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Diversity of Microorganisms Related to Biocorrosion in Ethanol Samples

Diogo Azevedo CoutinhoNational Institute of Technology

Av. Venezuela, 82 - 616Rio de Janeiro/RJ, 20081-312

Brazil

Mariana Machado GalvoNational Institute of Technology


Brazil

Viviane de OliveiraNational Institute of Technology

Av. Venezuela, 82 - 616Rio de Janeiro/RJ, 20081-312Brazil

Thas AbrantesNational Institute of Technology


Brazil

Marcelo ArajoPETROBRAS

Horcio Macedo, 950 - 2034Rio de Janeiro/RJ, 21941-598

Brazil

Carlos Alexandre Martins da SilvaTranspetro

Av. Presidente Vargas, 328Rio de Janeiro/RJ, 20091-060

Brazil

Gutemberg de Souza PimentaPETROBRAS

Horcio Macedo, 950 - 2034Rio de Janeiro/RJ, 21941-598Brazil

Mrcia Teresa Soares LutterbachNational Institute of Technology


Brazil

ABSTRACT

Ethanol is the most common alcohol and a renewable energy source that can be produced frombiomass. Brazil and the United States are world leaders in ethanol production, using as raw materialsugar cane and corn, respectively. In Brazil, ethanol is used as automotive fuel in two forms: hydrated,used in ethanol-using cars and flex fuel cars; or anhydrous alcohol, which is added to gasoline. Ethanolis capable of being metabolized by various microorganisms serving as a carbon source for them. Underfavorable conditions, bacteria and fungi can form biofilms and participate actively in the process ofbiocorrosion. The presence of some microorganisms in the biofilms may enhance corrosion by theproduction of corrosive metabolites such as organic acids. Given the scarcity of studies in the area, thiswork aimed to study the diversity of microorganisms present in samples of ethanol in order to identifypossible causes of the process of biocorrosion. For this, several samples of ethanol from differentorigins were received and analyzed. The main groups of cultivable microorganisms related tobiocorrosion were quantified. Furthermore, analyses of non-cultivable microorganisms were performed


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using molecular biology techniques. The results allow a better assessment of the susceptibility ofstorage tanks and pipelines to microbiological corrosion in the presence of ethanol.

Keywords: biocorrosion, ethanol, cloning, Acetobacter aceti.

INTRODUCTION

Brazil is one of the most advanced countries in production and use of ethanol as fuel, followed by U.S.Unlike petroleum, ethanol is a renewable resource that can be produced from cultivated plants, sugarcane, corn, beet, wheat, and cassava. In Brazil, ethanol is produced by the fermentation of sugar cane,which is one of the most efficient materials for commercial use. In 2003 the flexible-fuel car engines,called flex, started to be sold and today almost 90% of the cars in Brazil are flex.

The use of fuels containing ethanol in vehicles and its storage represents an additional challenge toindustries. As water and ethanol are high miscible, transportation by pipelines may result in the loss offuel octane rating and leave residual amounts of ethanol, water, and organic matter in the lines. Thesecompounds can provide all the nutrients needed for microbial communities, which could lead to

corrosion of the transportation infrastructure and storage facilities. Ethanol can also dissolve andincorporate impurities present within polyducts when it is carried by these systems.

At lower concentrations ethanol can be used as a carbon source by aerobic and anaerobicmicroorganisms 1. Therefore, the infrastructure that stores and transports fuel containing ethanol canprovide nutrients and the necessary conditions for microorganism growth (i.e. water, carbon source,and electron donors and acceptors). Environments that have favorable conditions for microbial growthare susceptible to microbiologically influenced corrosion (MIC). MIC has been already found in aqueousenvironments, oil wells and systems of transportation of refined fuels 2.

In a recent study conducted by the National Institute of Standards and Technology (NIST)(1),researchers have associated increased corrosion of metallic pipelines to the presence of the bacterium

Acetobacter aceti in an ethanol sample 3. This bacterial species is usually found in environments withethanol once it is able to convert ethanol into acetic acid which would have a direct influence on thematerials corrosion.

As ethanol is produced from natural materials, that are greatly variable, MIC of materials andbiodegradation of the fuel and its mixtures is perhaps higher than in conventional fuels. To prevent orcontrol the occurrence of these problems, a better understanding of the types of microorganisms foundin ethanol is fundamental. Given the scarcity of studies on the microorganisms present in the ethanolfuel and the importance to maintain the quality of the product from the production and storage sites tothe end consumer, the Laboratory of Biocorrosion and Biodegradation (LABIO)(2) analyzed severalsamples of ethanol from different sources with the aim of studying and detecting microorganismsinvolved in biocorrosion processes.

