Applied Soil Ecology 55 (2012) 27–35

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Applied Soil Ecology

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Biodegradability of soy biodiesel in microcosm experiments using soil from

the Atlantic Rain Forest
Gislaine S. Silva a , Eric L.S. Marques a , João C.T. Dias a , Ivon P. Lobo b , Eduardo Gross c ,
Martin Brendel a , Rosenira S. da Cruz b , Rachel P. Rezende a,∗
a
Department of Biological Sciences, State University of Santa Cruz – UESC Rod. BR 415, Km 16, CEP: 45662-900, Ilhéus, BA, Brazil
b
Department of Mathematical Sciences and Technology, State University of Santa Cruz – UESC Rod. BR 415, Km 16, CEP: 45662-900, Ilhéus, BA, Brazil
c
Department of Agricultural Sciences, State University of Santa Cruz – UESC Rod. BR 415, Km 16, CEP: 45662-900, Ilhéus, BA, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Biodiesel is a promising alternative energy source to diesel fuel and determining its biodegradability is
Received 12 August 2011 an important aspect of addressing potential environmental spills. The aim of this study was to compare
Received in revised form 9 December 2011 the biodegradability of biodiesel and its effect on soil microbial diversity with that of normal diesel using
Accepted 2 January 2012
blends of biodiesel with diesel in microcosm experiments with soil from the Atlantic Rain Forest. Degra-
dation was monitored by respirometry and GC analysis of the substrates. The highest respiration rates
Keywords:
were observed in soil contaminated with the B50 (41.47 mg CO2 /kg) and B100 (42.35 mg CO2 /kg) blends.
Biofuels
Soil contaminated with blends B5, B20, B50 and B100 showed higher numbers of culturable heterotrophic
Bioremediation
Biodegradability
microbes than control soil. Chromatographic analyses showed that microcosms contaminated with diesel
DGGE fuel and the B5 blend had less biodegradation than soil contaminated with the B20, B50 and B100 blends
Gas chromatography (80%, 62% and 84% of biodegradation, respectively). DGGE analysis showed that samples contaminated
with the B5, B20, B50 blends and D100 showed changes in microbial community after the incubation
period. Interestingly, soil contaminated with B100 decreased the microbial community compared with
the other contaminated samples. While the degradation of pure biodiesel (B100) was efficient, microbial
diversity of soil was reduced, which could compromise the overall activity of microorganisms involved
in the bioremediation of contaminated soils.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction Protection Agency (EPA 560/6-82-003). This protocol determines

Biodiesel derived from vegetable oils is a promising alternative
fuel and, as petroleum reserves are depleted, is already attract-
ing attention in the fuel market. Biodiesel has some advantages
over diesel as it is a source of renewable energy and reduces
environmental pollution caused by SO2 and soot (Mishra et al.,
2001; Vieira et al., 2007). It is free of sulfur and emits less CO
than diesel (Kaieda et al., 1999; Noureddine et al., 2005; Birchall
and Newman, 1995). Furthermore, biodiesel is more biodegradable
because it consists of alcohol esters of short-chain fatty acids, com-
pounds that exist naturally in the environment (Mudge and Pereira,
1999).
Louwrier (1998) and Zhang et al. (1998) showed that biodiesel
is degraded rapidly in aquatic environments. The biodegradability
studies in water used procedures established by the Environmental
∗ Corresponding author at: Dept. Ciências Biológicas, UESC – Universidade Estad-
ual de Santa Cruz, Rod. Ilhéus-Itabuna, Km 16, Ilhéus 45650-000, BA, Brazil.
Tel.: +55 73 3680 5033; fax: +55 73 3680 5226.
E-mail address: rezende.rachel@gmail.com (R.P. Rezende).
the extent of biodegradability of a substance by measuring CO2
release. However, relatively little is known about the effects of
biodiesel in soil. We, therefore, examined the biodegradability
of biodiesel fuels in natural soil and the potential impacts of
spills.
In Brazil, biodiesel is already commercialized as a minor com-
ponent in a blend with diesel. Various studies have addressed the
search for renewable resources for biodiesel production so that
it may serve as an alternative to conventional diesel. Brazil is a
leading producer of soybeans, a commodity that is used predomi-
nantly for food, feed and oil production. Soy-derived biodiesel has
gained much attention, as soybeans may be the only oilseed avail-
able for immediate large-scale production of biodiesel (Carneiro,
2003; Parente, 2003).
In this work, we investigated soy biodiesel biodegradation by
indigenous microorganisms in forest soil microcosm experiments.
We measured the production of CO2 and chromatographically ana-
lyzed the biodiesel components over a 60-day biodegradation trial.
We also studied the impact of biodiesel contamination on the
microbial community and thus obtained important information on
the ecological impact of biodiesel blends on soil microorganisms.

