Characterization of corrosive bacterial consortia isolated from petroleum-product-transporting pipelines

Microbiologically influenced corrosion is a problem commonly encountered in facilities in the oil and gas industries. The present study describes bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, using traditional cultivation technique and 16S rDNA gene sequencing. Phylogenetic analysis of 16S rRNA sequences of the isolates was carried out, and the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species namely Serratia marcescens ACE2, Bacillus subtilis AR12, Bacillus cereus ACE4, Pseudomonas aeruginosa AI1, Klebsiella oxytoca ACP, Pseudomonas stutzeri AP2, Bacillus litoralis AN1, Bacillus sp., Bacillus pumilus AR2, Bacillus carboniphilus AR3, and Bacillus megaterium AR4. Sulfate-reducing bacteria were not detected in samples from both pipelines. The dominant bacterial species identified in the petroleum pipeline samples were B. cereus and S. marcescens in the diesel and naphtha pipelines, respectively. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. In addition, localized (pitting) corrosion of the pipeline steel in the presence of the consortia was observed by scanning electron microscopy analysis. The potential role of each species in biofilm formation and steel corrosion is discussed.     

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non‐heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%–42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.     

Effect of substrate type on bacterial community composition in biofilms from the Great Barrier Reef

Natural and anthropogenic impacts such as terrestrial runoff, influence the water quality along the coast of the Great Barrier Reef (GBR) and may in turn affect coral reef communities. Associated bacterial biofilms respond rapidly to environmental conditions and are potential bioindicators for changes in water quality. As a prerequisite to study the effects of water quality on biofilm communities, appropriate biofilm substrates for deployment in the field must be developed and evaluated. This study investigates the effect of different settlement substrates (i.e. glass slides, ceramic tiles, coral skeletons and reef sediments) on bacterial biofilm communities grown in situ for 48 days at two locations in the Whitsunday Island Group (Central GBR) during two sampling times. Bacterial communities associated with the biofilms were analysed using terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of 16S rRNA genes. Findings revealed that substrate type had little influence on bacterial community composition. Of particular relevance, glass slides and coral skeletons exhibited very similar communities during both sampling times, suggesting the suitability of standardized glass slides for long-term biofilm indicator studies in tropical coral reef ecosystems.     

Phylogenetic characterization of bacterial consortia obtained of corroding gas pipelines in Mexico

Characterization of the microbial populations formed in gas pipelines is essential to understand the metallic surface-microbe interaction, their role in metal corrosion, and to implement efficient monitoring and control strategies. Microbial community analysis in a corroded gas pipeline in a petroleum-producing facility in the Southeast region in Mexico was performed by traditional cultivation techniques and identification based on 16S rRNA gene sequence. In all samples, thin bacterial biofilms were observed and pitting corrosion was reveled after removing the biofilms. Six pure or mixed cultures of anaerobic bacteria were obtained and their 16S rRNA libraries were constructed, respectively. At least two members of each RFLP profile were sequenced and the phylogenetic affiliations of cloned bacterial 16S rRNA genes indicated that native biofilms were mainly colonized by Desulfovibrio vulgaris and Desulfovibrio desulfuricans, sulfate-reducing bacteria members; Citrobacter freundii, an Enterobacteriaceae member; Clostridium celerecrescens and Clostridium sporogenes, spore-forming anaerobic species and Cetobacterium somerae, a microaerotolerant, non-spore-forming fusobacteria. Some of these species have been observed consistently in other steel pipelines previously, but Cetobacterium members and C. celerecrescens are described for the fist time in this corroded gas pipeline. The potential role of each species in biofilm formation and steel corrosion is discussed.     

Hydrocarbon-degrading bacteria and the bacterial community response in gulf of Mexico beach sands impacted by the deepwater horizon oil spill

A significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize the in situ response of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C₈ to C₄₀) concentrations ranging from 3.1 to 4,500 mg kg⁻¹ in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarily Gammaproteobacteria, including representatives of genera with known oil degraders (Alcanivorax, Marinobacter, Pseudomonas, and Acinetobacter). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 10⁷ to 10.2 × 10⁷ copies g⁻¹) versus clean (0.024 × 10⁷ to 1.4 × 10⁷ copies g⁻¹) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genus Alcanivorax showed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of the Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) as key players in oil degradation there.     

