Diversity of Microorganisms Related to Biocorrosion in Ethanol Samples

Diogo Azevedo Coutinho

National Institute of Technology
Av. Venezuela, 82 - 616
Rio de Janeiro/RJ, 20081-312
Brazil

Mariana Machado Galvo
National Institute of Technology
Av. Venezuela, 82 - 616
Rio de Janeiro/RJ, 20081-312
Brazil

Viviane de Oliveira
National Institute of Technology
Av. Venezuela, 82 - 616
Rio de Janeiro/RJ, 20081-312
Brazil

Thas Abrantes
National Institute of Technology
Av. Venezuela, 82 - 616
Rio de Janeiro/RJ, 20081-312
Brazil

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

Carlos Alexandre Martins da Silva
Transpetro
Av. Presidente Vargas, 328
Rio de Janeiro/RJ, 20091-060
Brazil

Gutemberg de Souza Pimenta
PETROBRAS
Horcio Macedo, 950 - 2034
Rio de Janeiro/RJ, 21941-598
Brazil

Mrcia Teresa Soares Lutterbach
National Institute of Technology
Av. Venezuela, 82 - 616
Rio de Janeiro/RJ, 20081-312
Brazil

ABSTRACT

Ethanol is the most common alcohol and a renewable energy source that can be produced from
biomass. Brazil and the United States are world leaders in ethanol production, using as raw material
sugar 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. Ethanol
is capable of being metabolized by various microorganisms serving as a carbon source for them. Under
favorable conditions, bacteria and fungi can form biofilms and participate actively in the process of
biocorrosion. The presence of some microorganisms in the biofilms may enhance corrosion by the
production of corrosive metabolites such as organic acids. Given the scarcity of studies in the area, this
work aimed to study the diversity of microorganisms present in samples of ethanol in order to identify
possible causes of the process of biocorrosion. For this, several samples of ethanol from different
origins were received and analyzed. The main groups of cultivable microorganisms related to
biocorrosion were quantified. Furthermore, analyses of non-cultivable microorganisms were performed

using molecular biology techniques. The results allow a better assessment of the susceptibility of
storage 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, sugar
cane, 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 to
industries. As water and ethanol are high miscible, transportation by pipelines may result in the loss of
fuel octane rating and leave residual amounts of ethanol, water, and organic matter in the lines. These
compounds 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 and
incorporate 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 anaerobic
microorganisms
1
. Therefore, the infrastructure that stores and transports fuel containing ethanol can
provide 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 growth
are susceptible to microbiologically influenced corrosion (MIC). MIC has been already found in aqueous
environments, 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 with
ethanol once it is able to convert ethanol into acetic acid which would have a direct influence on the
materials corrosion.

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









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


EXPERIMENTAL PROCEDURE


Samples

Several samples of ethanol were collected from different sources and analyzed by the LABIO from the
National 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-
Aldrich
Control _
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
Ethanol
Park of pumps Pipeline scraping End of the
operation
03/20/2011
Ethanol Pipeline Refinery 12/1/2010
Corn Ethanol Imported 12/1/2007
Ethanol
Terminal / Refinery pipeline Beginning of the
operation
03/20/2011
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 cultivable
microorganism 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 30
o
C/ 48 hours
Ferric ammoniacal citrate
broth
Iron-precipitating bacteria 30
o
C/ 14 days
Tioglicolate broth Anaerobic bacteria 30
o
C/ 28 days
Postgate E Sulfate-reducing bacteria (SRB) 30
o
C/ 28 days


Besides the direct inoculation in culture medium, 50 mL of each ethanol sample was filtered through a
polytetrafluorethylene membrane (millipore) to concentrate the number of microorganisms present in
the sample. After filtration, the membranes were cut into four pieces with sterile scalpel and inoculated
in 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 an
ethanol sample was also present in the samples analyzed by our laboratory.

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

A sample of corn ethanol was chosen to study the diversity of non-culturable microorganisms present in
this fuel. A 50 mL aliquot sample was filtered through PTFE membrane with the aid of a syringe. The
membrane with the retained microorganisms was then used to extract the community genomic DNA
using an enzymatic cells lysis protocol followed by purification with phenol/chloroform and subsequent
precipitation 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)
). The
clones 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.

3
TA Cloning

with pCR

2.1
4
Innvitrogen by Lifetechnologies - 3175 Staley Road Grand Island, NY 14072 USA


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

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

Growth curve.

After the strain adaptation in MRS culture medium with 2% of ethanol, a growth curve measuring optical
density (OD) versus time (total of 90 hours) in increasing concentrations of alcohol in MRS medium was
made (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 carbon
steel, metal coupons specimen were immersed in MRS medium at concentrations of 2 and 4% of
ethanol.

The weight loss experiment was conducted to measure if there were MIC or corrosion caused by the
MRS medium and A. aceti on the metal surfaces (Figure 3). Samples were prepared according to
ASTM G1 standard method and the corrosion rate according to ASTM
(5)
G31
8
. For SEM, the samples
were fixed in 2.5% glutaraldehyde buffered with 0.2 M sodium cacodylate. After fixation, the samples
were dehydrated in ethanol and were stored in 100% ethanol in air-tight sealed glass vials before

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

critical point drying in liquid CO
2
. Immediately following drying, they were coated with gold-palladium
and 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 different
points as ethanol production plants, terminals, rail cars, and gas stations to verify the possible source of
fuel contamination.

