Stainless steel mesh supports high density cell growth and production of recombinant Mullerian Inhibiting Substances

Summary Stainless steel mesh supported the high density growth of anchorage dependent CHO fibroblasts without the use of a special culture system. CHO cells, designated B-9, containing an amplified genomic construct of the human gene for Mullerian Inhibiting Substance (MIS), grew to a high confluent density on stainless steel meshwork while producing substantial amounts of human recombinant MIS over a long period of time. The mesh could be easily coated with various extracellular matrix proteins, such as Laminin, Fibronectin, Collagen or Matrigel, which permitted the testing of the effects of surface modifications on cell yield and recombinant protein production. Since the amount of medium per surface area required for optimal cell growth is lower than for some large volume cell culture methods, media costs can be reduced using mesh. In addition, no special cell culture equipment or complex manipulations are required. Thus, the use of meshwork for anchorage-dependent cells can increase the efficiency of growth and decrease the cost of recombinant protein production. This work is supported by NIH grant CA 17393 and American Cancer Society grant PDT 221A to P. K. D. and NIH grant EY 06535 to J. E. Editor's Statement This approach to large scale, high density cultivation of cells, one of several which are based on increasing surface area of the cultures, allows the production of large amounts of recombinant product within a research laboratory with modest bulk culture capability.     

Effects of selection and fate of substrates supplied to anaerobic bacteria involved in the corrosion of pipe-line steel

Summary The corrosion of AISI C1020 carbon steel in an anoxic, marine, sulphide-containing environment was examined as a function of bacterial physiology and consortial complexity. The carbon steel was exposed to three organism;Eubacterium limosum, Desulfovibrio sp. andDesulfobacter sp. which were provided with H₂/CO₂, butanol, glucose, and acetate as carbon and electron sources. A consortium of these bacteria utilizing hydrogen gave rise to relatively high corrosion rates (5.7×10^–4 mhos cm^–2) with respect to corrosion resulting from bacteria supplied with organic electron sources (0.6–1.6×10^–4 mhos cm^–2). Disproportionation of electrons between sulphate reduction and fermentation had a significant effect on the corrosion rate in the case ofDesulfovibrio. Surface examination using scanning electron microscopy coupled with electrochemical impedance spectroscopy supported the hypothesis that the corrosion rate was controlled by the relative intactness of a ferrous sulphide film in which the bacteria were embedded.     

Physico-chemical and hygienic property modifications of stainless steel surfaces induced by conditioning with food and detergent

The effect of repeated conditioning procedures (25 runs), consisting of soiling (milk and meat products) and cleaning steps, on the hygienic status, physico-chemical properties and surface chemical composition of stainless steel (SS) surfaces, was investigated. Five SSs differing in grade and finish were used. Both soiling and surface cleaning/conditioning procedures resulted in a similar increase in the surface contamination with carbon, while the changes in the basic component of the surface free energy depended on the conditioning procedure. The passive film was also affected, the Fe/Cr ratio in particular. The hygienic status was also changed, especially with milk as shown by monitoring the number of residual adhering Bacillus cereus spores after contaminating the surface with spores followed by cleaning. The results show that in food environments, the presence and the nature of conditioning molecules play a major role in the hygienic status of SS surfaces.     

Demonstration of biofilm removal from type 304 stainless steel using pulsed-waveform electropolishing

Abstract

This article describes an electrochemical method to remove bacterial biofilm from a stainless steel (SS) surface using a potential pulse/reverse pulse technique. This technique employs a periodic waveform that consists of anodic and cathodic pulses. The pulses can effectively strip a thin layer of metal off the SS surface, along with the adherent biofilm, in a saline solution. Not only can the pulses effectively remove biofilm from the SS surface, but they also regenerate the original mirror-like shiny surface. The importance of this electrochemical biofilm removal method is its wide applicability for any types of biofilms. That is, instead of directly removing the biofilm, it removes a very thin layer of the metal under the biofilm. Thus, the removal process is independent to the nature of the biofilms. Furthermore, this electrochemical biofilm removal method is rapid (less than 30?s of potential pulse time) and does not require hazardous chemicals.     

Development of silver-containing austenite antibacterial stainless steels for biomedical applications Part I: microstructure characteristics,mechanical properties and antibacterial mechanisms

The as-quenched (AQ) microstructure of the Ag-containing alloys was found to be essentially a mixture of austenite (γ) and Ag phases. The Ag phase precipitates had a face-centered-cubic structure and lattice parameter a = 4.09 Å. When the alloy contained Ag ≥0.2 wt%, the mechanical properties were slightly enhanced because of the precipitate strengthening by the Ag phase precipitates. Moreover, the Ag-containing alloys exhibited ductile fracture after tensile testing. The results of an antibacterial test revealed that the Ag phase precipitates play a key role in the antibacterial mechanism of Ag-containing alloys: Ag⁺ ions released from the Ag phase precipitates can kill bacteria. It is suggested that as AISI 316L alloy has an Ag content ≥0.2 wt%, it will have excellent antibacterial properties against both Staphylococcus aureus and Escherichia coli, with an antibacterial rate of nearly 100%.     