CARD-FISH and confocal laser scanner microscopy to assess successional changes of the bacterial community in freshwater biofilms

Bacterial community composition was assessed during riverine biofilm development by the Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) in combination with Confocal Laser Scanning Microscopy. Using artificial substrates, it was possible to follow the dynamics of specific bacterial clusters, while maintaining the unaltered structure and architecture of the biofilm.     

Identification and characterization of microbial biofilm communities associated with corroded oil pipeline surfaces

Microbially influenced corrosion (MIC) has long been implicated in the deterioration of carbon steel in oil and gas pipeline systems. The authors sought to identify and characterize sessile biofilm communities within a high-temperature oil production pipeline, and to compare the profiles of the biofilm community with those of the previously analyzed planktonic communities. Eubacterial and archaeal 16S rRNA sequences of DNA recovered from extracted pipeline pieces, termed ‘cookies,’ revealed the presence of thermophilic sulfidogenic anaerobes, as well as mesophilic aerobes. Electron microscopy and elemental analysis of cookies confirmed the presence of sessile cells and chemical constituents consistent with corrosive biofilms. Mass spectrometry of cookie acid washes identified putative hydrocarbon metabolites, while surface profiling revealed pitting and general corrosion damage. The results suggest that in an established closed system, the biofilm taxa are representative of the planktonic eubacterial and archaeal community, and that sampling and monitoring of the planktonic bacterial population can offer insight into biocorrosion activity. Additionally, hydrocarbon biodegradation is likely to sustain these communities. The importance of appropriate sample handling and storage procedures to oilfield MIC diagnostics is highlighted.     

Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of alginate genes in bacterial isolates from corroded metal surfaces

The distribution of alginate genes encoding biosynthesis of alginate was examined for bacterial isolates associated with corrosive biofilms recovered from source water, cooling lines, and reactor surfaces of a nuclear power plant. A total of 120 diverse Gram-positive and -negative isolates were obtained. Using DNA:DNA hybridization, 11 isolates were shown to contain sequences homologous to structural (algD, algG, alg-76) and/or regulatory (albB) alginate biosynthetic genes derived from an alginate-producing cystic fibrosis isolate of Pseudomonas aeruginosa (FRD1). Identification of isolates was accomplished by fatty acids methyl esters (FAME) analysis and the Biolog identification system. Nine of the twelve isolates were identified as various Pseudomonas spp., and two additional Gram-negative isolates were tentatively identified as Aeromonas veronii and Stenotrophomonas maltophilia. The remaining isolate was identified as a Gram-positive Bacillus pumilus. The results of the investigation extend current knowledge on the distribution of alginate biosynthetic genes in environmental isolates and permits the development of a more environmentally realistic model system to investigate the role of exopolymer production in biofilm formation and biocorrosion processes. Correspondence to: G.S. Sayler.     

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.     

Characterization of the bacterial community of a zinc-polluted soil.

