Mid-stationary Growth Phase Research Articles

Components Secreted by <i>Lactobacillus Fermentum</i> PC1 Inhibit <i>Salmonella Enterica</i> Serotype Typhimurium Induced IL-8 from Intestinal Epithelial Cells

Lactobacillus strains have been shown to confer health benefits to the host including attenuation of intestinal inflammatory responses. However, the health benefits of lactobacilli are strain specific. The aim of this study was to determine whether <i>Lactobacillus fermentum</i> PC1 (PC1) cell wall extract (CW) and the spent culture supernatant (SCS) had the capacity to inhibit IL-8 production by <i>Salmonella</i> <i>enterica </i>serotype Typhimurium (<I>S</I>. Typhimurium) infected epithelial cells. Epithelial cell line, HT-29 was treated with CW or SCS of PC1 both pre- and post-infection with <I>S</I>. Typhimurium and the resultant levels of IL-8 protein was assayed. Both the CW and SCS of PC1 was shown to inhibit <I>S</I>. Typhimurium induced IL 8 production in HT-29 cells in the therapeutic and prophylactic models. Furthermore, a secreted molecule produced by PC1 responsible for this effect was identified and characterized. The molecule was produced in mid-stationary phase of growth. This active component was present in both the cell wall extracts and spent culture medium of PC1. The bioactive molecule(s) had a size of Mr 2-30KDa, was heat stable at 90°C for 30 min, insensitive to lipase, distinct from acetic and lactic acid, and optimal function at pH 4.5. The activity of the molecules was inactivated by Proteinase K, Na-metaperiodate and Trypsin indicating that the molecule(s) was a glycoprotein. The isolation of an immunomodulatory molecule that could be used in the treatment of <I>S</I>. Typhimurium infection would be of great value.

Validation of a cationic polyacrylamide flocculant for the harvesting fresh and seawater microalgal biomass

Abstract A simple, efficient, and fast settling flocculation technique to harvest microalgal biomass was demonstrated using a proprietary cationic polyacrylamide flocculant for a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal culture at their mid-stationary growth phase. The optimal flocculant doses were 18.9 and 13.7 mg/g of dry algal biomass for C. vulgaris and P. tricornutum, respectively (equivalent to 7 g per m3 of algal culture for both species). The obtained optimal dose was well corroborated with changes in cell surface charge, and culture solution optical density and turbidity. At the optimal dose, charge neutralization of 64 and 86% was observed for C. vulgaris and P. tricornutum algal cells, respectively. Algae recovery was independent of the culture solution pH in the range of pH 6 to 9. Algal biomass recovery was achieved of 100 and 90% for C vulgaris and P. tricornutum respectively, and over 98% medium recovery was achievable by simple decanting.

Decimal reduction energies of UV-C-irradiated spoilage yeasts in coconut liquid endosperm

The ultraviolet-C (UV-C) decimal reduction energy (DUV-C) values of 17 spoilage yeasts and their composited inoculum were determined in coconut liquid endosperm (pH 5.26, 5.8 °Brix, 0.04% malic acid, 0.17% w/v insoluble solids). Growth kinetic parameters of all the test yeast strains were first established to standardize the growth stage of the cells prior to inactivation studies. Approximately 4.0 to 5.0 log CFU/mL cells in the mid-stationary growth phase (30.3 to 39.9 h, 25 °C) were suspended in 4 mL turbulent flowing juice and subjected to UV-C irradiation at a surface irradiance range of 3.42 to 4.99 mW/cm2. Survivor populations after exposure to predetermined UV-C energy were enumerated, and were used to derive the DUV-C values using the linear regression and Baranyi and Roberts (1994) model fitting. Results show that the yeast strains exhibited either log-linear or biphasic inactivation behavior with inactivation lag. The most UV-C resistant spoilage yeast was found to be Cryptococcus albidus (LJY1) with DUV-C values of 122.72 and 214.89 mJ/cm2 determined from linear regression and model-fitting, respectively. The least UV-C resistant was Torulaspora delbrueckii (LYJ5) with a DUV-C of 17.34 (linear regression) and 17.35 mJ/cm2 (model-fitting). The DUV-C values determined from the model fitting were generally greater than those calculated from linear regression, although only those determined for C. albidus were significantly different. To the investigators' knowledge, this is the first report of the UV-C inactivation kinetic parameters of Kluyveromyces marxianus, Trichosporon cutaneum, Pichia anomala, and Meyerozyma guilliermondii and C. albidus in coconut liquid endosperm. The results of this study can be used in the establishment and validation of UV-C process schedules for coconut liquid endosperm and other similar commodities.

