Biodiversity analysis by polyphasic study of marine bacteria associated with biocorrosion phenomena

Aims: A polyphasic approach was used to study the biodiversity bacteria associated with biocorrosion processes, in particular sulfate‐reducing bacteria (SRB) and thiosulfate‐reducing bacteria (TRB) which are described to be particularly aggressive towards metallic materials, notably via hydrogen sulfide release. Methods and Results: To study this particular flora, an infrared spectra library of 22 SRB and TRB collection strains were created using a Common Minimum Medium (CMM) developed during this study and standardized culture conditions. The CMM proved its ability to allow for growth of both SRB and TRB strains. These sulfurogen collection strains were clearly discriminated and differentiated at the genus level by fourier transform infrared (FT‐IR) spectroscopy. In a second step, infrared spectra of isolates, recovered from biofilms formed on carbon steel coupons immersed for 1 year in three different French harbour areas, were compared to the infrared reference spectra library. In parallel, molecular methods (M13‐PCR and 16S rRNA gene sequencing) were used to qualitatively evaluate the intra‐ and inter‐species genetic diversity of biofilm isolates. The biodiversity study indicated that strains belonging to the Vibrio genus were the dominant population; strains belonging to the Desulfovibrio genus (SRB) and Peptostreptococcaceae were also identified. Conclusion: Overall, the combination of the FT‐IR spectroscopy and molecular approaches allowed for the taxonomic and ecological study of a bacterial flora, cultivated on CMM, associated with microbiology‐induced corrosion (MIC) processes. Significance and Impact of the Study: Via the use of the CMM medium, the culture of marine bacteria (including both SRB and TRB bacteria) was allowed, and the implication of nonsulforogen bacteria in MIC was observed. Their involvement in the biocorrosion phenomena will have to be studied and taken into account in the future.     

Microbiologically influenced corrosion: looking to the future.

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).     

Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system

District heating systems (DHS) are extreme aqueous environments characterized by high temperatures, high pH (9.5-10.0), and low nutrient availability. Culture-independent and culture-dependent techniques showed that DHS may nevertheless harbour geno- and phenotypically diverse bacterial biofilm communities. Approximately 50% of the cells in biofilms growing on mild steel coupons in rotortorque reactors connected to the return line (40 degrees C) of a Danish DHS were detectable by FISH analysis and thus were probably metabolically active. A bacterial 16S rRNA gene clone library generated from the biofilms was dominated by proteobacterial phylotypes (closely related to known aerobic species) and by phylotypes affiliated to the anaerobic class Clostridia. Anoxic enrichment cultures derived from biofilms primarily contained 16S rRNA gene and dsrAB (encoding major subunits of dissimilatory sulfite reductase) phylotypes affiliated to the latter class. Alkalitolerant and neutrophilic anaerobic bacteria were isolated from the DHS, including novel Gram-positive and deltaproteobacterial sulfate-reducers and sulfite-reducers constituting novel Gram-positive lineages. In total, 39 distinct 16S rRNA gene phylotypes representing ten classes were identified. The detection of several alkalitolerant, sulfide-producing, and, thus, potentially biocorrosive species underlines the need to maintain a high water quality in the DHS in order to prevent the proliferation of these species.     

Phylogenetic characterization of a corrosive consortium isolated from a sour gas pipeline

Biocorrosion is a common problem in oil and gas industry facilities. Characterization of the microbial populations responsible for biocorrosion and the interactions between different microorganisms with metallic surfaces is required in order to implement efficient monitoring and control strategies. Denaturing gradient gel electrophoresis (DGGE) analysis was used to separate PCR products and sequence analysis revealed the bacterial composition of a consortium obtained from a sour gas pipeline in the Gulf of Mexico. Only one species of sulfate-reducing bacteria (SRB) was detected in this consortium. The rest of the population consisted of enteric bacteria with different characteristics and metabolic capabilities potentially related to biocorrosion. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. The low abundance of the detected SRB was evidenced by environmental scanning electron microscopy (ESEM). In addition, the localized corrosion of pipeline steel in the presence of the consortium was clearly observed by ESEM after removing the adhered bacteria.     

