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NiCrAlY overlay coatings were applied on Ni-base single-crystal superalloy CMSX-4 substrates by cathodic arc deposition. The coated specimens were subjected to one- and two-step heat treatments performed under 870°C/20h/AC (AC stands for air cooling) and 1140°C/6h/AC+870°C/20h/AC conditions, respectively. The microstructure of the original NiCrAlY cathodes fabricated by spark plasma sintering and the as-deposited as well as heat-treated coatings were characterized using the scanning electron microscopy, energy-dispersive spectroscopy and X-ray diffraction techniques. It was observed that the cathodes contained multiple phases uniformly distributed across the cathode volume. The as-deposited coatings exhibited a duplex microstructure consisting of splats and inter-splat regions with the latter showing internal porosity and a significantly lower Al concentration compared to that of the cathodes. The post-deposition heat treatments eliminated the porosity but further reduced the Al content, resulting in a coating microstructure dominated by the solid solution γ-Ni phase. The total thickness of the coating including the interdiffusion zone increased significantly during the two-step heat treatment. The oxide scale formed on the coating surface during heat treatment consisted primarily of α-Al2O3, with Y2O3 pegs underpinning the scale to the coating. The oxide scale was continuous on the smooth sections of the coating surface, but became fragmented at the protruding macroparticles present on the coating surface., ASME Turbo Expo 2014: Turbine Technical Conference and Exposition, GT 2014, 16 June 2014 through 20 June 2014

MKES Tools is a microbial kinetics expert system for developing food production systems and assessing product safety. The specific information required as input are: (1) a flowchart of the production system, (2) the factors affecting the survival and growth of food-borne pathogens and (3) the ranges of variation for each factor's parameters. With this information, MKES Tools simulates the growth and survival of pathogenic microorganisms when subjected to many different factor/parameter situations. The responses obtained are then used to estimate the significance of each factor's parameters.

To determine how stress response and virulence gene expression of stationary phase (SP) Escherichia coli O157:H7 are affected by nutrient levels.A targeted microarray (n=125 genes) was used to determine the impact of nutrient deprivation [15 min in 3-(N-Morpholino)propanesulfonic acid buffer] on SP E. coli O157:H7. In total, 24 genes were significantly affected (1·5-fold; P 0·05) with 17 induced and seven attenuated. Additionally, 11 genes belonging to significantly affected stress response regulons were significantly induced (P0·05), though1·5-fold. Induced genes included global and specific stress response regulators, the mar operon, iron acquisition and virulence genes. In contrast, transcript for major porins and replicative genes were repressed. Comparison of the nutrient deprived transcriptome to that derived from nutrient replenished cells revealed a disparate transcriptome, with 44 genes expressed at significantly elevated levels in nutrient replenished cells, including all queried global and specific stress response regulators and key virulence genes. Genes expressed at elevated levels in nutrient deprived cells were related to σ(S) . The microarray data were validated by qRT-PCR. SP E. coli O157:H7 were affected by nutrient deprivation, with both starvation-related and unrelated networks induced, thereby demonstrating how the E. coli O157:H7 stress response transcriptome is fine-tuned to environmental conditions. Further, by comparison of starved cells to cells provided with fresh nutrients, it is clear starved E. coli O157:H7 undergo massive physiological reprogramming dominated initially by stress response induction to adapt to a nutrient rich environment. This study demonstrated how σ(S) -induced SP E. coli O157:H7 remain highly sensitive and adaptable to environmental conditions. Further, by examining how starved cells respond to nutrient-rich conditions, we show preliminary adaptation to a nutrient rich environment is dominated by the induction of diverse stress response networks. Combined, this provides E. coli O157:H7 stress physiology-based knowledge that can be used to design more effective food safety interventions.