1National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD.2Laboratory of Biocorrosion and Biodegradation, National Institute of Technology, Av. Venezuela, 82 sala 616 Rio deJaneiro, Brazil


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EXPERIMENTAL PROCEDURE

Samples

Several samples of ethanol were collected from different sources and analyzed by the LABIO from theNational Institute of Technology since 2007 (Table 1).

Table 1

Ethanol samples

Sample Source Collection date

Corn Ethanol In API Medium _

Ethanol To be distributed at the gas stations 12/1/2007

Ethanol Imported - stained _Ethanol- Sigma-

AldrichControl _

Ethanol Ethanol production plant 11/09/2007

Ethanol Gas Station _

Ethanol Wagon Beginning of the unloading process 03/20/2011

Ethanol Wagon End of the unloading process 03/20/2011

Ethanol Park of pumps Beginning of the operation 03/20/2011

Ethanol Park of pumps During the operation 03/20/2011

Ethanol Park of pumps End of the operation 03/20/2011

EthanolPark of pumps Pipeline scraping End of the

operation03/20/2011

Ethanol Pipeline Refinery 12/1/2010

Corn Ethanol Imported 12/1/2007

EthanolTerminal / Refinery pipeline Beginning of the

operation03/20/2011

Ethanol Terminal / Refinery pipeline During the operation 03/20/2011

Ethanol Terminal / Refinery pipeline End of the operation 03/20/2011

In the laboratory the samples were inoculated in different culture media to quantify the main cultivablemicroorganism groups that could participate in biocorrosion processes (Table 2).

Table 2

Culture media used on microorganism quantification

Culture Medium Microorganism Temperature/Incubation Time

Nutrient Broth Anaerobic bacteria 30oC/ 48 hours

Ferric ammoniacal citratebroth

Iron-precipitating bacteria 30oC/ 14 days

Tioglicolate broth Anaerobic bacteria 30oC/ 28 days

Postgate E Sulfate-reducing bacteria (SRB) 30oC/ 28 days


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Besides the direct inoculation in culture medium, 50 mL of each ethanol sample was filtered through apolytetrafluorethylene membrane (millipore) to concentrate the number of microorganisms present inthe sample. After filtration, the membranes were cut into four pieces with sterile scalpel and inoculatedin the culture media described above.

A second membrane was inoculated directly onto a Petri dish containing agar Manitol (specific culture

medium to isolate A. aceti) in order to verify if the bacteria A. aceti found by NIST researchers in anethanol sample was also present in the samples analyzed by our laboratory.

The cultivation of microorganisms in laboratory does not reflect the real environmental conditions. Onlya minor portion of bacterial species are able to grow in culture media. In environmental samples thisfraction may correspond to less than 1% of total bacteria 4-6. Thus, techniques that only use growthmedia may underestimate the complexity of microbial communities. To circumvent the drawbacks ofcultivation, molecular techniques that do not require the cultivation of microorganisms have been usedto characterize bacterial communities, generally based on the sequence of the gene encoding the 16SrRNA. The development and application of biomolecular methods have allowed major advances in thestudy of microbial ecology.

A sample of corn ethanol was chosen to study the diversity of non-culturable microorganisms present inthis fuel. A 50 mL aliquot sample was filtered through PTFE membrane with the aid of a syringe. Themembrane with the retained microorganisms was then used to extract the community genomic DNAusing an enzymatic cells lysis protocol followed by purification with phenol/chloroform and subsequentprecipitation of DNA with ethanol 7.

The extracted DNA was amplified by PCR of the 16S gene rRNA using universal primers for the region.The PCR product of approximately 1500 bp was cloned into the vector pCR (3) 2.1 (Invitrogen(4)). Theclones were sequenced to identify the diversity of bacteria present in the sample (Figure 1).

Figure 1: Methodology for the analysis of the diversity of non-culturable microorganisms in ethanol

samples.

3TA Cloningwith pCR2.14Innvitrogen by Lifetechnologies - 3175 Staley Road Grand Island, NY 14072 USA


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One strain of A. acetiwas acquired from a culture collection in order to study the influence of thisbacterium on metals corrosion in the presence of ethanol. Therefore, the bacteria were inoculated inMRS broth added of 2% ethanol according to the instructions provided by the Institute from which thestrain was acquired. Increasing concentrations of ethanol were added to the broth in order to adapt thebacterium to the ethanol-enriched medium.

Aiming to observe the relationship of A. acetiwith corrosion, the following experiments were performed:

Growth curve.

After the strain adaptation in MRS culture medium with 2% of ethanol, a growth curve measuring opticaldensity (OD) versus time (total of 90 hours) in increasing concentrations of alcohol in MRS medium wasmade (Figure 2).