No viable microorganisms were recovered from the samples. In just one sample of corn ethanol was it
possible to isolate aerobic bacteria. The bacterial strain was identified as Staphylococcus epidermidis
by genetic sequencing. S. epidermidis is part of human endogenous microbial flora
9
. Thus, this
microorganism has not been characterized as belonging to the sample analyzed but as an
anthropogenic 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 a
rich 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, molecular
techniques were used to study the microbial diversity. A cloning assay was used because it allows a
complete 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.







Table 3
Microorganisms 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 toxic
compounds 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 of
organic and inorganic toxic compounds
43 98% Alcaligenes sp.
Able to grow using compounds such as
phenol. Resistant to Hg2 +
50 99% Burkholderia cepacia
Commonly found in soil, water and plants.
Bacterium of interest in agriculture
58 98% Serratia marcescens
Widely found in drinking water in
developing 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. This
organism 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 is
considered the precursor of the corrosion process, since it is an important producer of
exopolysaccharide (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 other
microorganisms
12
. The EPS can create physical and nutritional conditions for the development of
sessile SRB. Moreover, it protects the cells against the flow and hinders the diffusion of biocides which
can help the biofilm formation. The presence of EPS in ethanol samples can also increase the viscosity
of 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 a
great metabolic versatility being able to metabolize toxic compounds like those found in pesticides.
Many Burkholderia species are capable of degrading petroleum which makes them of great interest in
bioremediation studies.

The bacterium Sphingobium olei was 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 due
to its relatively recent discovery.

Bacteria of Alcaligenes genus were isolated from oil-contaminated environments
15
. Similar to the
Sphingobium and Burkholderia genera, Alcaligenes are able to tolerate large amounts of toxic
substances 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 of
metallic pipelines used for transporting petroleum products
16
. This species was able to grow using
diesel as carbon source. Its influence on the corrosion of API 5LX steel was investigated by the
authors.


The Acinetobacter genus has been found in samples of ethanol fuel in one of the few published studies
on ethanol microbial diversity
17
. Researchers found this genus in samples collected from the bottom of
ethanol storage tanks. Some species are capable of using ethanol as carbon source and tolerate high
concentrations of solvents
18, 19
.

All identified organisms have diverse metabolic characteristics, being able to grow and tolerate
environments considered harsh for many microorganisms. Most bacteria identified in this phase are
known for the ability to tolerate harsh chemical compounds like solvents and pesticides.

The bacterium Acetobacter aceti, detected by researchers in ethanol samples
4
was not observed in any
of the analyzed samples. Therefore, a strain of this microorganism was acquired from a culture
collection to evaluate its influence on materials corrosion exposed to ethanol. With the intention of
adapting the bacteria to an environment rich in ethanol, the strain was inoculated into MRS broth with
2% of alcohol so that the corrosion tests and the ethanol consumption assay could be performed. After
the observation of turbidity of the culture medium, which indicates bacterial growth, an aliquot of the
broth was inoculated into the medium with 4% of ethanol. However, 4% was the maximum
concentration of ethanol in which A. aceti growth was observed. Thus, the continuous adaptation of the
strain was not possible to go further on since it did not grow on higher concentrations of ethanol.

In order to observe the growth rate and to obtain the growth curve of the A. aceti strain acquired by our
laboratory, an experiment was performed in a microplate reader. The A. aceti growth curve was similar
in both MRS medium (2% ethanol) and in MRS medium (2% ethanol) with 2% of corn ethanol (Figure 4
A, B). From the growth curves we infer that as corn ethanol concentration increases, the alcohol
becomes toxic to the cells (Figure 4).


Figure 4: A. aceti growth curves by optical density versus time in MRS medium (2% ethanol) (A); MRS
medium (2% ethanol) with 2% of corn ethanol (B); MRS medium (2% ethanol) with 4% of corn ethanol (C)
and MRS medium (2% ethanol) with 6% of corn ethanol.



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 seen
between the coupons exposed to the A. aceti and those that were exposed only to the sterile culture
media. Probably, the 3-day-long assay was not enough to see any alteration on the surface of the metal
caused by the bacteria. Longer assays should be performed to confirm whether or not A. aceti is 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
for
uniform corrosion in the four assays (Figure 6). These high corrosion rates were most likely caused by
the presence of water in MRS culture medium since the corn ethanol has only 1-2% of that element in
its composition. As no significant difference was observed in the corrosion rates and on the metal
surfaces, the active participation of A. aceti on microbial corrosion could not be determined.


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


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, biofilm
formation was visualized through SEM especially in the coupons inoculated with A. aceti and 2% of
corn ethanol (Figure 7B). This result corroborates the growth curves; the coupons immersed in MRS
with 2% of corn ethanol demonstrated to be a better medium for the growth of A. aceti than with 4% of
corn 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).



CONCLUSIONS

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

The bacterium A. aceti was not detected in the analyzed samples;


The experiments of SEM and the growth curves demonstrated that the A. aceti strain
acquired by the laboratory was not able to grow in MRS medium (2% ethanol) with more than
4% of corn ethanol;

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

Molecular biology tools allowed the identification of bacteria that are able to use ethanol as a
carbon source in the sample. Microorganisms related to biocorrosion cases and
microorganisms 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 in
the study of microorganisms diversity in ethanol samples. This methodology will be used to
study another ethanol sample in order to compare the results between populations.


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