PYG Broth Research Articles

Metal-Pseudomonas veronii 2E Interactions as Strategies for Innovative Process Developments in Environmental Biotechnology.

The increase of industrial discharges is the first cause of the contamination of water bodies. The bacterial survival strategies contribute to the equilibrium restoration of ecosystems being useful tools for the development of innovative environmental biotechnologies. The aim of this work was to study the Cu(II) and Cd(II) biosensing, removal and recovery, mediated by whole cells, exopolymeric substances (EPS) and biosurfactants of the indigenous and non-pathogenic Pseudomonas veronii 2E to be applied in the development of wastewater biotreatments. An electrochemical biosensor was developed using P. veronii 2E biosorption mechanism mediated by the cell surface associated to bound exopolymeric substances. A Carbon Paste Electrode modified with P. veronii 2E (CPEM) was built using mineral oil, pre-washed graphite power and 24 h-dried cells. For Cd(II) quantification the CPEM was immersed in Cd(II) (1–25 μM), detected by Square Wave Voltammetry. A similar procedure was used for 1–50 μM Cu(II). Regarding Cd(II), removal mediated by immobilized EPS was tested in a 50 ml bioreactor with 0.13 mM Cd(II), pH 7.5. A 54% metal retention by EPS was achieved after 7 h of continuous operation, while a 40% was removed by a control resin. In addition, surfactants produced by P. veronii 2E were studied for recovery of Cd(II) adsorbed on diatomite, obtaining a 36% desorption efficiency at pH 6.5. Cu(II) adsorption from a 1 mM solution was tested using P. veronii 2E purified soluble EPS in 50 mL- batch reactors (pH = 5.5, 32°C). An 80% of the initial Cu(II) was retained using 1.04 g immobilized EPS. Focusing on metal recovery, Cu nanoparticles (NPs) biosynthesis by P. veronii 2E was carried out in Cu(II)-PYG Broth at 25°C for 5 days. Extracellular CuNPs were characterized by UV-Vis spectral analysis while both extracellular and intracellular NPs were analyzed by SEM and TEM techniques. Responses of P. veronii 2E and its products as biosurfactants, bound and soluble EPS allowed Cu(II) and Cd(II) removal, recovery and biosensing resulting in a multiple and versatile tool for sustainable wastewater biotreatments.

Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O’Toole 1935) Prévot 1938

The recent proposal by Lawson and Rainey (2015) to restrict the genus Clostridium to Clostridium butyricum and related species has ramifications for the members of the genera that fall outside this clade that should not be considered as Clostridium sensu stricto. One such organism of profound medical importance is C. difficile that is a major cause of hospital-acquired diarrhea and mortality in individuals. Based on 16S rRNA gene sequence analysis, the closest relative of Clostridium difficile is Clostridium mangenotii with a 94.7% similarity value and both are located within the family Peptostreptococcaceae that is phylogenetically far removed from C. butyricum and other members of Clostridium sensu stricto. Clostridium difficile and Clostridium mangenotii each produce abundant H2 gas when grown in PYG broth and also produce a range of straight and branched chain saturated and unsaturated fatty acids with C16:0 as a major product. The cell wall peptidoglycan contains meso-DAP as the diagnostic diamino acid. Based on phenotypic, chemotaxonomic and phylogenetic analyses, novel genus Clostridioides gen. nov. is proposed for Clostridium difficile as Clostridioides difficile gen. nov. comb. nov. and that Clostridium mangenotii be transferred to this genus as Clostridioides mangenotii comb. nov. The type species of Clostridioides is Clostridioides difficile.

Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces.

An obligately anaerobic, rod-shaped (0.5-1.0 × 2.0-10.0 μm), Gram-stain-positive bacterium, occurring mainly singly or in pairs, and designated BLPYG-8T, was isolated from faeces of a healthy human volunteer aged 56 years. Cells were non-motile. Oval, terminal spores were formed that swell the cells. The strain was affiliated with the genus Clostridium sensu stricto (Clostridium rRNA cluster I) as revealed by 16S rRNA gene sequence analysis. Strain BLPYG-8T showed 97.3 to 97.4 % 16S rRNA gene sequence similarity with Clostridium sulfidigenes DSM 18982T, Clostridium subterminale DSM 6970T and Clostridium thiosulfatireducens DSM 13105T. DNA-DNA hybridization and phenotypic analysis showed that the strain was distinct from its closest relatives, C. sulfidigenes DSM 18982T, C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T with 54.2, 53.9 and 53.3 % DNA-DNA relatedness, respectively. Strain BLPYG-8T grew in PYG broth at temperatures between 20 and 40 °C (optimum 37 °C). The strain utilized a range of amino acids as well as carbohydrates as a source of carbon and energy. Glucose fermentation resulted in the formation of volatile fatty acids mainly acetic acid, n-butyric acid and organic acids such as succinic and lactic acid. The DNA G+C content of strain BLPYG-8T was 44.1 mol%. The major fatty acids (>10 %) were C14 : 0, iso-C15 : 0, C16 : 1ω7c and C16 : 0. Phylogenetic analysis and specific phenotypic characteristics and/or DNA G+C content differentiated the strain from its closest relatives. On the basis of these data, strain BLPYG-8T represents a novel species of the genus Clostridium, for which the name Clostridium punense sp. nov. is proposed. The type strain is BLPYG-8T ( = DSM 28650T = CCUG 64195T = MCC 2737T).