Ultraviolet-C resistance of selected spoilage yeasts in orange juice

This study determined the ultraviolet-C (UV-C) dose necessary to reduce 90% population (DUV-C) of 17 spoilage yeasts and their composited inoculum in orange juice (pH 3.71, 11.60 °Brix, 0.55% citric acid, 2.46% w/v insoluble solids). Growth parameters of all test yeasts were first established to standardize the growth stage of the cells prior to harvesting and eventual UV-C challenge studies. Approximately 4–5 log CFU/ml cells in the mid-stationary growth phase (30.3 t0 39.9 h, 25 °C) were suspended in 4 ml turbulent flowing juice and subjected to UV-C irradiation at an incident surface irradiance of 3.64–4.97 mW/cm2. The inactivation rates of each yeast and their composited inoculum were determined using 2 methods namely, the linear regression and Baranyi and Roberts (1994) model-fitting. Results showed that the yeasts exhibited either log-linear or biphasic inactivation behavior with downward concavity or inactivation lag. Regardless of the method of determination, Cryptococcus albidus (LJY1) exhibited the significantly greatest (p < 0.05) UV-C resistance with DUV-C values of 1924.31 and 2174.63 mJ/cm2. On the other hand, Candida parapsilosis was determined to be least resistant with a DUV-C values of 245.83 and 357.88 mJ/cm2. Majority of the DUV-C values determined from the model-fitting were greater than those calculated from linear regression. However, only those determined for the composited inoculum were significantly different. The results of this study address knowledge gaps pertinent to the UV-C resistance of less studied spoilage yeast, and help in better understanding the utility of this non-thermal food processing technology.

Streptomyces sp. AT37 isolated from a Saharan soil produces a furanone derivative active against multidrug-resistant Staphylococcus aureus.

A novel actinobacterium strain, named AT37, showed a strong activity against some multidrug-resistant Staphylococcus aureus, including methicillin-resistant S. aureus MRSA ATCC 43300, other clinical isolates of MRSA and vancomycin resistant S. aureus VRSA S1. The strain AT37 was isolated from a Saharan soil by a dilution agar plating method using chitin-vitamin agar medium supplemented with rifampicin. The morphological and chemical studies indicated that this strain belonged to the genus Streptomyces. Its 16S rRNA gene sequence was determined and a database search indicated that it was closely associated with the type strain of Streptomyces novaecaesareae NBRC 13368T with 99.6% of similarity. However, the comparison of the morphological and the physiological characteristics of the strain with those of the nearest species showed significant differences. The strain AT37 secreted the antibiotic optimally during mid-stationary phase of growth. One active compound (AT37-1) was extracted from the culture broth with dichloromethane, separated on silica gel plates and purified by HPLC. Based on spectroscopic analysis of UV-Visible, infrared, and 1H and 13C NMR spectra and spectrometric analysis, the chemical structure of the compound AT37-1 was identified as 5-[(5E,7E,11E)-2,10-dihydroxy-9,11-dimethyl-5,7,11-tridecatrien-1-yl]-2-hydroxy-2-(1-hydroxyethyl)-4-methyl-3(2H)-furanone. Minimum inhibitory concentrations and minimum biofilm inhibitory concentration (MBIC50) of this compound showed significant activity against multidrug-resistant S. aureus with 15-30 and 10-15μg/mL, respectively.