Heat resistance experiments were carried out with Listeria monocytogenes which had been grown at three different temperatures (30, 39, and 43°C). Heated whole milk was inoculated with L. monocytogenes and then passed through a high-temperature short-time system at 72, 69, 66, and 63°C for a minimum holding time of 16.2 s. Heated cells were recovered both aerobically and anaerobically using four different methods: direct plating, most probable number, cold enrichment, and warm enrichment. Significant differences in recovery of L. monocytogenes were observed depending on the growth temperature. Cells grown at 43, 39, or 30°C, held 1 d at 4°C, and then heated at 69°C showed an overall decrease in numbers of approximately 2.1, 2.8, and 4.1 logs, respectively. Cells grown at 39°C and then held 3 d at 4°C appeared to be the most heat sensitive. Although cells grown at 43 and 39°C were capable of surviving at the minimum high-temperature short-time temperature (72°C), those grown at 30°C were not. In some instances, anaerobic incubation enhanced the recovery of L. monocytogenes , as compared to cells recovered aerobically, although these differences were not statistically significant. While L. monocytogenes can survive minimum pasteurization treatment (71.7°C/16 s) under certain conditions, common methods of handling, processing, and storing fluid milk will provide an adequate margin of safety.

SummaryThe effect of arginine on the production of extracellular lipase byPseudomonas fluorescens32A was studied. Arginine repressed lipase production when N was supplied partially or totally as arginine. Proteinase production was unaffected under the same conditions. Arginine did not repress lipase secretion when cells were pregrown in an arginine-containing medium; however, repression was found with uninduced cells. Several arginine-analogues were tested for the ability to repress lipase secretion and. of these, L-homoarginine and L-canavanine were the most effective. D-Arginine, L-arginine methyl ester, and L-ornithine did not cause repression. With the exception of glutamic acid and methionine, those amino acids that supplied N but not C for growth were found to repress lipase secretion. It is suggested that the accumulation of metabolic intermediate(s) may cause repression of lipase production.

It has proven difficult to develop adequate mathematical models for the lag phase (lambda) which characterizes the adaptation period prior to the initiation of exponential growth by microorganisms. This is due, in part, to our incomplete understanding of the nature of the initial physiological state of cells (defined as h0 or p0 depending on the model), and changes taking place during adaptation. The objectives of the present study were to characterize p0 using data from growth of Listeria monocytogenes in an automated turbidimetric instrument (Bioscreen), and to determine the influence of limiting growth pH. A model was developed for individual cells which combined a continuous adaptation phase (defined by p0) with a discrete step marking the transition to a continuous exponential growth phase (the CDC model). Parameters of the new model were: p0; the specific growth rate (mu); the initial cell number (N0); and the maximum cell density (Nmax). Progressive reduction of the growth pH in the Bioscreen to 4.7 decreased the p. It was noted that the regression lines for all trials at all pH values appeared to have a common x-intercept (20.086+/-1.092), and it was deduced that, when the Bioscreen detection limit (15.07 In cfu well(-1)) was subtracted, the resulting value represented the "true" value for the initial physiological state of the cells.

— Some of the factors influencing the production of extracellular proteinase by three strains of Pseudomonas fluorescens were studied. Tryptic Soy Broth supported good growth and enzyme production by all three strains. Poor enzyme yields resulted from growth in Nutrient Broth, Brain Heart Infusion and Peptone Yeast Extract. Addition of 0.2% (w/v) Skim Milk Powder to citrate mineral salts or Nutrient Broth stimulated enzyme production. Maximum yields of enzyme on mineral salts medium containing skim milk were obtained when pyruvate was the source of carbon; activity was low when glucose or citrate were used. Glutamine, when added as the sole carbon and/or nitrogen source, supported enzyme synthesis. At 5, 10, 15 and 20°C enzyme levels increased with increasing skim milk concentration. At 5°C enzyme activity was approximately 55% of that found at 20°C. A filter-sterilized or autoclaved high-speed centrifuged supernatant from skim milk strongly induced the proteinase. A small molecular weight (