The bacterial community of a zinc-contaminated soil (Maatheide soil in Lommel, Belgium) was studied using cultivation as well as cultivation-independent techniques. Colony-forming units (CFU) were determined by plating on media with or without metals. Dominant isolates were characterized by fatty acid methyl ester analysis (FAME analysis) and PCR fingerprinting using repetitive extragenic palindromic sequences as primers. DNA was directly extracted from soil samples and used as a template for the PCR amplification of the 16S rDNA (8-1511) or a 16S rDNA fragment (968-1401). Clones resulting from cloning the 16S rDNA from soil DNA were sequenced. Temperature gradient gel electrophoresis (TGGE analysis) was performed for 16S rDNA fragments (968-1401) amplified from the dominant isolates, the clones, and the total soil DNA extracted according to two protocols differing in strength of lysis. Total CFU ranged from 10(4) to 10(5)/g soil. The majority of the isolates were identified by FAME analysis as Arthrobacter spp. (18 out of 23). None of the isolates were identified as a Ralstonia eutropha like strain (formerly Alcaligenes eutrophus). Metalloresistant Rastomia eutropha like strains were previously shown to be dominant in the analyzed biotope. Most of the isolates were zinc tolerant but only seven could be considered zinc resistant. Sequences of the 16S rDNA clones obtained from total soil DNA were affiliated with genes of different bacteria such as alpha-proteobacteria, beta-proteobacteria, and the Cytophaga-Flexibacter-Bacteroides group. None of the sequenced clones aligned with the Ralstonia eutropha 16S rRNA gene. TGGE analysis of the 16S rDNA fragments (968-1401) amplified from the dominant strains, the clones, and the total soil DNA showed that isolates and clones represented only a part of the bands present in the TGGE pattern from total DNA. The 968-1401 fragment amplified from all Arthrobacter strains had a similar electrophoretic mobility. This band was seen as a major band in the pattern of DNA extracted from soil using a harsh cell lysis, whereas it did not appear, or appeared only as a weak band, in patterns obtained from soil DNA extracted using gentle lysis. The previously reported predominance of a Ralstonia eutropha like strain in this soil was no longer observed. This may suggest a population replacement by less resistant bacteria, concomitant with a progressive decrease of the zinc toxicity in the Maatheide soil.     

The bacterial community in 'taberna' a traditional beverage of Southern Mexico

Aims: To characterize the bacterial community of taberna, an alcoholic traditional beverage from the Southern part of Mexico produced by the fermentation of the coyol palm sap (Acrocomia aculeate). Methods and Results: Bacterial 16S rDNA libraries were constructed from metagenomic DNA extracted during the fermentation process at 0, 60 and 108 h. A total of 154 clones were sequenced, and 13, 10 and nine unique sequences were found at each sampling time. At the onset of the fermentation, Zymomonas mobilis, Fructobacillus spp., Pantoea agglomerans and other Gammaproteobacteria were detected. After 60 h, lactic acid bacteria were found and 30% of clones in the library were related to Lactobacillus nagelii, L. sucicola and L. sp. By the end of the experiment, i.e. after 108 h, the bacterial community included Z. mobilis, Lact. nagelii and Acetobacter pasteurianus. Conclusions: Our results suggest that Z. mobilis population represented an important proportion of the bacterial community (60–80%), as well as the lactobacilli during the fermentation process. The bacterial diversity was low and decreased as the fermentation progressed. Significance and Impact of the Study: This culture‐independent study suggests that Z. mobilis and lactobacilli play an important role in the alcoholic fermentation of the taberna beverage.     

Characterization of bacterial strains able to grow on high molecular mass residues from crude oil processing

Oil residues containing high molecular mass hydrocarbons, rich in polyaromatic compounds, are frequent end-products of crude oil processing and are poorly biodegradable. Their disposal poses an environmental problem. Through batch-enrichments from contaminated soils we have isolated and characterized seven bacterial strains that can use a residue from crude oil processing as a source of carbon and energy. The residue was a complex mixture of high molecular mass compounds, including saturated, aromatic and polycyclic aromatic hydrocarbons (PAHs). Analysis of the metabolic profiles of the strains isolated showed that they could all metabolize long-chain-length alkanes efficiently, but not PAHs. Strains degrading naphthalene, a simple PAH, did exist in the soil inocula used, but could be isolated only when enrichments were performed using pure naphthalene as the sole carbon source. All strains tested emulsified the oil residue and their ability to produce surfactants was studied.     