Atmospheric pressure plasma jet inactivation of Pseudomonas aeruginosa biofilms on stainless steel surfaces

Abstract This study established the efficacy of atmospheric pressure plasma jet on Pseudomonas aeruginosa on stainless steel types 316 and 304; with different finishes namely, mirror (MR), hairline (HL) and 2B surfaces. A cocktail of four strains of P. aeruginosa in the mid-stationary growth phase were allowed to attach on the test surfaces, and subjected to atmospheric pressure plasma jet treatment with an air injection rate of 5 l/min, output power of 360 W at 4.22 cm source-to-surface distance. Attachment rates were significantly affected by surface finish, rather than by stainless steel type. The D-values on the 316 stainless steel type ranged from 2.53 s (MR) to 3.16 s (2B); while those on the 304 type ranged from 1.95 s (HL) to 3.27 s (2B). Variations in D-values were observed between surface finishes within a specific stainless steel type. However, significant variations were not observed between the same surface finish of different steel types. The observed antimicrobial efficacy was attributed to the generation of reactive oxygen species, ultraviolet-C rays, and rapid temperature increase (final temperature of 143.42 °C to 174.05 °C) within 15 s of treatment. In the absence of heating, the D-value increased to 16.45 s, but a 5-log (99.999%) reduction in the population was observed in a relatively short treatment time of 90 s. Industrial relevance The results obtained in this work demonstrated the potential of using atmospheric pressure plasma jet technology as a non-chemical, non-thermal, and thermal stainless steel food contact surface decontamination against Pseudomonas aeruginosa, a common biofilm-producing bacterium. Such a technology shall help the industry address the challenge of cross contamination in the food manufacturing and food service settings.

Reference organism selection for microwave atmospheric pressure plasma jet treatment of young coconut liquid endosperm

This study identified a reference organism for the establishment of microwave atmospheric pressure plasma jet treatment of young coconut liquid endosperm. Three different, predominant spoilage bacteria were isolated from spoiled liquid endosperm and identified through 16S rDNA sequencing, prior to propagation and eventual challenge studies. Basic Local Alignment Search Tool comparisons revealed that the isolated spoilage bacteria were Klebsiella sp., Staphylococcus sp., and Kluyvera sp (%Identity 97.34–97.82%). The isolates were propagated to mid-stationary growth phase (17 h) and thereafter suspended in liquid endosperm. The inactivation rates of the spoilage isolates in liquid endosperm subjected to plasma jet at 450 and 650 W input power settings were determined and compared to those of pathogens Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes. Inactivation rates of all test organisms at 450 W (2.76–5.98 min) were significantly less (p < 0.05) than those at 650 W (1.86–3.11 min). In both power settings, S. enterica exhibited the significantly (p < 0.05) greatest resistance towards plasma treatment, while Staphylococcus sp. exhibited the least. Heat penetration studies in both power settings revealed that the temperature of the liquid endosperm did not significantly increase and did not reach bactericidal ranges (<30 °C) during plasma jet treatment, hence the observed inactivation were attributed to antimicrobial species generated by the plasma. Optical emission spectroscopy revealed that these antimicrobial species included atomic oxygen (777.41 and 844.67 nm) and ultraviolet radiation (200–280 nm). Thus, this study determined S. enterica as an appropriate target organism for the establishment of plasma jet process for coconut liquid endosperm, the inactivation rate of which shall be soon established in higher scale treatment process.

Purification and characteristics of bifidocin A, a novel bacteriocin produced by Bifidobacterium animals BB04 from centenarians' intestine

Bacteriocin-producing strain BB04, isolated from centenarians' intestine and identified as Bifidobacterium animals, produced the maximum activity of bifidocin A (640 AU/ml) in MRS medium supplemented with 0.05% (w/v) l-cysteine hydrochloride in the mid-stationary phase of growth (i.e. after 32 h at 37 °C). The bacteriocin, with a molecular mass of 1198.68 Da, was successfully purified by a four-step procedure consisting of adsorption and desorption onto/from producer cells, cation-exchange chromatography, gel filter chromatography and reverse phase high-performance liquid chromatography (RP-HPLC). Purified bifidocin A exhibited a wide range of antimicrobial activity against many food-borne spoilage and pathogenic bacteria such as Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, as well as some yeast. The mode of action of bifidocin A was bactericidal with concomitant cell lysis. Also, bifidocin A was heat stable, pH resistant and protease sensitive. EDTA, Tween 80, Tween 20 and urea did not decrease the activity, whereas, SDS induced 36% activity loss. This is the first report on a bacteriocin produced by B. animals. And the characterization, antimicrobial spectrum and molecular mass of bifidocin A suggested that it was a novel bacteriocin with potential research and application value in food industry.

Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.

Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7–40% from log phase growth to late log – early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20–31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60–90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC–MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.

Is it possible “to cancel” the aging process of cell cultures under optimal cultivation conditions?

The characteristics of epigenotypes of Dunaliella viridis Teod. cells have been investigated during chronological and replicative aging. It was shown that by the 40th day of cumulative cultivation, which coincided with the stationary phase of growth, the DNA content doubled, the TG content tripled, the content of β-carotin doubled, carbonilated proteins doubled, and the content of RNA decreased in the cells of Dunaliella viridis compared to the cells in the exponential phase of growth; i.e., by the 40th day of cultivation, age-dependent epigenotype develops. Four subcultures were obtained which were transplanted for two years in a midlogarithmic growth phase (subculture-10), early stationary phase (subculture-20), a midstationary growth phase (subculture-30), and a late stationary phase (subculture-40). The epigenotype of subculture-10 was shown to remain unchanged for two years of cultivation, i.e., there was no replicative aging. At the same time, despite the fact that subculture-20 maintained the epigenotype characteristic of a young culture for quite a long time (no less than 40 passages), it displayed age-dependent changes. Marked age-dependent changes in the epigenotype during the cultivation were revealed in subculture-30; subculture-40 was characterized by an unstable epigenotype. The conditions for cultivation were shown to define the intensity of replicative aging in Dunaliella viridis.

A novel strain of Actinopolyspora mortivallis with antibacterial activity isolated from a Saharan soil

A new halophilic actinomycete strain, designated H16, has been isolated from a hypersaline Saharan soil in Ouargla province (southern Algeria) and characterized taxonomically using a polyphasic approach. The strain grew at 18–50°C, pH 5–9, and 7–32% (w/v) NaCl. It produced abundant aerial mycelia, which formed long chains of rod-shaped spores at maturity, and fragmented substrate mycelia. The strain contained chemotaxonomic markers that were diagnostic for the genus Actinopolyspora, such as meso-diaminopimelic acid, arabinose, and galactose, phosphatidylcholine as a diagnostic phospholipid, and predominant menaquinones MK-9(H4) and MK-10(H4). The predominant fatty acids were anteiso- and iso-C17:0, iso-C15:0, and 9-methyl-C16:0. Phylogenetic analysis, based on 16S rDNA gene sequences, confirmed that strain H16 is a member of the genus Actinopolyspora and most closely related to A. mortivallis (98.7% identity). DNA–DNA hybridization confirmed that it belonged to A. mortivallis. This strain showed high activity against Klebsiella pneumoniae and optimally secreted antibiotics during the mid-stationary growth phase in liquid culture. The antibiotics were extracted with n-butanol and separated on silica gel plates by ethanol–ammonia–water (mobile phase). The results of bioautography revealed the presence of five antibiotics. The chemical revelations showed that these antibiotics were glycosylated polycyclic aromatic compounds containing amine groups and hydroxamic acids. The UV-visible and mass spectra of the most active antibiotics were determined.

Cadmium effects on transcriptional expression of rhlB/rhlC genes and congener distribution of monorhamnolipid and dirhamnolipid in Pseudomonas aeruginosa IGB83

While variable production of the biosurfactant, rhamnolipid, by Pseudomonas aeruginosa has been shown to be dependent on growth conditions, no research has evaluated potential relationships between rhamnolipid production and the presence of heavy metals. The current investigation evaluates the influence of Cd(2+) on rhamnolipid synthesis. Cultures grown in the presence of 0.45 and 0.89 mM Cd(2+) were monitored for rhlB/rhlC expression, rhamnolipid yield, and the ratio of monorhamnolipid (RL1) and dirhamnolipid (RL2) produced. Results show a Cd-induced enhancement of rhlB expression in mid-stationary phase (53 h). In addition, sustained production of rhamnolipid through late stationary growth phase (96 h) was observed for Cd-amended cultures, unlike Cd-free control cultures that ceased rhamnolipid production by mid-stationary growth phase. Most significant was an observed increase in the ratio of RL2 to RL1 congeners produced by cultures grown in the presence of Cd(2+). Previous results have shown that the complexation constant for RL2-Cd is several orders of magnitude larger than that of RL1-Cd thus the preferential production of RL2 in the presence of Cd(2+) impacts its bioavailability and toxicity both for the cell and in the surrounding environment.