Dithiothreitol reacted, at pH 9.0, with the isolated cell walls of Methanospirillum hungatii, to release about 23% of the cell wall dry weight as a high molecular weight fraction (> 0.5 million daltons). Untreated walls consisted of 70% amino acids, 11% lipid, and 6.6% carbohydrate. Sugars were identified as rhamnose, ribose, glucose, galactose, and mannose. The wall material that was released contained only 47% amino acids and was enriched in lipid, glucose, and phosphate. These results support data from electron micrographs, showing the localized release of cell wall material by the disulfide bond-breaking reagent at alkaline pH. In amino acid composition the untreated walls did not differ greatly from the material released by dithiothreitol, but differed considerably from the walls of another strain of M. hungatii. The ratios of the amino acids found in the cell wall proteins of several archaebacteria and of Bacillus cereus spore coats were similar.

Bifidobacterium adolescentis ATCC 15703, B. longum ATCC 15707, and B. thermophilum ATCC 25525 were examined for the ability to grow with fructo-oligosaccharides (FOS) as carbohydrate sources. The three species produced cell-associated β-fructosidases (inulinases) capable of hydrolysing FOS. Maximum activity was obtained with short-chain FOS with degrees of polymerization (DP) of between three and five (neosugars). The B. thermophilum inulinase was induced by inulin, a long-chain FOS with DP=35, while the enzymes from the other two strains were constitutive. Production of inulinase by all three strains was regulated by catabolite repression. Inulinase activity of the three Bifidobacterium spp. was similar when grown with 0.5% inulin as the carbohydrate source; however, B. thermophilum grew much more rapidly. All three strains utilized crude Jerusalem artichoke flour (JAF) as a carbohydrate source, suggesting that JAF might have commercial application as a food or feed additive to stimulate bifidobacteria in the gut.

A method of determining extracellular lipase activity from Pseudomonas fluorescens based on the hydrolysis of β-naphthyl caprylate has been developed. Optimum activity was determined as a function of temperature, pH, β-naphthyl caprylate and sodium taurocholate concentrations using a stepwise super-simplex optimization procedure. Optimization was performed with a Sharp PC-1500A computer and appropriate ranges for each experimental factor were determined by prior experimentation. A maximum A540 response of 0.38 units was obtained at 50°C, pH 7.2, 0.1 mM β-naphthyl caprylate and 17.6 mM sodium taurocholate. This response was 61% better than a previous response obtained by the one-factor-at-a-time method.

Heat treatment of raw milk in an HTST pasteurizer operated at 60.0 to 72.0°C for a minimum holding time of 16.2s rapidly inactivated mixtures of hemorrhagic Escherichia coli O157:H7, Yersinia enterocolitica and Campylobacter spp. ( C. fetus, C. coli , and C. jejuni ). Each of the three genera in the mixture was inoculated at a level of approximately 1.0×10 5 cfu/ml. At 60.0°C, hemorrhagic E. coli showed a maximum 2 log 10 reduction in counts and no viability at ≥64.5°C. Yersinia enterocolitica and Campylobacter spp. showed greater heat sensitivity with a 4 log 10 reduction in counts at 60.0°C and absence of viable cells at ≥63.0°C. These findings reiterate the need for stringent control of thermal processes in the manufacture of dairy products from raw or heat-treated (non-pasteurized) milk.