0929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2012.01.001
28 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35
2. Materials and methods
2.1. Soil
The soil sample used for this work was collected from a pre-
served area of the Atlantic Rain Forest of Universidade Estadual de
Santa Cruz (UESC), Ilhéus, Bahia. Soil samples were collected at a
depth of 10 cm, sieved through 2-mm mesh and analyzed imme-
diately. The physico-chemical analysis of the soil was performed
by the Laboratório de Solos e Nutrição de Plantas of Embrapa. The
soil was classified as sandy clay with a pH of 6.0, 39 mg/dm3 P,
0.82 cmolc /dm3 K, 18.45 cmolc /dm3 S and 68.82 g/kg organic mat-
ter. Soybean oil-derived biodiesel, produced by the Laboratório
Bioenergia e Meio Ambiente of UESC, was used in all experiments.
2.2. Basal respiration
Microcosms were set up in triplicate in tightly closed Bartha
flasks. To determine metabolic activity in each microcosm,
respirometric analyses were periodically performed by monitor-
ing CO2 emissions. Each flask contained 100 g of soil, 20 mL of
Bushnell–Hass medium (g/L: MgSO4 , 0.2; CaCl2 , 0.02; KH2 PO4 , 1.0;
NH4 NO3 , 1.0; FeCl3 , 0.05) and 5% of fuel. Ten milliliters of NaOH
(0.1 M) was added to plastic tubes inserted into each microcosm.
The following fuels were used: biodiesel B100 (100% biodiesel),
diesel D100 (100% diesel), and biodiesel mixed with diesel in
varying percentages (B5—5% biodiesel and 95% diesel; B20—20%
biodiesel and 80% diesel; and B50—50% biodiesel and 50% diesel).
Microcosms containing sterilized and natural uncontaminated soil
were used as negative and positive controls (C−, C+). The micro-
cosms were incubated in the dark for a period of 60 days, and
every five days, the basal respiration was measured by titration. The
NaOH (0.1 M) present in the microcosms was removed on reading
days to be titrated with HCl (0.1 M), and phenolphthalein was used
as indicator. The amount of CO2 released in each microcosm during
microbial respiration was inversely proportional to the volume of
HCl used in the titration.
2.3. Number of heterotrophic microorganisms in soil
Identical sets of microcosms were set up in triplicate to deter-
mine the total number of heterotrophic microorganisms. The
samples were collected from these microcosms on days 0, 30 and
60. One gram of soil was removed and mixed in 9 ml of 0.85%
saline (NaCl). The suspensions were serially diluted to 10−6 and
plated on Plate Count Agar (PCA; g/L: casein peptone, 5.0; yeast
extract, 2.5; dextrose, 1.0; agar, 15.0). The number of cultivable het-
erotrophic microorganisms was determined by measuring colony
forming units per gram of soil (CFU g−1 ) after incubation at 30 ◦ C
for 48 h.
2.4. Gas chromatographic analysis of biodiesel degradation
Soil samples contaminated with diesel (D100) and biodiesel
blends B5, B20, B50, B100 were removed from the microcosms on
days 0, 30 and 60 for degradation analysis. Diesel and biodiesel
were extracted from 15 g of soil with 20 mL of solvent (hexane) in a
separate flask. The resulting suspensions were ultrasound-treated
(Ultrasonic Cleaner, Maxiclean 1600, Unique) for 30 min, and the
supernatant was removed. The extraction procedure was repeated
three times. The three supernatants were combined, and the sol-
vent was removed by rotary evaporation (RV06-ML IKA® Werke),
leaving fuel oil residue. Five microliters of the oil extract was col-
lected and redissolved in 495 ␮L of hexane. Chromatographic anal-
yses were performed on a Shimadzu Gas Chromatograph, using an
RTX® -WAX column (30 m, 0.25 mm; 0.30 ␮m film) and hydrogen
as the carrier gas. One microliter of each sample was injected.
The temperature of the injector was 280 ◦ C. The programmed col-
umn temperature was as follows: 80 ◦ C for 1 min; heating speed of
15 ◦ C/min up to 230 ◦ C; hold for 24 min. The percentage of degra-
dation of the compounds was calculated as the difference between
the peak areas of the experimental and control samples, where the
peak area of the control was used as the 100% point of reference.
2.5. Soil DNA extraction, PCR and DGGE analysis
Total DNA was extracted from soil samples using the PowerSoil
DNA Isolation Kit (MoBio), following the manufacturer’s instruc-
tions. The DNA was subjected to electrophoresis in 1% (w/v) agarose
gels, stained with ethidium bromide and visualized under UV light.
DNA was amplified using primers for the yeast 26S rRNA gene,
NL1GC (5 -GC-GCCATATCAATAAGCGGAGGAAAAG-3 ) and LS2 (5 -
GCCATATCAATAAGCGGAGGAAAAG-3 ) (Cocolin et al., 2000). A
fragment of the V3 region of the bacterial 16S rRNA gene was
amplified using primers F385 (5 -GC-ACTCCTACGGGAGGCAGCAG-
3 ) and R518 (5 -ATTACCGCGGCTGCTGG-3 ) (Lane, 1991; Muyzer
et al., 1993). All PCR reactions used a mix containing 1.25 U of Taq
DNA polymerase (Invitrogen), 5 ␮L of 10× reaction buffer, 200 ␮M
deoxyribonucleotide, 3.0 mM MgCl2 and 1 ␮L DNA and sterile milli-
Q water, for a final volume of 25 ␮L. Amplification was performed
using an automated thermal cycler (Mastercycler personal, Eppen-
dorf). The amplified rDNA gene sequences were analyzed in an 8%