Marine sulfate-reducing bacteria cause serious corrosion of iron under electroconductive biogenic mineral crust

Iron (Fe(0) ) corrosion in anoxic environments (e.g. inside pipelines), a process entailing considerable economic costs, is largely influenced by microorganisms, in particular sulfate-reducing bacteria (SRB). The process is characterized by formation of black crusts and metal pitting. The mechanism is usually explained by the corrosiveness of formed H(2) S, and scavenge of 'cathodic' H(2) from chemical reaction of Fe(0) with H(2) O. Here we studied peculiar marine SRB that grew lithotrophically with metallic iron as the only electron donor. They degraded up to 72% of iron coupons (10?mm?×?10?mm?×?1?mm) within five months, which is a technologically highly relevant corrosion rate (0.7?mm?Fe(0) year(-1) ), while conventional H(2) -scavenging control strains were not corrosive. The black, hard mineral crust (FeS, FeCO(3) , Mg/CaCO(3) ) deposited on the corroding metal exhibited electrical conductivity (50?S?m(-1) ). This was sufficient to explain the corrosion rate by electron flow from the metal (4Fe(0) →?4Fe(2+) +?8e(-) ) through semiconductive sulfides to the crust-colonizing cells reducing sulfate (8e(-) +?SO(4) (2-) +?9H(+) →?HS(-) +?4H(2) O). Hence, anaerobic microbial iron corrosion obviously bypasses H(2) rather than depends on it. SRB with such corrosive potential were revealed at naturally high numbers at a coastal marine sediment site. Iron coupons buried there were corroded and covered by the characteristic mineral crust. It is speculated that anaerobic biocorrosion is due to the promiscuous use of an ecophysiologically relevant catabolic trait for uptake of external electrons from abiotic or biotic sources in sediments.     

Microbial communities in bulk fluids and biofilms of an oil facility have similar composition but different structure

The oil-water-gas environments of oil production facilities harbour abundant and diverse microbial communities that can participate in deleterious processes such as biocorrosion. Several molecular methods, including pyrosequencing of 16S rRNA libraries, were used to characterize the microbial communities from an oil production facility on the Alaskan North Slope. The communities in produced water and a sample from a 'pig envelope' were compared in order to identify specific populations or communities associated with biocorrosion. The 'pigs' are used for physical mitigation of pipeline corrosion and fouling and the samples are enriched in surface-associated solids (i.e. paraffins, minerals and biofilm) and coincidentally, microorganisms (over 10(5) -fold). Throughout the oil production facility, bacteria were more abundant (10- to 150-fold) than archaea, with thermophilic members of the phyla Firmicutes (Thermoanaerobacter and Thermacetogenium) and Synergistes (Thermovirga) dominating the community. However, the structure (relative abundances of taxa) of the microbial community in the pig envelope was distinct due to the increased relative abundances of the genera Thermacetogenium and Thermovirga. The data presented here suggest that bulk fluid is representative of the biofilm communities associated with biocorrosion but that certain populations are more abundant in biofilms, which should be the focus of monitoring and mitigation strategies.     

Bacterial corrosion of iron in seawater in situ, and in aerobic and anaerobic model systems

Abstract A screening of twenty-two marine isolates was made to examine their effects on corrosion of carbon steel ASTM A619. In batch cultures, sixteen of the isolates gave a lower corrosion than the control. Aerobic and anaerobic biofilm populations were formed by immersing iron coupons in natural seawater under aerobic and anaerobic conditions. The effects of the biofilms depended on a balance between the presence of oxygen and the type of population. An anaerobic population attached to the surface increased the corrosion rate if immersed in a suspension of Vibrio sp. DW1. The vibrio population probably 'protected' the anaerobic population from oxygen and may have provided nutrients, thereby creating conditions that allowed production of corrosive metabolites close to the metal. In contrast, coupons without a biofilm showed a decrease in the corrosion when immersed in the same vibrio suspension. The protective effect of a dense suspension of bacteria found earlier [5,6] was tested in situ in seawater. Iron coupons were immersed in dialysis bags with a suspension of Vibrio sp. DW1. Coupons immersed in dialysis bags with DW1 showed a lower degree of corrosion than coupons immersed in bags with seawater.     

Nitrate treatment effects on bacterial community biofilm formed on carbon steel in produced water stirred tank bioreactor

To better understand the impact of nitrate in Brazilian oil reservoirs under souring processes and corrosion, the goal of this study was to analyse the effect of nitrate on bacterial biofilms formed on carbon steel coupons using reactors containing produced water from a Brazilian oil platform. Three independent experiments were carried out (E1, E2 and E3) using the same experimental conditions and different incubation times (5, 45 and 80 days, respectively). In every experiment, two biofilm-reactors were operated: one was treated with continuous nitrate flow (N reactor), and the other was a control reactor without nitrate (C reactor). A Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis approach using the 16S rRNA gene was performed to compare the bacterial groups involved in biofilm formation in the N and C reactors. DGGE profiles showed remarkable changes in community structure only in experiments E2 and E3. Five bands extracted from the gel that represented the predominant bacterial groups were identified as Bacillus aquimaris, B. licheniformis, Marinobacter sp., Stenotrophomonas maltophilia and Thioclava sp. A reduction in the sulfate-reducing bacteria (SRB) most probable number counts was observed only during the longer nitrate treatment (E3). Carbon steel coupons used for biofilm formation had a slightly higher weight loss in N reactors in all experiments. When the coupon surfaces were analysed by scanning electron microscopy, an increase in corrosion was observed in the N reactors compared with the C reactors. In conclusion, nitrate reduced the viable SRB counts. Nevertheless, the nitrate dosing increased the pitting of coupons.     

