High‐throughput sequencing approach in analysis of microbial communities colonizing natural gas pipelines

Abstract This study provides a deep modern insight into the phylogenetic diversity among bacterial consortia found in working and nonworking high‐methane natural gas pipelines located in Poland. The working pipeline was characterized by lower biodiversity (140–154 bacterial genera from 22 to 23 classes, depending on the source of the debris) in comparison to the off‐gas pipeline (169 bacterial genera from 23 classes). The sediment recovered from the working pipeline contained mostly DNA identified as belonging to the phylum Firmicutes (66.4%–45.9% operational taxonomic units [OTUs]), predominantly Bacillus (41.4%–31.1% OTUs) followed by Lysinibacillus (2.6%–1.5% OTUs) and Clostridium (2.4%–1.8% OTUs). In the nonworking pipeline, Proteobacteria (46.8% OTUs) and Cyanobacteria (27.8% OTUs) were dominant. Over 30% of the Proteobacteria sequences showed homologies to Gammaproteobacteria, with Pseudomonas (7.1%), Enhydrobacter (2.1%), Stenotrophomonas (0.5%), and Haempohilus (0.4%) among the others. Differences were noted in terms of the chemical compositions of deposits originating from the working and nonworking gas pipelines. The deposits from the nonworking gas pipeline contained iron, as well as carbon (42.58%), sulphur (15.27%), and oxygen (15.32%). This composition can be linked to both the quantity and type of the resident microorganisms. The presence of a considerable amount of silicon (17.42%), and of aluminum, potassium, calcium, and magnesium at detectable levels, may likewise affect the metabolic activity of the resident consortia in the working gas pipeline. All the analyzed sediments included both bacteria known for causing and intensifying corrosion (e.g., Pseudomonas, Desulfovibrio, Shewanella, Serratia) and bacteria that can protect the surface of pipelines against deterioration (e.g., Bacillus). Biocorrosion is not related to a single mechanism or one species of microorganism, but results from the multidirectional activity of multiple microbial communities. The analysis presented here of the state of the microbiome in a gas pipeline during the real gas transport is a particularly valuable element of this work.

is referred to as Microorganisms Induced Corrosion or biodeterioration. It results in the development of biofilms and the formation of "black powder" deposits. It has been estimated that approximately 40% of all corrosion cases in industry are of biological origin (Jan-Roblero et al., 2008;Rajasekar, Anandkumar, Maruthamuthu, Ting, & Rahman, 2010). Damage due to biological corrosion is responsible for up to 20%-30% of total costs of service, predominantly because of the corrosion of gas pipelines (AlAbbas et al., 2012).
No previous study of the microbiome in gas pipelines has been conducted in Poland. The identification of microbial consortia in these ecological niches may be associated with the corrosion of natural gas transmission lines. The aim of our study was to investigate the phylogenetic diversity of bacterial environments in working and nonworking natural gas pipelines located in Poland. Furthermore, the composition of the microbiomes was discussed in the aspects of internal chemical changes to the surface of the pipelines.

| Sampling
Four samples of sediments were collected from the inner surfaces of two DN 350 natural gas pipelines in Poland: M1-Piotrków Trybunalski, the Warta River; M2-Piotrków Trybunalski, Sworzyce.
Sediments were scraped from a surface area of 25 cm 2 and placed in sterile plastic vessels. The samples were collected during the dismantlement of the M1 transmission line and the overhaul of the M2 gas pipeline by the network operator. The deposits from the nonworking gas pipeline (sample M1-1) and the working pipeline (samples M2-1, M2-2, and M2-3) were subjected to metagenomic and scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) analysis. Fragments from the carbon steel gas pipelines on both the M1 and M2 lines were collected and their chemical compositions submitted to SEM/EDS analysis.

| Natural gas pipeline characteristics
The gas pipelines had been protected against external factors by a bituminous coating and cathodic protection. The gas lines transported high-methane gas with a maximum working pressure of 3.2 MPa, from domestic deposits in the Podkarpacie region. Detailed descriptions of the gas pipelines and collected samples are given in Table 1.