polyacrylamide gel (w/v) (37.5:1 acrylamide:bis-acrylamide) com-
posed of a denaturing gradient of 40–70%. The gels were run in
0.5× TAE buffer (20 mM Tris acetate, pH 7.4, 10 mM sodium acetate,
0.5 mM EDTA disodium) with a constant voltage of 60 V for 18 h at
60 ◦ C (16S), and 200 V for 4 h at 60 ◦ C (26S). The mutation detection
system (MAXFILL, BioAgency, USA) was used for the DGGE analysis.
The bands were visualized by staining with silver nitrate. Each band
was considered an operational taxonomic unit (OTU). The DGGE
profile was analyzed from the matrix of presence and absence of
bands to calculate the similarity values among samples.
2.6. Multivariate analysis
Three methods of multivariate analysis were applied to our
results: 1. Cluster analysis; 2. Cluster analysis preceded by principal
component analysis (PCA); 3. Venn diagram. In our first method,
cluster analysis was based on a binary matrix, representing the
presence or absence of bands in each treatment. The similarity
matrix was obtained by calculating the coefficient of Dice (1945).
In the second method, PCA was applied to reduce the dimension
of the original variables. In the third method, Venn diagrams were
constructed manually, taking into account the intersections of the
obtained DGGE bands.
2.7. Statistical analysis
Basal respiration levels, heterotrophic microorganism counts
and gas chromatographic analyses of biodiesel degradation were
analyzed in triplicate. Statistical significance of the data was eval-
uated by analysis of variance (ANOVA); the means were then
compared by Tukey’s test. The means between treatments with
P ≤ 0.05 were considered significantly different.
3. Results and discussion
3.1. Basal respiration
Production of CO2 indicated that the microbial community was
capable of using biodiesel as an energy source. When compared
with the control (non-oil-contaminated) soil, which showed the
 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35
Fig. 1. Respiration (mg CO2 kg−1 ) during experiments in microcosms with B100 and D100 (a) and B5, B20, and B50 (b). The positive control (C+) contained uncontaminated
soil. The negative control (C−) contained autoclaved contaminated soil.
highest respiration rate at day 35, active microorganisms needed to
adapt to the presence of the oil contaminants. In contaminated sam-
ples, respiration began to increase after 15 days, stabilized after 25
days and decreased after 45 days. Fig. 1a shows the CO2 production
in soil contaminated with B100 and D100 and in uncontaminated
soil; both contaminated samples had a higher CO2 emission rate
than the control sample. B100 contamination led to the highest
respiration, confirming that biodiesel is more easily biodegrad-
able than diesel. Fig. 1b compares B5, B20 and B50 contaminated
soil with uncontaminated soil and again showed that biodiesel-
contaminated soil had the highest respiration rate. Among all
biodiesel blends, B5 and B50 had the best biodegradation. B5, B50
and B100 showed maximum respiration values of 38.39, 41.47 and
42.35 mg kg−1 , respectively. The B20 blend produced significantly
lower CO2 than other blends (P ≤ 0.05). When D100 was compared
with the sample blends B5, B20 and B50, only B20 was not signifi-
cantly different from the D100-contaminated sample (P ≤ 0.05).
Miller and Mudge (1997) and Mudge and Pereira (1999) showed
that the addition of biodiesel increased diesel biodegradation. It
is hypothesized that biodiesel acts as a non-volatile solvent that
dissolves diesel and increases its availability for cellular absorption
and use as a carbon source. Lapinskiene et al. (2006) also verified
that the extent of respiration in soil contaminated with biodiesel
was higher than that of diesel-contaminated soil, showing that
29
diesel fuel is more resistant to microbial decomposition than
biodiesel.
3.2. Total counts of heterotrophic microorganisms
Comparisons of the amount of heterotrophic microorganisms
in contaminated soil and uncontaminated soil (Fig. 2) showed that
biodiesel blends and diesel favored the growth of microorganisms.
We found a considerable increase of total heterotrophic microor-
ganisms from day 0 to day 30 in soil treated with biodiesel blends
(B20, B50) and pure diesel (D100); only B5 treatment did not appear
to increase heterotrophic counts in this time frame. After 60 days
of incubation, all samples maintained higher population densities
of microorganisms except the B100 sample. Pure biodiesel seems
to interfere with the microbial community, as the sample contam-
inated with B100 exhibited a decrease in microbial growth after 60
days of incubation. Biodiesel, although presenting a simpler struc-
ture than diesel, is a xenobiotic compound. Initially (in the period of
0–30 days), the sample containing B100 showed an increase in the
number of total heterotrophic microorganisms, but after this initial
period, there was a large reduction of heterotrophs in the sample.
Although pure biodiesel is clearly used by the microorganisms, it
seems to negatively affect their vitality.
30 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35

