprays with individual chemical fungicides (copper oxychloride and hexaconazole). Spray application of O. anthropi BMO-111 to tea bushes improved the biochemical parameters such as the levels of chlorophyll, polyphenols, and catechins in the harvestable tea shoots. Moreover, in the microplot and large scale trials, the integrated treatment of every two O. anthropi BMO-111 sprays followed by a single fungicides spray was found to be more efficient than the stand alone O. anthropi BMO-111 or chemicals sprays. Further, pathogenicity study employing Swiss albino mice showed no mortality in the test animals when challenged with O. anthropi BMO-111 through oral, intravenous and intranasal routes. The field trials clearly established that O. anthropi BMO-111 has capability to reduce incidence in integrated management of blister blight disease of tea and safe to use in the field. The results indicate that O. anthropi BMO-111 can be used as an agricultural input in the integrated crop protection systems.

The aim of this work is to study the expression of stress genes and those involved in pediocin and nisin production in Pediococcus acidilactici UL5 and Lactococcus lactis ATCC11454 under simulated gastrointestinal (GI) physiological conditions. The two strains were fed to a dynamic GI model (TIM-1). Samples were taken from different compartments and analysed for strain survival as well as for the expression of pediocin PA-1 operon, nisin A production gene and stress genes using RT-qPCR. Ileal-delivered efflux showed a survival rate of 17 and 0·0007% for Ped. acidilactici and La. lactis, respectively. Pediocin operon genes from stressed cells were generally expressed at least at the same level as for unstressed cells. However, pedA is up-regulated in the effluent at 120 and 180 min. Nisin A genes were always up-regulated with particularly in the stomach after 70 min compared with control. Bacteriocin production of Ped. acidilactici UL5 and Lc. lactis ATCC 11454 are not affected by upper GI simulated conditions and thus could be considered as relevant probiotic candidates. This study demonstrates the capacity of lactic acid bacteria to survive and express their bacteriocins genes under simulated GI conditions.

To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.

The movement of populations shapes the patterns and distribution of infectious diseases globally. The consequences of travel are seen in the traveller and in places and populations visited and may persist long after travel. The traveller can be seen as an interactive biological unit who picks up, processes, carries and drops off microbial genetic material. A traveller can introduce potential pathogens in the absence of signs or symptoms of illness. Travellers can serve as a sentinel population; study of them can provide insights into the presence and level of risk of transmission of infections in other geographical regions. Travellers can also be seen as couriers who inadvertently ferry pathogens and microbial genetic material to regions where researchers can carry out detailed analyses that can help to map the location and movement of strains, genotypes and resistance patterns. The laboratory plays a key role in the identification and characterization of pathogens, which can inform management of individual patients and the public health response. The connectedness and mobility in the world today facilitate the emergence of infectious diseases in humans and also in animals and plants. Many traditional barriers have been breached by travel, roads and technology. Population size and density favour spread of many infections. The rapid generation time of microbes and their capacity to adapt to changes in the physico-chemical and immunological environment will pose continuing challenges.

To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis' acid in the medium influences its biodegradability. From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l(-1) of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0.1 mmol l(-1) AlCl(3) added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al(3+) : TMA complex. Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl(3). The use of Lewis' acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.

Ceragenin CSA-13 is a synthetic mimic of cationic antibacterial peptides, with facial amphiphilic morphology reproduced using a cholic acid scaffold. Previous data have shown that this molecule displays broad-spectrum antibacterial activity, which decreases in the presence of blood plasma. However, at higher concentrations, CSA-13 can cause lysis of erythrocytes. This study was designed to assess in vitro antibacterial and haemolytic activity of CSA-13 in the presence of pluronic F-127. CSA-13 bactericidal activity against clinical strains of bacteria associated with topical infections and in an experimental setting relevant to their pathophysiological environment, such as various epithelial tissue fluids and the airway sputum of patients suffering from cystic fibrosis (CF), was evaluated using minimum inhibitory and minimum bactericidal concentration (MIC/MBC) measurements and bacterial killing assays. We found that in the presence of pluronic F-127, CSA-13 antibacterial activity was only slightly decreased, but CSA-13 haemolytic activity was significantly inhibited. CSA-13 exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, including methicillin-resistant strains, Pseudomonas aeruginosa present in CF sputa, and biofilms formed by different Gram (+) and Gram (-) bacteria. CSA-13 bactericidal action is partially compromised in the presence of plasma, but is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. The synergistic action of CSA-13, determined by the use of a standard checkerboard assay, reveals an increase in CSA-13 antibacterial activity in the presence of host defence molecules such as the cathelicidin LL-37 peptide, lysozyme, lactoferrin and secretory phospholipase A (sPLA). These results suggest that CSA-13 may be useful to prevent and treat topical infection. Combined application of CSA-13 with pluronic F-127 may be beneficial by reducing CSA-13 toxicity.

CSA-13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F-127 could interfere with the toxicity of CSA-13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa. The addition of pluronic acid F-127 slightly decreased the number of dead cells after exposure to CSA-13. Pluronic acid F-127 blocked the permeabilizing effect of CSA-13 on the plasma membrane of HUVEC (uptake of ethidium bromide, release of lactate dehydrogenase) without modifying its toxic effect on their mitochondrial function (MTT test, uptake of tetramethyl rhodamine ethyl ester). Pluronic acid F-127 decreased the toxicity of CSA-13 against eukaryotic cells without completely protecting them from mitochondrial damage at high concentrations of the drug. This work establishes that studies on the toxic effects of synthetic antimicrobials on eukaryotic cells should not only focus on the permeability of the plasma membrane but also on the integrity of the mitochondria.

To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.