| SEM/EDS microstructure analysis
The surfaces of the pipeline and their chemical compositions were analyzed using X-ray microanalysis. The microstructure was TA B L E 1 Description of gas pipelines and collected samples examined using an S-3000N SEM (Hitachi, Japan) equipped with an EDS microanalyzer (Thermo NORAN, Madison), according to the method described by Burnat, Walkowiak-Przybyło, Błaszczyk, and Klimek (2013) and Pietnicki, Wołowiec, and Klimek (2014). Tests were carried out on uncoated samples, prepared in the form of fracture surfaces. The results were presented as the percentage of individual elements in the surface layer of the carbon steel (the average values of the elemental composition in % wt).

| PCR amplification and sequencing
The obtained amplicons, c. 450 bp in size, were analyzed in 1% (w/v)

| Sequencing data analysis
The sequence reads were filtered by quality using a FastX toolkit and processed using a QIIME pipeline (http://qiime.org/). Paired reads were merged into contigs. Sequences were grouped based on their taxonomic classification and highly similar sequences were clustered into operational taxonomic units (OTUs

| RE SULTS AND D ISCUSS I ON
Two natural gas pipelines, one nonworking (M1) and another work-     (Beech & Gaylarde, 1999). In addition to creating a protective EPS or stabilizing a pre-existing biofilm on the surface of metal, microorganisms can also contribute to inhibit corrosion by neutralizing the action of corrosive substances and decreasing the cathodic rate, by consuming oxygen thought respiratory activity (Kip & van Veen, 2015;Videla & Herrera, 2005). to the best knowledge of the authors, this is the first study to report the detection of this species in a gas pipeline. To date, these bacteria have been noted only in oil-polluted or petroleum-contaminated sediments (Allen et al., 2007).

SRB belonging to Desulfosporosinus and Desulfotomaculum
In the sample of sediment taken from the bottom of the gas pipeline (M2-1), the presence of anaerobic acetogenic bacteria Sporomusa was identified. These bacteria are gram negative and also express the ability to form endospores. The occurrence of both these features in one microorganism is extremely rare. Sporomusa (mainly S. ovata) was isolated from an UGS reservoir in Russia (Balk et al., 2010). The Sporomusa isolates were found to be capable of perchlorate reduction. According to Drake, Küsel, and Matthies (2006), Sporomusa bacteria derive energy by CO 2 reduction in acetate via acetogenesis.
Their acetogens utilize the acetyl-CoA pathway for the energy-conserving CO 2 -fixing process (Drake, 2009 Zhu et al. (2003) in microbial communities collected from different three gas pipelines, were also detected.
In gas pipelines, a dewaterer is used to collect condensate water released from gas and solid contaminants. The dewaterer is installed at the lowest point in the high pressure line, to ensure the proper operation. Due to its structure and functions, the dewaterer in the analyzed pipeline was found to serve as a specific ecological niche for bacterial growth. Sample M2-2 from sediment collected from the dewaterer was slightly different from the other samples in terms of the microbial species it contained. As in the case of sample M1-1, the largest percentage of the analyzed sequences was recorded for the The macro-and microelements of steel also affect the environmental conditions and the microbiome in gas pipelines. The chemical compositions of the pipelines are presented in Table 2. The most striking differences between the tested pipelines concern the phosphorus and the sulphur contents. Even small differences in the compositions of steel pipelines can affect the diversity of microorganisms.
Therefore, the 4-and 6-times lower content of P and S in the composi-  Table 2). Impurities carried with the flowing gas may also have been a source of the microelements detected in these sediments.
The compounds detected in the samples are also described as the elements of the "black powder" created on the inner surfaces of the gas pipes. "Black powder" is composed mostly of iron oxides, iron hydroxides and siderite, and contains impurities such as elemental sulphur,  (Videla & Herrera, 2005). It should also be emphasized that biocorrosion is not related to a single mechanism or the occurrence of one species of microorganism, but most often results from the multidirectional activity of multiple microbial species. Therefore, estimating the progress of biocorrosion on the basis of data regarding the diversity and abundance of individual microorganisms is not straightforward, and requires further research.

ACK N OWLED G EM ENT
This work has been supported by the National Science Centre Poland, PRELUDIUM9-2015/17/N/NZ9/03683. Special thanks to Polska Spółka Gazownictwa-Lodz division for providing raw materials used in this research.

CO N FLI C T O F I NTE R E S T
No conflict of interests is declared. K.Z. involved in experiment design and manuscript preparation.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
Raw sequence data have been deposited to NCBI SRA under the Bioproject number PRJNA431861.