Figure 2: Scheme of the growth curve assay in a microplate reader.

Weight loss and Scanning Electron Microscopy (SEM).

In order to check whether biofilm formation and MIC can occur on the surface of API5L X60 carbonsteel, metal coupons specimen were immersed in MRS medium at concentrations of 2 and 4% ofethanol.

The weight loss experiment was conducted to measure if there were MIC or corrosion caused by theMRS medium and A. aceti on the metal surfaces (Figure 3). Samples were prepared according toASTM G1 standard method and the corrosion rate according to ASTM(5)G318. For SEM, the sampleswere fixed in 2.5% glutaraldehyde buffered with 0.2 M sodium cacodylate. After fixation, the sampleswere dehydrated in ethanol and were stored in 100% ethanol in air-tight sealed glass vials before

5ASTM International, 100 Barr Harbor Dr., PO Box C700, West Conshohocken, PA, 19428


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critical point drying in liquid CO2. Immediately following drying, they were coated with gold-palladiumand viewed under a scanning electron microscope.

Figure 3: Microplate assay for API5L X60 specimen immersed for 120 hours to observe the biofilm in SEM(B), the surface (C), and weight loss (D, E and F). Specimen immersed in MRS medium without the

bacteria, MRS and ethanol was observed as control (A).

RESULTS AND DISCUSSION

A total of 17 ethanol samples were analyzed over 5 years. These samples were collected from differentpoints as ethanol production plants, terminals, rail cars, and gas stations to verify the possible source offuel contamination.

No viable microorganisms were recovered from the samples. In just one sample of corn ethanol was itpossible to isolate aerobic bacteria. The bacterial strain was identified as Staphylococcus epidermidisby genetic sequencing. S. epidermidis is part of human endogenous microbial flora 9. Thus, thismicroorganism has not been characterized as belonging to the sample analyzed but as ananthropogenic contamination that probably occurred during the sample collection.

The fact that no viable organisms were recovered from the ethanol samples was not a surprise. It is

well known that when microorganisms are in an oligotrophic environment and then are inoculated in arich culture medium they probably will not growth due to the stress imposed by the medium change 10.

Since it was not possible to detect the presence of culturable microorganisms in the samples, moleculartechniques were used to study the microbial diversity. A cloning assay was used because it allows acomplete analysis of the microorganisms present in the sample, including the uncultured.

The identification of the microorganisms found in the sample analyzed is shown in Table 3.


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Table 3Microorganisms identified by cloning of a sample of ethanol

Clone Similarity Identification Characteristics

5 97% Pseudomonas putida Exopolysaccharide producer

10 e 49 98% Burkholderia vietnamiensis Capable of degrading various toxiccompounds as pesticides

19 98% Burkholderia sp. Found in flower nectar

24 97% Sphingobium olei Isolated from soil contaminated with oil

29 98% Acinetobacter sp.Related to biodegradation and removal oforganic and inorganic toxic compounds

43 98% Alcaligenes sp.Able to grow using compounds such as

phenol. Resistant to Hg2 +

50 99% Burkholderia cepaciaCommonly found in soil, water and plants.

Bacterium of interest in agriculture

58 98% Serratia marcescensWidely found in drinking water indeveloping countries due to poor

chlorination

70 99% Acinetobacter radioresistens Radiation resistant

Among the organisms that were found, it is worth mentioning the bacterium Pseudomonas putida. Thisorganism is ubiquitously present in a variety of environments and is capable of precipitating iron,characterizing it as iron-precipitating bacteria. According to Beech & Gaylarde 11, this species isconsidered the precursor of the corrosion process, since it is an important producer ofexopolysaccharide (EPS). EPS is the substance with the highest concentration in biofilms, the main

structure of the biocorrosion process, responsible for adhesion and also used as nutrient for othermicroorganisms 12. The EPS can create physical and nutritional conditions for the development ofsessile SRB. Moreover, it protects the cells against the flow and hinders the diffusion of biocides whichcan help the biofilm formation. The presence of EPS in ethanol samples can also increase the viscosityof the fuel which can lead to clogging of valves and filters and the loss of quality.

Bacteria of the Burkholderia genus have been found in other fuels samples 13. Burkholderia have agreat metabolic versatility being able to metabolize toxic compounds like those found in pesticides.Many Burkholderiaspecies are capable of degrading petroleum which makes them of great interest inbioremediation studies.

The bacterium Sphingobium oleiwas first isolated from a soil sample contaminated with oil 14. There

are only few studies of this species and it is not yet known its participation and function in ethanol dueto its relatively recent discovery.

Bacteria of Alcaligenes genus were isolated from oil-contaminated environments 15. Similar to theSphingobium and Burkholderia genera, Alcaligenes are able to tolerate large amounts of toxicsubstances which make them candidates to survive in a hostile environment such as in ethanol fuel.

