Microbially-influenced corrosion: damage to prostheses, delight for bacteria.

In natural and man-made environments, microbial communities thrive as biofilms on living (e.g. tissue) and inanimate (e.g. plastic, metal, wood, mineral) surfaces. Biofilms are found in a wide range of aqueous habitats, including physiological fluids. Numerous types of microorganisms are able to colonize catheters, implants, prosthetics, and other medical devices manufactured from different metallic and non-metallic materials dwelling within a human body. The development of biofilm is facilitated by the production of extracellular polymeric substances (EPS). Biofilms formed on surfaces of metallic materials may alter interfacial electrochemical processes, which can lead to increased corrosion of the colonized substratum. Deterioration of metallic materials in the presence of a biofilm is termed biocorrosion or microbially-influenced corrosion (MIC). In the field of biomaterials, ""biocorrosion"" is commonly used when describing the effect of host tissue on the corrosion of implant metals and alloys. Therefore, to avoid confusion, we will here use the term MIC as a reference to biofilm-influenced corrosion. It is important to realise that although most metals are prone to microbial colonization, i.e. to biofouling, this does not imply that they are susceptible to MIC. For example, a metal such as titanium, accumulates biofilm, however, it still demonstrates excellent resistance against MIC. Corrosion is, by definition, an electrochemical process, therefore, electrochemical techniques are frequently employed to determine and measure the rate of abiotic, as well as biologically driven corrosion reactions. This communication addresses the use of electrochemical techniques for monitoring (i) biofilm formation on and (ii) MIC of implant metals and alloys.

The effect of super-oxidized water on Escherichia coli.

The mechanism of action of Sterilox, a non-toxic liquid biocide produced by electrolysis of a dilute saline solution, upon planktonic cells of Escherichia coli JM109 was investigated using protein and nucleic acid analysis. The results revealed total destruction of chromosomal and plasmid DNA, RNA and proteins of E. coli within 5 min of exposure. Our earlier investigation conducted using atomic force microscopy imaging revealed swelling and rupture of E. coli cells with release of cytoplasm. We propose that the biocidal properties of Sterilox are due to its effect upon constituents of the bacterial cell including proteins and nucleic acids.

The effect of Pseudomonas NCIMB 2021 biofilm on AISI 316 stainless steel.

A bioreactor system operating in a continuous mode was designed to generate biofilms on polished and as-received surfaces of AISI 316 stainless steel coupons exposed for 36 d to a pure culture of marine Pseudomonas NCIMB 2021. Scanning electron microscopy (SEM) and atomic force microscopy were employed to determine the degree of surface colonisation and to examine corrosion damage of the steel. X-ray photoelectron spectroscopy analysis was carried out to characterise the chemistry of the passive layers on polished steel stored for a period of time, freshly re-polished coupons, and as-received steel. The effect of biofilms on the composition of layers formed on the steel specimens was evaluated. SEM revealed that the surfaces of polished and stored steel appeared to accumulate more biofilm compared to as-received specimens. Micropitting of steel occurred underneath the biofilm, regardless of surface finish. The concentration of elements in the passive layers differed significantly between freshly re-polished and as-received or polished and stored coupons. In the presence of Pseudomonas NCIMB 2021 biofilm, the composition of the passive layer on the as-received steel surface was considerably altered compared to unexposed steel or steel exposed to abiotic medium.

The effect of extracellular polymeric substances on the attachment of Pseudomonas NCIMB 2021 to AISI 304 and 316 stainless steel.

Surfaces of AISI 304 and 316 stainless steels were pre-treated with three different types of extracellular polymeric substances, viz. (i) exopolymers released into the culture medium ("free"; or planktonic exopolymers), (ii) capsular exopolymers, and (iii) biofilm exopolymers, produced by continuous cultures of marine Pseudomonas NCIMB 2021. The initial attachment of Pseudomonas cells to exopolymer-conditioned steel surfaces varied with the exopolymer type and concentration. Results gained from wettability studies of exopolymer-treated steel using contact angle measurements, as well as from the surface roughness measurements conducted employing atomic force microscopy analysis, could not account for the observed, statistically significant differences (p < 0.1) in the level of bacterial surface colonisation. It is therefore proposed that neither surface hydrophobicity nor roughness play an important part in the early attachment of Pseudomonas NCIMB 2021 to the conditioned steel surfaces and that a difference in the chemistry of the exopolymers is most likely a key parameter influencing initial cell adhesion to pre-treated steel.

Study of the interaction of sulphate-reducing bacteria exopolymers with iron using X-ray photoelectron spectroscopy and time-of-flight secondary ionisation mass spectrometry.

Time-of-flight secondary ionisation mass spectrometry and X-ray photoelectron spectroscopy were employed to determine the interaction of crude extracellular polymeric substances recovered from static batch cultures of two isolates of marine sulphate-reducing bacteria of the genus Desulfovibrio, grown in the presence of and without mild steel surfaces, with Fe ions released from steel. The results demonstrated that exopolymers synthesised by different strains of sulphate-reducers varied in their ability to bind iron originating from steel. Based on the X-ray photoelectron spectroscopy analysis it is proposed that Fe released from steel was associated with bacterial exopolymers such as Fe(III) ion. The application of surface science techniques to study exopolymer/metal interaction allowed quantitative evaluation of Fe binding using small sample size.

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001.

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars--possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924-930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

Isolation and characterisation of a novel sulphate-reducing bacterium of the Desulfovibrio genus.

A novel sulphate-reducing bacterium (Ind 1) was isolated from a biofilm removed from a severely corroded carbon steel structure in a marine environment. Light microscopy observations revealed that cells were Gram-negative, rod shaped and very motile. Partial 16S rRNA gene sequencing and analysis of the fatty acid profile demonstrated a strong similarity between the new species and members from the Desulfovibrio genus. This was confirmed by the results obtained following purification and characterisation of the key proteins involved in the sulphate-reduction pathway. Several metal-containing proteins, such as two periplasmic proteins: hydrogenase and cytochrome c3, and two cytoplasmic proteins: ferredoxin and sulphite reductase, were isolated and purified. The latter proved to be of the desulfoviridin type which is typical of the Desulfovibrio genus. The study of the remaining proteins revealed a high degree of similarity with the homologous proteins isolated from Desulfovibrio gigas. However, the position of the strain within the phylogenetic tree clearly indicates that the bacterium is closely related to Desulfovibrio gabonensis, and these three strains form a separate cluster in the delta subdivision of the Proteobacteria. On the basis of the results obtained, it is suggested that Ind 1 belongs to a new species of the genus Desulfovibrio, and the name Desulfovibrio indonensis is proposed.

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy.

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.

Short Communication: Lipopolysaccharide composition of Desulfovibrio cell wall.

The lipopolysaccharide of the sulphate-reducing bacterium Desulfovibrio desulfuricans was analysed by GC, combined either with flame-ionization detection or with MS, and by standard chemical tests. The major sugar of the polysaccharide portion of the macromolecule was glucose (56%). Low amounts of mannose, galactose, rhamnose and amino sugar were also found. Six fatty acids were identified in the lipid A fraction: 9-octadecenoic, tetracosenoic, heptadecenoic, 10-octadecenoic, eicosenoic and 8-octadecynoic.