Megasphaera indica sp. nov., an obligate anaerobic bacteria isolated from human faeces

Two coccoid, non-motile, obligately anaerobic, Gram-stain-negative bacteria, occurring singly or in pairs, or as short chains, with a mean size of 1.4-2.5 µm were isolated from the faeces of two healthy human volunteers, aged 26 and 56 years, and were designated NMBHI-10(T) and BLPYG-7, respectively. Both the strains were affiliated to the sub-branch Sporomusa of the class Clostridia as revealed by 16S rRNA gene sequence analysis. The isolates NMBHI-10(T) and BLPYG-7 showed 99.1 and 99.2% 16S rRNA gene sequence similarity, respectively, with Megasphaera elsdenii JCM 1772(T). DNA-DNA hybridization and phenotypic analysis showed that both the strains were distinct from their closest relative, M. elsdenii JCM 1772(T) (42 and 53% DNA-DNA relatedness with NMBHI-10(T) and BLPYG-7, respectively), but belong to the same species (DNA-DNA relatedness of 80.9 % between the isolates). According to DNA-DNA hybridization results, the coccoid strains belong to the same genospecies, and neither is related to any of the recognized species of the genus Megasphaera. Strains NMBHI-10(T) and BLPYG-7 grew in PYG broth at temperatures of between 15 and 40 °C (optimum 37 °C), but not at 45 °C. The strains utilized a range of carbohydrates as sources of carbon and energy including glucose, lactose, cellobiose, rhamnose, galactose and sucrose. Glucose fermentation resulted in the formation of volatile fatty acids, mainly caproic acid and organic acids such as succinic acid. Phylogenetic analysis, specific phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The DNA G+C contents of strains NMBHI-10(T) and BLPYG-7 are 57.7 and 54.9 mol%, respectively. The major fatty acids were 12 : 0 FAME and 17 : 0 CYC FAME. On the basis of these data, we conclude that strains NMBHI-10(T) and BLPYG-7 should be classified as representing a novel species of the genus Megasphaera, for which the name Megsphaera indica sp. nov. is proposed. The type strain is NMBHI-10(T) ( = DSM 25563(T) = MCC 2481(T)).

Parabacteroides chartae sp. nov., an obligately anaerobic species from wastewater of a paper mill

A bacterial strain, designated NS31-3(T), was isolated from the wastewater of a paper mill. Cells of the isolate were obligately anaerobic, non-pigmented, non-motile, Gram-negative, short rods (0.7-1.0 × 1.4-2.5 µm). The isolate was able to grow on media containing 20% bile salts. API 20A tests showed that acid was produced from glucose, lactose, sucrose, maltose, D-xylose, L-arabinose, cellobiose, D-mannose, D-melezitose, D-raffinose, D-trehalose, D-mannitol, salicin and D-sorbitol. The main fermentation products from PYG broth were lactic acid, propionic acid, formic acid and acetic acid. Chemotaxonomic analysis showed that the major fatty acids were anteiso-C(15:0), C(15:0) and iso-C(17:0) 3-OH and the predominant respiratory quinones were MK-9 and MK-10. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NS31-3(T) was related to members of genus Parabacteroides (91.2-93.2% sequence similarity); the isolate had the closest affinity with Parabacteroides merdae JCM 9497(T). The G+C content of the genomic DNA was 37.2 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, strain NS31-3(T) represents a novel species of the genus Parabacteroides, for which the name Parabacteroides chartae sp. nov. is proposed. The type strain is NS31-3(T) (=JCM 17797(T) =DSM 24967(T)).