Enterococcus faecium LM-2, a multi-bacteriocinogenic strain naturally occurring in “Byaslag”, a traditional cheese of Inner Mongolia in China

A bacteriocin-producing strain LM-2 was isolated from “Byaslag”, a traditional cheese in Inner Mongolia of China, and identified as Enterococcus faecium. The strain produced the maximum activity of enterocin (5120 AU ml −1) in MRS medium in the mid-stationary phase of growth (i.e. after 30 h at 37 °C). Purified enterocin LM-2 was obtained by using a two-step procedure consisting of ammonium sulfate precipitation and cation exchange chromatography. Tricine–SDS–PAGE of the purified enteriocin LM-2 contained two active protein bands between 3.5 and 6.4 kDa. It showed a broad spectrum of activity against most of the tested strains in the genera Listeria, Staphylococcus, Bacillus, Clostridium, Salmonella and Pseudomonas. All Listeria strains tested, including L. monocytogenes, were highly sensitive to this bacteriocin. The inhibitory activity of enterocin LM-2 against L. monocytogenes was bactericidal without concomitant cell growth, and the highest level of adsorption (87.5%) to the surface of L. monocytogenes 54002 cells was founded at pH 6.0. This strong inhibitory activity against L. monocytogenes, coupled with its broad pH-resistance and heat-stability indicated its potential use as a food preservative to enhance the safety of fermented foods. The presence of enterocin P and L50-like structural genes in E. faecium LM-2 was established by PCR and direct sequencing, which indicated strain LM-2 may be a new multi-bacteriocin producer strain.

Hydrogen production and transcriptional analysis of nifD, nifK and hupS genes in Rhodobacter sphaeroides O.U.001 grown in media with different concentrations of molybdenum and iron

Rhodobacter sphaeroides O.U.001 was grown in media with different concentrations of molybdenum and iron to study the expression level of nifD and nifK genes coding for the large subunit of Mo-nitrogenase and hupS gene coding for the small subunit of uptake hydrogenase. Hydrogen productions under the same growth conditions were also evaluated. Increasing concentrations of the molybdenum and iron stimulated hydrogen production and the highest total hydrogen accumulation was achieved in 16.5 μ M sodium molybdate (0.84 l H 2/l culture at t = 168 h ) and 0.1 mM ferric citrate (1.14 l H 2/l culture at t = 240 h ) containing media. Maximal expressions of nifD and nifK were observed from the late log phase until the mid-stationary phase of growth and hupS expression was seen as soon as the hydrogen was produced in the cells. Moreover, nifK, nifD and hupS expressions were significantly reduced in the molybdenum and iron starved cells.

Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa.

nfxC-type cells of Pseudomonas aeruginosa that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical beta-lactam antibiotics. We investigated the molecular mechanism of how the nfxC mutation causes beta-lactam hypersusceptibility. The MexAB-OprM extrusion pump transports and confers resistance to beta-lactam antibiotics. Interestingly, expression of the mexAB-oprM operon reached the highest level during the mid-stationary growth phase in both wild-type and nfxC-type mutant strains, suggesting that expression of the mexAB-oprM operon may be controlled by cell density-dependent regulation such as quorum sensing. This assumption was verified by demonstrating that exogenous addition of the quorum-sensing autoinducer N-butyryl-L-homoserine lactone (C4-HSL) enhanced the expression of MexAB-OprM, whereas N-(3-oxododecanoyl)-L-homoserine lactone had only a slight effect. Furthermore, this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon. It was concluded that beta-lactam hypersusceptibility in nfxC-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.