Fig. 2. Comparative analysis of microbial communities in contaminated and uncontaminated soils. Different letters in the same time indicate a significant difference among
samples (Tukey’s test, P ≤ 0.05). Asterisk (*) indicates samples without significant difference. There was no growth in the negative control (C−).

3.3. DGGE analysis
It is well known that conventional cultivation techniques
cannot evaluate all members of the natural microbial community.
Therefore, the impact of biodiesel on the microbial community in
soil was evaluated using DGGE analysis (Heuer et al., 1997). DGGE
results showed a quantitative change in operational taxonomic
units (OTUs). Different OTUs were observed after 60 days of
incubation (Figs. 3 and 4). Analysis with primers for the V3 region
of the 16S rRNA gene showed a clear alteration in the microbial
community of soils contaminated with B5, B20, B50, B100 and
D100 after 60 days of incubation when compared with B5 , B20 ,
B50 , B100 and D100 samples from time 0 days (Fig. 3). The
samples labeled as Cont , B20 , B50 and B100 formed a group with
100% similarity in the dendrogram, sharing all 35 OTUs that are
also present in the samples Cont , B5 and D100 . Based on PCA and
dendrogram analyses, the samples were divided into three groups:
Group 1 (G1) consisted of samples Cont , B50 , B100 and B20 , B5 ,
Cont, D100 ; Group 2 (G2) consisted of samples D100, B20, B50 and
B5; and Group 3 consisted of the sample B100. The Venn diagram
in Fig. 6a, comprised of the three groups (G1, G2 and sample B100),
shows the number of bands shared among the groups. All groups
shared 10 bands, while G1 and G2 shared a further 11; 14 bands
appeared in Group 1 alone. Sample B100 comprised a single clade
(Fig. 3) and showed the lowest number of bands.
Similar results were observed in DGGE-targeting yeasts (Fig. 4).
Samples D100 , B20 , B50 , Cont and B100 formed a group with
100% similarity in the dendrogram sharing all four OTUs each other