Efficacy of Lagenidium giganteum metabolites on mosquito larvae with reference to nontarget organisms

Lagenidium giganteum is a water mold and an effective mosquito control agent with limited use due to poor survival and contamination during storage. Invert extracellular metabolites of L. giganteum is easy to produce, long shelf life, and a potential candidate in tropical climates. This fungus was grown in PYG broth in the laboratory at 25 +/- 2 degrees C, and relative humidity was maintained at 75 +/- 5% for 15 +/- 2 days. Filtration process of metabolites was done using Whatman filter paper, column chromatograph, and range syringe filters techniques. Then 5-ml fractions were collected and used to assay larvicidal efficacies. Larvicidal efficacies were performed against Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti at five different concentrations, viz. 1.68, 1.99, 2.17, 2.30, and 2.40 ppm. And also, filtrates were assessed against four species of nontarget organisms named Daphnia pulex, Cyclopes, Lymnea auriculeta, and tadpoles of Rana tigrina with different concentrations. The mortality values were subjected by the Probit analysis. The complete mortalities that resulted from applying filtrates dosage on all instars of mosquitoes persisted for a period of 24, 48, and 72 h, respectively. The efficacies in killing instar of three important vectors and safer for nontarget organisms with good biological stability of extracellular metabolites make this a promising alternative to mycelium and conidial-based larvicides. It could be regarded as fungal-based natural larvicide for the use of vector control.

タイ王国でウシエビの卵および幼生から分離された<i>Lagenidium thermophilum</i>

ABSTRACT —A fungal infection was found in eggs and larvae of black tiger shrimp Penaeusmonodon at a hatchery in Chachensao Province, Thailand in August 2000. Fungi were isolatedfrom eggs and larvae with fungal infection, and studied on the morphological and biological charac-teristics. When it was transferred from PYGS broth to artificial seawater, discharge tubes devel-oped from the mycelia, and a vesicle for zoospore formation was produced at the top of eachdischarge tube. The characteristic feature of an asexual reproduction of the fungus was thatzoospores swam away in seawater after the vesicle separated from the discharge tube. Based onthese morphological characteristics, the fungus was identified as Lagenidium thermophilum.Some biological characteristics of the selected isolate NJM 0031 were compared with the otherspecies in the genus Lagenidium isolated from some crustaceans. As a result, the isolate NJM0031 showed similar characteristics to those of L. thermophilum ATCC 200318 isolated from man-grove crab Scylla serrata. The isolate was demonstrated to be pathogenic to larvae of black tigershrimp by artificial infection. This is the first report of L. thermophilum infection in black tigershrimp in Thailand.

Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study.

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of Parachlamydia bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of P. acanthamoeba within A. polyphaga.

Combinations of Heat Treatment and Sodium Chloride That Prevent Growth from Spores of Nonproteolytic Clostridium botulinum

The ability of spores of nonproteolytic Clostridium botulinum to survive heat treatment and subsequently produce turbidity at 10°C in the presence of NaCl was quantified for different incubation times. Spores of nonproteolytic C. botulinum strain Eklund 17B were heated at 75°C for up to 4 min or 90°C for up to 30 min and subsequently incubated at l0°C in PYGS broth containing 1.5%. 3.0%, or 4.0% NaCl (wt/vol) with or without 10 μg hen egg white lysozyme ml−1. Heat treatment at 90°C for 30 min or incubation at 10°C in the presence of 4.0% NaCl did not prevent growth from up to 1.4 × l05 spores. Heat treatment at 90°C for 15 min and incubation in the presence of 4.0% NaCl did prevent growth in the same conditions.

飼育水のｐＨ調整によるガザミ幼生真菌症の防除

Halocrusticida (=Atkinsiella) okinawaensis was isolated from diseased swimming crab larvae Portunus trituberculatus in the Hyogo Prefectural Mariculture Center, and pathogenicity of the isolates was confirmed by infection experiments with zoeal larvae of the crab. The isolates grew in PYGS broth at pH 5-9, but the growth at pH 9.25 was remarkably reduced.Experimental infections using zoea-I demonstrated that the infection with H. okinawaensis was controlled by keeping the pH of reaing water at 9.25. Acute toxicities of pH 9.25 to swimming crab zoea, rotifer Brachionus plicatilis rotundiformis, brine shrimp Artemia salina, and unicellular alga Nannochloropsis sp. were not observed. Survival rates of swimming crab larvae from zoea-I to juvenile crab-I in the center were apparently raised by applying this pH adjustment at 9.25.

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum.

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90 degrees C for between 0 and 60 min and subsequently incubated at 5 degrees, 10 degrees or 30 degrees C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90 degrees C for 1 or more min ensured a 10(6) reduction when spores were subsequently incubated at 5 degrees C for up to 23 weeks. Heating at 90 degrees C for 60 min ensured a 10(6) reduction over 23 weeks when subsequent incubation was at 10 degrees C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10(6) when subsequent incubation was at 30 degrees C.