Extracellular hemolytic activity in rapidly growing mycobacteria.

Little is known about virulence factors associated with rapidly growing mycobacteria. We evaluated 42 clinical isolates of Mycobacterium fortuitum and Mycobacterium chelonae and 4 reference strains of Mycobacterium smegmatis for the production of hemolysin (or hemolytic substance) as a possible contributor to the pathogenesis of disease caused by these organisms. All the strains tested possessed extracellular hemolytic activity that was stable after heating and proteinase treatment, and the active substance had a molecular weight less than 10,000. The activity accumulated in culture medium during the late exponential to mid stationary phase of growth. Hemolysis in vitro was relatively slow; incubation for 10 h at 35 degrees C was required to obtain maximal activity. Some specificity of the hemolysis with regard to red blood cells from different animals was observed.

Biosynthesis of acyclic methyl branched polyunsaturated hydrocarbons inPseudomonas maltophilia

The hydrocarbon composition ofPseudomonas maltophilia was determined by gas chromatography-mass spectrometry. Mono-, di- and tri-unsaturated alkenes were identified with a predominance of polyunsaturated components. The carbon chains of the alkenes contained single methyl branches iniso andanteiso position and double methyl branches in theiso-iso andanteiso-anteiso configurations. The composition of the hydrocarbons from cells grown in synthetic media enriched with amino acids or volatile fatty acids demonstrated that the probable precursors incorporated into individual hydrocarbons were branched and normal fatty acid chains in the range from C3 to C16. The probable fatty acid precursors which were connected together to form the major triunsaturated hydrocarbon chains were two monounsaturated chains, whereas the major liunsaturated chains resulted from condensation of saturated and monounsaturated chains. The probable precursors for the major monounsaturated hydrocarbons were C14 (C15) and C16 (C15) fatty acids. The accumulation of hydrocarbons was not detected until the cells were in the late exponential phase of growth; the maximal levels were reached at the mid-stationary phase of growth.

Characterization of a Selenium-Independent Glutathione Peroxidase From Euglena gracilis

Light or dark grown Euglena gracilis strains contain similar levels of glutathione (GSH) peroxidase. Cells in midstationary phase of growth contained the highest level of the enzyme. The enzyme was purified 280-fold to homogeneity from the permanently bleached strain, E. gracilis var bacillaris W(3)BUL. The native enzyme has a molecular weight of 130,000 as measured by gel permeation chromatography, and contains four subunits (mol wt 31,500) as measured by sodium dodecyl sulfate gel electrophoresis. A variable amount of a higher molecular weight form of the enzyme (approximate mol wt 250,000) was detected but not further characterized. The enzyme has an isoelectric point of 4.7. No selenium could be detected in the purified enzyme. The enzyme is active with H(2)O(2) and a variety of organic hydroperoxides, including 13-hydroperoxylinoleic acid, and is specific for GSH as the thiol substrate. Apparent K(m) values for H(2)O(2), t-butyl hydroperoxide, and GSH were 0.03, 1.5, and 0.7 millimolar, respectively. A comparison of selenium-dependent and selenium-independent GSH peroxidases from various eukaryotic sources is presented.

In situ and laboratory studies of bacterial survival using a microporous membrane sandwich.

A new device and procedure for the study of bacterial survival in an aquatic environment are described. The device uses two appressed presterilized microporous membranes to expose a bacterial cell suspension to the environment at a cell concentration that closely resembles those levels found in natural aquatic ecosystems. The device has been used under laboratory controlled conditions and in situ to study and compare bacterial survival times. In laboratory studies, Escherichia coli and Streptococcus faecalis survived the longest at 12 degrees C, pH 5, and in the presence of iron or calcium ions and cysteine. Cells in mid-stationary growth phase survived longer than those in mid- or late-logarithmic phase, whereas those maintained for a year or more as stock cultures survived for shorter period of time than did recent environmental isolates. In situ studies indicate that 5% of the starting number of E. coli and S. faecalis cells may survive longer than 96 h at 16 degrees C in potable lake water, whereas survival times in polluted lake water were approximately 12 h.