The bacterium Serratia marscences has been isolated from corrosion product and from inside ofmetallic pipelines used for transporting petroleum products 16. This species was able to grow usingdiesel as carbon source. Its influence on the corrosion of API 5LX steel was investigated by theauthors.


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There was no localized corrosion in the API5L X60 coupons immersed in MRS media with 4% and 6%of ethanol inoculated with A. aceti after a period of 120 h (Figure 5). No differences could be seenbetween the coupons exposed to the A. acetiand those that were exposed only to the sterile culturemedia. Probably, the 3-day-long assay was not enough to see any alteration on the surface of the metalcaused by the bacteria. Longer assays should be performed to confirm whether or not A. acetiis able to

cause MIC.

Figure 5: Images of coupons immersed in MRS medium with 2% of ethanol (A), 2% of ethanol + A. aceti(B), 4% of ethanol (C) and 4% of ethanol + A. aceti. Before the analyses, metals were cleaned and

observed in stereoscope at 25 X magnification.

The corrosion rates obtained were considered high according to NACE (6) standard RP-07-75 20 foruniform corrosion in the four assays (Figure 6). These high corrosion rates were most likely caused bythe presence of water in MRS culture medium since the corn ethanol has only 1-2% of that element inits composition. As no significant difference was observed in the corrosion rates and on the metalsurfaces, the active participation of A. acetion microbial corrosion could not be determined.

6NACE International, 1440 South Creek Drive Houston, TX.


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Figure 6: Corrosion rate of API5L X60 specimens in four different conditions acquired after 72 hours.

No corrosion was observed in both experiments designed for analysis of the surface. However, biofilmformation was visualized through SEM especially in the coupons inoculated with A. acetiand 2% ofcorn ethanol (Figure 7B). This result corroborates the growth curves; the coupons immersed in MRSwith 2% of corn ethanol demonstrated to be a better medium for the growth of A. acetithan with 4% ofcorn ethanol (Figure 7).

Figure 7 - SEM of API5L X60 coupons immersed in MRS medium (2% ethanol) (A), MRS (2% ethanol)inoculated with A. aceti(B), MRS (2% ethanol) + 2% of corn ethanol (C), and MRS (2% ethanol) + 4% of

corn ethanol inoculated with A. aceti(D).


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CONCLUSIONS

It was not possible to detect the presence of viable microorganisms in any of the 17 samplesof ethanol through the conventional culture media approach. However, alternative culturemedia should be tested;

The bacterium A. acetiwas not detected in the analyzed samples;

The experiments of SEM and the growth curves demonstrated that the A. aceti strainacquired by the laboratory was not able to grow in MRS medium (2% ethanol) with more than4% of corn ethanol;

The corrosion rate, considered high by NACE standard methods, was probably caused bythe water of the MRS medium since no significant difference was found between the corrosionrates of the coupons under all four conditions;

Molecular biology tools allowed the identification of bacteria that are able to use ethanol as acarbon source in the sample. Microorganisms related to biocorrosion cases andmicroorganisms tolerant to toxic substances were found in the ethanol sample;

The PCR of the 16s rRNA gene followed by cloning proved to be an efficiency approach inthe study of microorganisms diversity in ethanol samples. This methodology will be used tostudy another ethanol sample in order to compare the results between populations.

REFERENCES

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(16) RAJASEKAR, A.; BABU, T. G.; PANDIAN, S. T. K.; MARUTHAMUTHU, S.;PALANISWAMY, N.; RAJENDRAN, A. Role of Serratia marcescens ACE2 on dieseldegradation and its influence on corrosion. J Ind Microbiol Biotechnol., v. 34, p.589598, 2007.

(17) JAIN, L.; WILLIAMSON, C.; BHOLA, S. M.; BHOLA, R.; SPEAR, J. R.; MISHRA, B.;OLSON, D. L.; KANE, R. Microbiological and Electrochemical Evaluation of Corrosion andMicrobiologically Influenced Corrosion of Steel in Ethanol Fuel Environments. Corrosion 2012Conference & Expo. Paper 10070, 2010.

(18) ABBOTT; BERNARD, J.; LASKIN, A. I.; MCCOY, C. J. Growth of Acinetobacter calcoaceticuson Ethanol. Applied and Environmental Microbiology, v. 25, n. 5 p. 787-792, 1973.

(19) CHEN, H. L.; YAO, J., WANG, L.; WANG, F.; BRAMANTI, E., MASKOW, T.; ZARAY, G.Evaluation of Solvent Tolerance of Microorganisms by Microcalorimetry. Chemosphere, v. 74, n.

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