Gram-negative bacteria viable in ultrapure water: identification of bacteria isolated from ultrapure water and effect of temperature on their behavior

This study describes the effect of temperature on the behavior of bacteria viable in ultrapure water and the contamination of ultrapure water by bacteria. Three species of bacteria were isolated from ultrapure water (total organic carbon, 60 ppb and 5 ppb; effluent resistivity > 18 MΩ cm at 25°C) and identified by morphological and physiological characteristics. The three isolates were incubated in water for injection and PYG broth to check the growth profile at various temperatures. In PYG broth, temperature influenced the behavior of bacteria directly; however, it did not in water for injection. By checking both viable and non-viable bacterial numbers and endotoxin concentration in pure water, the water was found to be contaminated with non-viable bacteria and newly generated endotoxins besides viable bacteria. A column treatment, a mixed bed of fully regenerated strong acid cation exchange resin (SACER) and strong base anion exchange resin (SBAER), was used to remove bacteria from pure water. Bacteria could not grow on the surfaces of ion exchange resins in the mixed bed. The removal of bacteria was more effective as pure water was circulated through the mixed bed more rapidly.

A marine oomycete Atkinsiella panulirata sp. nov. from philozoma of spiny lobster, Panulirus japonicus

A marine oomycete fungus was isolated from philozoma of the spiny lobster,Panulirus japonicus. The fungus exhibited slow growth, occasionally submerged, with a creamy white, raised moist colony. Hyphae were stout, arranged in radiating pattern, irregularly branched, 10–22 µm diam, occasionally separated by cross walls into subthalli. Thalli occasionally consisted of swollen features. Sporangia formed from the subthalli had 1–3 wavy or partly coiled discharge tubes at the terminal or subterminal area. Zoospores were pyriform or reniform, biflagellate, isokont and diplanetic. Encysted spores germinated as a hairlike filament with a globular enlarged tip in sterilized synthetic seawater, and directly as stout initial hypha in PYGS broth. This fungus was identified as belonging to the genusAtkinsiella and was designatedAtkinsiella panulirata sp. nov. The optimal temperature for the fungus was 25°C. Hyphal growth occurred in both sterilized synthetic seawater and NaCl in the range of 1.5–6.0% and 2.0–4.0%, respectively. The fungus could utilize all carbohydrates and derivatives tested as the carbon sources. From in vitro tests of five fungicides against the fungus malachite green was found to be the most effective.

Atkinsiella awabi sp. nov. isolated from stocked abalone, Haliotis sieboldii

A fungal disease in the abalone,Haliotis sieboldii, stocked in Yamaguchi Prefecture, Japan, showed external signs of infection of tubercle-like swelling on the mantle and melanized lesions on the peduncle. The fungus responsible was isolated by inoculating materials taken from the lesions onto PYGS agar with streptomycin sulphate and ampicillin, and incubation at 20°C. For morphological observation and spore formation study, the fungus was transferred respectively into PYGS broth and sterilized artificial seawater and incubated at 20°C. Resulting, hyphae were stout, irregular, branched, 16–140µm diam, sporadically consisting of dense cytoplasmic swollen hyphae. Sporangia were formed through the formation of septa and lateral or terminal discharge tubes which were wavy or coiled. Zoospores were pyriform, biflagellate and diplanetic. The encysted spore generally developed a hairlike filament with globular enlarged tip in PYGS broth. Direct germination without filament formation also occurred occasionally. This fungus was identified as belonging to the genusAtkinsiella, and was designatedAtkinsiella awabi sp. nov. The fungus was exclusively a marine fungus and grew best in shrimp extract medium at 20°C. Five chemicals were tested for their effects against fungal zoospores.

Atomic radiation spurs growth of “whiskers” on metals

A fungal disease in the abalone, Haliotis sieboldii, stocked in Yamaguchi Prefecture, Japan, showed external signs of infection of tubercle-like swelling on the mantle and melanized lesions on the peduncle. The fungus responsible was isolated by inoculating materials taken from the lesions onto PYGS agar with streptomycin sulphate and ampicillin, and incubation at 20°C. For morphological observation and spore formation study, the fungus was transferred respectively into PYGS broth and sterilized artificial seawater and incubated at 20°C. Resulting, hyphae were stout, irregular, branched, 16-140μm diam, sporadically consisting of dense cytoplasmic swollen hyphae. Sporangia were formed through the formation of septa and lateral or terminal discharge tubes which were wavy or coiled. Zoospores were pyriform, biflagellate and diplanetic. The encysted spore generally developed a hairlike filament with globular enlarged tip in PYGS broth. Direct germination without filament formation also occurred occasionally. This fungus was identified as belonging to the genus Atkinsiella, and was designated Atkinsiella awabi sp. nov. The fungus was exclusively a marine fungus and grew best in shrimp extract medium at 20°. Five chemicals were tested for their effects against fungal zoospores.