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.     

Study of parameters implicated in the biodeterioration of mild steel in the presence of different species of sulphate-reducing bacteria

Two different species of sulphate-reducing bacteria, strain classified by NCIMB as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (8313) isolated from the corroding heat exchanger, and SRB species recovered from a corroding ship hull anchored off the Indonesian coast (Indo isolate) were grown as laboratory batch cultures. Several factors such as the surface finish of substratum, metabolic activity of planktonic and sessile bacterial populations, initial attachment of cells to surfaces and subsequent formation of biofilms on the process of biodeterioration of mild steel in the presence of these two different species of SRB were investigated. The corrosion rates of mild steel were estimated by weight loss measurements and correlated with the density of sessile SRB population. The yield and composition of extracellular polymers released into the bulk phase of culture media were determined and the amount of dissolved hydrogen sulphide was monitored. The results revealed differences between SRB species in their aggressiveness towards mild steel under identical growth conditions, emphasising the importance of biochemistry and physiology of SRB for the biocorrosion process. Biochemical and genetic characterisation of SRB isolates chosen for this study are currently in progress.     

Microbial activity in drinking water-associated biofilms

Microbial biofilms from surfaces in contact with water may play a beneficial role in drinking water treatment as biological filters. However, detrimental effects such as biofouling (i.e., biocorrosion and water quality deterioration) may also occur. In this study microbiological processes and factors influencing the activity of bacteria in biofilms were investigated by conventional cultivation methods. The presence of bacteria belonging to different ecophysiological groups was assessed during drinking water treatment, in biofilms developed on concrete, steel and sand surfaces. Influences of the treatment process, type of immersed material and physico-chemical characteristics of raw/bulk water and biofilms upon the dynamics of bacterial communities were evaluated. Results revealed intense microbial activity in biofilms occurring in the drinking water treatment plant of Cluj. Ammonification, iron reduction and manganese oxidation were found to be the predominant processes. Multiple significant correlations were established between the evolution of biofilm bacteria and the physico-chemical parameters of raw/ bulk water. The type of immersed material proved to have no significant influence upon the evolution of microbial communities, but the treatment stage, suggesting that the processes applied restrict microbial growth not only in bulk fluid but in biofilms, too.     

Sulfato/thiosulfato reducing bacteria characterization by FT-IR spectroscopy: a new approach to biocorrosion control

Sulfato and Thiosulfato Reducing Bacteria (SRB, TRB) can induce corrosion process on steel immersed in seawater. This phenomenon, called biocorrosion, costs approximatively 5 billion euros in France each year. We provide the first evidence that Fourier Transformed InfraRed (FTIR) spectroscopy is a competitive technique to evaluate the sulfurogen flora involved in biocorrosion in comparison with time consuming classical identification methods or PCR analyses. A great discrimination was obtained between SRB, TRB and some contamination bacteria known to be present in seawater and seem to be able to reduce sulfate under particular conditions. Moreover, this preliminary study demonstrates that FTIR spectroscopic and genotypic results present a good correlation (these results are confirmed by other data obtained before or later, data not shown here). The advantages gained by FTIR spectroscopy are to give information on strain phenotype and bacterial metabolism which are of great importance in corrosion processes.     

The potential use of atomic force microscopy for studying corrosion of metals in the presence of bacterial biofilms — an overview

This communication outlines the principles of application of scanning probe microscopy (SPM) as a tool for studying physico-chemical and biological phenomena and discusses the potential use of atomic force microscopy (AFM) , a form of SPM, for investigation of bacterial biofilms formed on metal surfaces and for studying corrosion of these surfaces in the presence of such biofilms. AFM images showing biofilms developed in pure cultures of either Pseudomonas species on copper, or by a marine isolate of sulphate-reducing bacterium on 304 stainless steel are presented to demonstrate usefulness of the SPM technique for both quantitative and qualitative determination of biocorrosion.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

Interaction of Desulfovibrio desulfuricans biofilms with stainless steel surface and its impact on bacterial metabolism