A porous elastic model for bacterial biofilms: application to the simulation of deformation of bacterial biofilms under microfluidic jet impingement

The presence of bacterial biofilms is detrimental in a wide range of healthcare situations especially wound healing. Physical debridement of biofilms is a method widely used to remove them. This study evaluates the use of microfluidic jet impingement to debride biofilms. In this case, a biofilm is treated as a saturated porous medium also having linear elastic properties. A numerical modeling approach is used to calculate the von Mises stress distribution within a porous medium under fluid-structure interaction (FSI) loading to determine the initial rupture of the biofilm structure. The segregated model first simulates the flow field to obtain the FSI interface loading along the fluid-solid interface and body force loading within the porous medium. A stress-strain model is consequently used to calculate the von Mises stress distribution to obtain the biofilm deformation. Under a vertical jet, 60% of the deformation of the porous medium can be accounted for by treating the medium as if it was an impermeable solid. However, the maximum deformation in the porous medium corresponds to the point of maximum shear stress which is a different position in the porous medium than that of the maximum normal stress in an impermeable solid. The study shows that a jet nozzle of 500 μm internal diameter (ID) with flow of Reynolds number (Re) of 200 can remove the majority of biofilm species.     

Exploring the bovine rumen bacterial community from birth to adulthood

The mammalian gut microbiota is essential in shaping many of its host''s functional attributes. One such microbiota resides in the bovine digestive tract in a compartment termed as the rumen. The rumen microbiota is necessary for the proper physiological development of the rumen and for the animal''s ability to digest and convert plant mass into food products, making it highly significant to humans. The establishment of this microbial population and the changes occurring with the host''s age are important for understanding this key microbial community. Despite its importance, little information about colonization of the microbial populations in newborn animals, and the gradual changes occurring thereafter, exists. Here, we characterized the overall bovine ruminal bacterial populations of five age groups, from 1-day-old calves to 2-year-old cows. We describe the changes occurring in the rumen ecosystem after birth, reflected by a decline in aerobic and facultative anaerobic taxa and an increase in anaerobic ones. Some rumen bacteria that are essential for mature rumen function could be detected as early as 1 day after birth, long before the rumen is active or even before ingestion of plant material occurs. The diversity and within-group similarity increased with age, suggesting a more diverse but homogeneous and specific mature community, compared with the more heterogeneous and less diverse primary community. In addition, a convergence toward a mature bacterial arrangement with age was observed. These findings have also been reported for human gut microbiota, suggesting that similar forces drive the establishment of gut microbiotas in these two distinct mammalian digestive systems.     

Stratification of activity and bacterial community structure in biofilms grown on membranes transferring oxygen

Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s(-1)) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s(-1), AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s(-1), AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.     

Characterization of the bacterial community structure of Sydney Tar Ponds sediment

The Sydney Tar Ponds is one of the largest toxic waste sites in Canada. The bacterial diversity and abundance in the Sydney Tar Ponds sediment was examined using a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) with four different primer sets. The clone library was grouped into 19 phylotypes that could be divided into five phyla: Proteobacteria (56.9%), Actinobacteria (35%), Acidobacteria (4.9%), Firmicutes (2.4%), and Verrucomicrobia (0.8%). Members of the phyla Actinobacteria (represented mainly by Mycobacterium spp.) and Alphaproteobacteria (represented by Acidocella spp.) comprised the majority of the clone library. This study also revealed that the phylogenetic results obtained from clone library analysis and from DGGE analysis, with all the primer sets, showed some variability. However, similar Mycobacterium spp. and Acidocella spp. were found in all the different DGGE analyses, again suggesting that these two genera are dominant in the Sydney Tar Ponds sediment. In addition, DGGE analysis indicated that primer sets targeting the V3 region produced results that were the most similar to those obtained with the clone library.     

Resistance of bacterial biofilms to disinfectants: a review

A biofilm can be defined as a community of microorganisms adhering to a surface and surrounded by a complex matrix of extrapolymeric substances. It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. Although the precise origin of such resistance remains unclear, different studies have shown that it is a multifactorial process involving the spatial organization of the biofilm. This review will discuss the mechanisms identified as playing a role in biofilm resistance to disinfectants, as well as novel anti-biofilm strategies that have recently been explored.