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-β- d -glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77°C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5′-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p -hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from Thiobacillus novellus was purified 54-fold. The preparation was, on the basis of densitometer scans of polyacrylamide gels, at least 85% pure. The optimum pH in the forward direction (2 ADP ⇌ATP+AMP) was about 8.7, and in the reverse 8.2 The enzyme was specific for AMP, ADP and ATP with apparent K m values of 0.04, 0.34 and 0.09 mM respectively. A double reciprocal plot of specific activity vs. (ADP)2 was linear. Both AMP and ATP inhibited the forward reaction with AMP the more inhibitory of the two. AMP inhibition was competitive with respect to ADP with a K i of 0.125 mM. Although Mg2+ was necessary for maximal activity, about 20% of this was obtained in its absence. Co2+ and Mn2+ at similar concentrations gave 46% and 26% respectively of the activity found with Mg2+. Apparent K m for Mg2+ was about 0.054 mM in the forward and 0.15 mM in the reverse direction. pHMB and HgCl2 were potent inhibitors. Inhibition by pHMB but not HgCl2 was reversible by GSH or cysteine. Arrhenius plots gave an E a of 3.25 kcal/mole/degree C in the forward direction without discontinuity. In the reverse, there was a “break” at 26.7°C with an E a of 3.62 kcal/mole/degree C for lower temperatures and 3.92 kcal/mole/degree C for higher temperatures.Molecular weights of the enzyme were 46,300±300 by SDS PAGE and 47,800±200 by SDS PAGE after treatment with 5 M urea and about 40,000 by sucrose density gradient centrifugation.

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH 4 Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.

The thermal resistance of Salmonella senftenberg 775W, Salmonella muenster previously isolated from raw fluid milk, and two mixtures each consisting of ten Salmonella strains commonly isolated from human or non-human sources was tested. Cells were suspended in whole milk at a final concentration of 105 cells/ml. The inoculated milk was thermally processed at temperatures ranging from 60°C to 74°C using a pilot-scale plate pasteurizer unit. The mean and minimum residence time of milk in the holding tube of the pasteurizer was 17.6 and 16.2 s, respectively. The maximum temperature at which viable salmonellae were detected in the human (61.5°C) and non-human (64.5°C) mixtures was considerably lower than that obtained with S. senftenberg 775 W (67.5°C). S. muenster failed to show any milk-adapted response and could not be recovered at temperatures greater than 63.0°C. Treatment at 63°C produced a 4 log10 or greater reduction in the number of viable Salmonella including the heat resistant S. senftenberg 775 W, and a minimum 2 log10 decrease at 60°C. These findings warrant caution in the use of subpasteurizing temperatures for thermal processing of fluid milk.

The influence of a shift in temperature from 20 to 32 degrees C on extracellular proteinase synthesis by Pseudomonas fluorescens B52 was examined. When cells actively synthesizing proteinase at 20 degrees C were shifted to 32 degrees C, enzyme synthesis ceased immediately. After 30 min at 32 degrees C, cells recovered at 20 degrees C after a lag of 30 min. Rifampin and chloramphenicol prevented recovery of synthesis at 20 degrees C. Rifampin-insensitive proteinase synthesis (an indirect measure of proteinase-specific mRNA pools) decreased after the exposure of cells to 32 degrees C for 30 min but was recovered during incubation at 20 degrees C. Controls not exposed to a temperature shift experienced no loss of rifampin-independent synthesis. Cells experienced a 50% reduction in mRNA pools after 15 min at 32 degrees C. The data support the working hypothesis that the loss of mRNA pools after treatment at 32 degrees C is responsible for the lag before the recovery of extracellular proteinase synthesis.

Strains of Methanospirillum hungatii, when treated with dithiothreitol at alkaline pH, formed spheroplasts which lysed in the absence of an osmotic stabilizer. Other methanogenic bacteria showed no response to this treatment, including Methanobacterium thermoautotrophicum. Methanobacterium strain M.o.H., Methanobacterium strain G2R, and Methanosarcina barkeri. The reaction with M. hungatii proceeded slowly at pH 8.0, but increased dramatically up to a pH of 10. Spheroplasts did not form in the presence of MgCl2 or if large amounts of FeS precipitate had been deposited on the cells during growth. Ultra-thin sections of M. hungatii GPI confirmed that individual cells, surrounded by a membrane and an inner wall, were contained within an outer wall or sheath. Adhesive material, which stained in the region of the cell spacer, was observed to bind together the inner and outer wall layers. After treatment with dithiothreitol, the spheroplasts, which retained the flexible inner wall and membrane, were released from the rigid outer wall. Examination of the isolated outer cell wall revealed that dithiothreitol (and dodecyl sodium sulfate) dissolved the adhesive material and damaged the cell spacer at the attachment site to the outer wall. It is proposed that dithiothreitol at alkaline pH loosens both the cell spacer and inner wall attachments to the outer wall, thereby allowing the osmotic pressure of the cytoplasm to eject the spheroplast. Resistance of the cells to lysis by Triton X-100 is lost upon spheroplast formation, indicating that protection to the cell is conferred by the outer cell wall.