Fig. 3. DGGE of the V3 region of the 16S rRNA gene using an 8% polyacrylamide gel and a denaturant gradient from 40 to 70%. The dendrogram represents the similarities
between the communities present in samples analyzed at 0 days (Cont , D100 , B100 , B50 , B20 , B5 ) and at 60 days (Cont, D100, B100, B50, B20, B5).
 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35 31

Fig. 4. DGGE of the eukaryotic 26S rRNA gene amplified using primers NL1GC and LS2 and run on an 8% polyacrylamide gel. The dendrogram plots the similarities between
the communities present in samples analyzed at 0 days (Cont , D100 , B100 , B50 , B20 , B5 ) and at 60 days (Cont, D100, B100, B50, B20, B5).

and with the sample B5 . These data corroborate the PCA and the
dendrogram (Fig. 5b). Based on PCA and dendrogram analyses, the
samples were grouped into the same three groups as in bacteria
but with different band sharing among the samples (Fig. 6b). Thus,
the sample B100 showed one unique, unshared band. G1 and G2
samples shared three bands, two of which were in all the samples of
the G1 and B5 and belonged to Cont, B5 , B5, B20 and B50 (Fig. 6b).
However, there is significantly less band sharing in yeast than in
bacteria between groups G1 and G2; these groups have 7 and 14
unique, unshared bands, respectively.
In both gels, the samples of the initial time point showed fewer
bands than in the final time point, with the exception of samples
B100 and B100. In sample B100, total bands decreased with time,
indicating that the presence of pure biodiesel selected (i.e., favored
growth of) a smaller group of bacteria. Thus, despite being highly
biodegradable, B100 reduced the microbial diversity of the soil,
suggesting that pure biodiesel negatively affected the soil yeast
community. These effects caused by pure biodiesel corroborated
the results obtained by the total heterotrophic counts from the
B100 soil sample (Fig. 2). These data show that the addition of fuel
caused changes in community structure of bacteria and yeast.
3.4. Analysis of biodiesel degradation
Biodiesel and biodiesel/diesel blends have been shown to be
more easily and quickly biodegraded than pure diesel (Zhang et al.,
1998; Pasqualino et al., 2006; Lapinskiene et al., 2006). For rapid
degradation, the appropriate enzymes must be available in the
soil microbes. Biodiesel consists of pure fatty acid esters, and the
enzymes responsible for their degradation exist naturally (Zhang
et al., 1998). The presence of two atoms of oxygen makes biodiesel
vulnerable to biological degradation. In contrast, the composition of