AIMS: To study the influence of some metallic elements of stainless steel 304 (SS 304) on the development and activity of a sulfate-reducing bacterial biofilm, using as comparison a reference nonmetallic material polymethylmethacrylate (PMMA). METHODS AND RESULTS: Desulfovibrio desulfuricans biofilms were developed on SS 304 and on a reference nonmetallic material, PMMA, in a flow cell system. Steady-state biofilms were metabolically more active on SS 304 than on PMMA. Activity tests with bacteria from both biofilms at steady state also showed that the doubling time was lower for bacteria from SS 304 biofilms. The influence of chromium and nickel, elements of SS 304 composition, was also tested on a cellular suspension of Des. desulfuricans. Nickel decreased the bacterial doubling time, while chromium had no significant effect. CONCLUSIONS: The following mechanism is hypothesized: a Des. desulfuricans biofilm grown on a SS 304 surface in anaerobic conditions leads to the weakening of the metal passive layer and to the dissolution in the bulk phase of nickel ions that have a positive influence on the sulfate-reducing bacteria metabolism. This phenomenon may enhance the biocorrosion process. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the interactions between metallic surfaces such as stainless steel and bacteria commonly implied in the corrosion phenomena which is primordial to fight biocorrosion.     

Selected chitinase genes in cultured and uncultured marine bacteria in the alpha- and gamma-subclasses of the proteobacteria

PCR primers were patterned after chitinase genes in four gamma-proteobacteria in the families Alteromonadaceae and Enterobacteriaceae (group I chitinases) and used to explore the occurrence and diversity of these chitinase genes in cultured and uncultured marine bacteria. The PCR results from 104 bacterial strains indicated that this type of chitinase gene occurs in two major groups of marine bacteria, alpha- and gamma-proteobacteria, but not the Cytophaga-Flavobacter group. Group I chitinase genes also occur in some viruses infecting arthropods. Phylogenetic analysis indicated that similar group I chitinase genes occur in taxonomically related bacteria. However, the overall phylogeny of chitinase genes did not correspond to the phylogeny of 16S rRNA genes, possibly due to lateral transfer of chitinase genes between groups of bacteria, but other mechanisms, such as gene duplication, cannot be ruled out. Clone libraries of chitinase gene fragments amplified from coastal Pacific Ocean and estuarine Delaware Bay bacterioplankton revealed similarities and differences between cultured and uncultured bacteria. We had hypothesized that cultured and uncultured chitin-degrading bacteria would be very different, but in fact, clones having nucleotide sequences identical to those of chitinase genes of cultured alpha-proteobacteria dominated both libraries. The other clones were similar but not identical to genes in cultured gamma-proteobacteria, including vibrios and alteromonads. Our results suggest that a closer examination of chitin degradation by alpha-proteobacteria will lead to a better understanding of chitin degradation in the ocean.     

Identification of Bacterial Strains Isolated from the Mediterranean Sea Exhibiting Different Abilities of Biofilm Formation

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.     

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor was investigated over a 10-month period in a hotel. Planktonic and sessile numbers of sulphate reducing bacteria (SRB) and heterotrophic bacteria were monitored. The corrosion rate was determined by the weight loss method. The corrosion products were analyzed by energy dispersive X-ray spectroscopy and X-ray diffraction. A mineralized, heterogeneous biofilm was observed on the coupons. Although a biocide and a corrosion inhibitor were regularly added to the cooling water, the results showed that microorganisms, such as SRB in the mixed species biofilm, caused corrosion of galvanized steel. It was observed that Zn layers on the test coupons were completely depleted after 3?months. The Fe concentrations in the biofilm showed significant correlations with the weight loss and carbohydrate concentration (respectively, p?<?0.01 and p?<?0.01).     

Bacterial community structure, compartmentalization and activity in a microbial fuel cell

AIMS: To characterize bacterial populations and their activities within a microbial fuel cell (MFC), using cultivation-independent and cultivation approaches. METHODS AND RESULTS: Electron microscopic observations showed that the fuel cell electrode had a microbial biofilm attached to its surface with loosely associated microbial clumps. Bacterial 16S rRNA gene libraries were constructed and analysed from each of four compartments within the fuel cell: the planktonic community; the membrane biofilm; bacterial clumps (BC) and the anode biofilm. Results showed that the bacterial community structure varied significantly between these compartments. It was observed that Gammaproteobacteria phylotypes were present at higher numbers within libraries from the BC and electrode biofilm compared with other parts of the fuel cell. Community structure of the MFC determined by analyses of bacterial 16S rRNA gene libraries and anaerobic cultivation showed excellent agreement with community profiles from denaturing gradient gel electrophoresis (DGGE) analysis. CONCLUSIONS: Members of the family Enterobacteriaceae, such as Klebsiella sp. and Enterobacter sp. and other Gammaproteobacteria with Fe(III)-reducing and electrochemical activity had a significant potential for energy generation in this system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that electrochemically active bacteria can be enriched using an electrochemical fuel cell.