A mutant of Pseudomonas fluorescens strain B52 deficient in the synthesis of the fluorescent pigment, pyoverdine, was isolated. Absence of pyoverdine and other siderophores was confirmed by gel filtration, a specific siderophore assay, and inhibition studies with the iron chelator EDDA. Both parent and mutant synthesized additional outer membrane proteins in response to iron-limitation. Mutant cells cultured in the absence of iron(III) accumulated 55Fe-labeled pyoverdine. The mutant produced extracellular proteinase normally on various media, but was deficient in lipase secretion. Growth of the mutant with partially-purified pyoverdine resulted in a 2.5-fold stimulation of lipase secretion. The mutant grew poorly in deferrated medium; however, the addition of iron(III) stimulated growth. Proteinase secretion in deferrated medium was stimulated over a narrow range of iron(III) concentration, while lipase secretion was only slightly affected. The data suggest that separate regulatory mechanisms exist for the control of proteinase and lipase secretion by iron(III).

Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined. Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A600 of 0.3–0.9) Resting suspensions of younger cells (A600

The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined. Synthesis was monitored using cells resuspended at high density in fresh growth medium. Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l). Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation. Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis. Sephadex G-100 chromatography of L-[4,5-3H]leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000–14000 daltons) irreversibly inactive ‘precursor’ of the proteinase was formed. The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.

Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenoiindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 μM for NADH and 0.2 μM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in this reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 °C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.

Heat treatment of raw milk in an HTST pasteurizer operated at 60.0 to 72.0 degrees C for a minimum holding time of 16.2 s rapidly inactivated mixtures of hemorrhagic Escherichia coli O157:H7, Yersinia enterocolitica and Campylobacter spp. (C. fetus, C. coli, and C. jejuni). Each of the three genera in the mixture was inoculated at a level of approximately 1.0 x 10(5) cfu/ml. At 60.0 degrees C, hemorrhagic E. coli showed a maximum 2 log10 reduction in counts and no viability at greater than or equal to 64.5 degrees C. Yersinia enterocolitica and Campylobacter spp. showed greater heat sensitivity with a 4 log10 reduction in counts at 60.0 degrees C and absence of viable cells at greater than or equal to 63.0 degrees C. These findings reiterate the need for stringent control of thermal processes in the manufacture of dairy products from raw or heat-treated (non-pasteurized) milk.

Raw whole milk inoculated with 10(5) CFU/ml of Listeria monocytogenes was thermally processed at 60-72 degrees C for a minimum holding time of 16.2 s with survival being observed at temperatures up to 67.5 degrees C. In addition, milk naturally contaminated with L. monocytogenes serotype 1 (around 10(4) CFU/ml) was pooled for 2 to 2.5 days and then run through an HTST pasteurizer at temperatures ranging from 60-78 degrees C. Viable L. monocytogenes were detected in the temperature range of 60-66 degrees C. No viable Listeria were detected after treatment at temperatures of 69 degrees C and above in any of five trials. Efficacy of pasteurization and widespread use of processing conditions well above the minimum HTST guidelines ensure the absence of Listeria in pasteurized milk products. However, survival of Listeria at sub-pasteurization temperatures (60-67.5 degrees C) is of concern with regard to heat-treated or raw-milk cheeses.

A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, α-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by α-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. α-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to α-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of initial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was preferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells with respect to molecular weight (61 700) and reaction mechanism, but may be regulated differently.