Analysis of Microfouling Products Formed on Metallic Surfaces Exposed in a Marine Environment

Mild steel (MS), stainless steel (SS) and copper (Cu) test panels were immersed in the surface water of Dona Paula Bay over a period of 15 d. During the immersion period data on the hydrography, nutrients and suspended matter were also collected. The suspended matter and fouling products on the MS, SS and Cu panels were analysed for organic carbon (OC), organic nitrogen (ON), chlorophyll a (chl a), protein and carbohydrate concentration and composition, and the dry weight (DW) was recorded. Compared to suspended matter, the chemical and biochemical components of the fouling products showed strong temporal and substratum related differences. The microfouling biomass (as DW, OC, ON, chl a and protein) on all the test panels generally increased over the period of immersion. Carbohydrates were more abundant in the suspended matter whereas fouling products were enriched in proteins. The contribution of protein-carbon to the total carbon increased over the period of immersion for the microfouling products on MS and SS whilst it did not show a consistent trend on Cu. Whereas, the carbohydrate-carbon contribution to the total carbon increased for the fouling products on MS, it did not exhibit a particular pattern on SS or Cu over the period of immersion. Capillary gas chromatographic analysis showed the presence of glucose, galactose, mannose, arabinose, xylose, fucose and ribose in both the fouling products and suspended matter. However, there were differences in the relative distribution of these monosaccharides in the suspended matter and the fouling products. Glucose was the most abundant monosaccharide, which showed strong temporal variations in suspended matter. In contrast, the wt % concentrations of individual monosaccharides showed large temporal differences for the fouling products, which were strongly influenced by the period of immersion and the type of test substratum. Glucose and fucose were relatively more abundant in the fouling products on SS and Cu, whilst glucose was the most abundant monosaccharide on MS. The monosaccharide and chemical composition data suggest strong temporal changes in the composition of the fouling products.     

The use of biocides to control sulphate‐reducing bacteria in biofilms on mild steel surfaces

Three different types of biocides, viz. formaldehyde (FM), glutaraldehyde (GA) and isothiozolone (ITZ) were used to control planktonic and sessile populations of two marine isolates of sulphate‐reducing bacteria (SRB). The influence of these biocides on the initial attachment of cells to mild steel surfaces, on subsequent biofilm formation and on the activity of hydrogenase enzymes within developed biofilms was evaluated. In the presence of biocides the rate and degree of colonization of mild steel by SRB depended on incubation time, bacterial isolate and the type of biocide used. Although SRB differed in their susceptibility to biocides, for all isolates the biofilm population was more resistant to the treatment than the planktonic population. GA showed highest efficiency in controlling planktonic and sessile SRB compared with the other two biocides. The activity of the enzyme hydrogenase measured in SRB biofilms varied between isolates and with the biocide treatment. No correlation was found between the number of sessile cells and hydrogenase activity.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.     

Metal corrosion by aerobic bacteria isolated from stimulated corrosion systems: Effects of additional nitrate sources

Many microorganisms are reported to influence the corrosive behaviour of mild steel and stainless steel in different habitats. In this study, 40 bacterial strains were isolated from corroded mild steel and stainless steel coupons in the nitrate supplemented environments. The corrosion abilities of the isolates against the mild steel and stainless steel coupons were tested with or without additional nitrate sources. The presence of bacterial isolates alone stimulated the corrosion of mild steel coupons. Most of the bio-corrosion processes of mild steel coupons were mitigated by adding nitrate supplement with bacterial isolates. The effects of bacterial isolates and additional nitrogen sources on corrosion of stainless steels were varied. Not all bacterial isolates stimulated the corrosion on stainless steel during the study period. Unlike the effects on mild steel coupons, additional NaNO₃ might stimulate, retard the corrosion rate by the bacterial isolates or have limited effects. Similar results were obtained when NH₄NO₃ was used. Phylogenetic analysis demonstrated that all isolates were closely related. The majority of the bacterial isolates from corroded metal coupons were identified as Bacillus species. Others were identified as Pseudomonas sp., Marinobacter sp., and Halomonas species. The results prove that the isolated aerobic microorganisms do play a role in the corrosion process of stainless and mild steel. Adding additional nitrate sources might be a tool to mitigate corrosion of mild steel which was stimulated by the presence of bacteria. However, to prevent the corrosion of stainless steels, it might need a trial and errors approach in each case.