Fig. 5. Principal component analysis (PCA) on the DGGE profiles of the bacteria (a) and yeast (b). The microbial communities at time 0 (Cont , D100 , B100 , B50 , B20 , B5 )
are more clustered; after 60 days of incubation, the profile of the communities (Cont, D100, B100, B50, B20, B5) was greatly changed.
32 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35

Fig. 6. Venn diagram showing the DGGE profile band shared among all samples when (a) the bacterial 16S and (b) eukaryotic 26S DNA were analyzed. Cont , B50 , B100 ,
B20 , B5 , B100 and Cont were grouped into one group labeled G1. The bands from treatments D100, B200, B50 and B5 were grouped in G2. The group denominated as B100
correspond to the B100 sample after 60 days of contamination.

diesel is chemically more complex and less susceptible to biological
degradation (Zhang et al., 1998; Pitter and Chudoba, 1990).
Chromatographic analysis confirmed that biodiesel is more
biodegradable than diesel (Figs. 7–9). Even in diesel blends,
biodiesel was more easily and quickly degraded. Thus, while the
total B5 blend was not completely degraded, the biodiesel compo-
nent was degraded (Fig. 7). The B5 blend is composed of only 5%
biodiesel; the biodiesel present in this mixture was 100% degraded,
while diesel was 29% degraded. However, because the diesel in the
mixture was not degraded as extensively, the overall degradation
of the mixture was 51%. In the blends B20 and B50 (Table 1), the
overall degradation was 80% and 62%, respectively, as the amount of
diesel in these mixtures is lower than in the B5 blend. Although the
percentage of biodiesel in the blends decreased, as shown in Table 1,

Fig. 7. Chromatograms of B5 and B20 blends after 0, 30 and 60 days of incubation, showing the extent of degradation of these mixtures (C16:0, palmitate; C18:0, stearate;
C18:1, oleate; C18:2, linoleic acid; C18:3, linolenic acid).
 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35 33

Fig. 8. Chromatograms of mixtures B50 and B100, after 0, 30 and 60 days of incubation (C16:0, palmitate; C18:0, stearate; C18:1, oleate; C18:2, linoleic acid; C18:3- linolenic
acid).

the degradation percentage of the diesel component only slightly the bioavailability of diesel to microorganisms (Table 1). The B50
decreased. The B50 had a lower overall percentage of degradation blend, however, showed less biodegradation than the B20 blend,
than the B5 and B20 blends but showed better diesel degradation suggesting something unusual occurred in this mixture. Table 1
than the other samples. It is known that blending biodiesel with shows that the extent of degradation of total fatty ester blends in
diesel can promote diesel biodegradation. In fact, some studies sug- B5, B20 and B50 decreased, while the percentage of degradation of
gest the use of biodiesel as a solvent for removal of diesel from diesel increased.
contaminated environments (Miller and Mudge, 1997; Taylor and This observation supports the general notion that biodiesel can
Jones, 2001; Pasqualino et al., 2006). promote and accelerate the biodegradability of diesel by means of
When calculating total degradation (Table 1), we observed stimulating co-metabolism of the two fuels. This would explain the
that the blends B20 and B50 were more degraded than B5. observation that, in some cases, biodiesel may be applied in diesel-
The ranking of biodegradability by soil microorganisms is contaminated areas as a bioremediation-stimulating agent (Mudge
B100 > B20 > B50 > B5 > D100. and Pereira, 1999; Fernández-Álvarez et al., 2006; Zhang et al.,
In analyzing the degradation of the blends, we confirmed a 1998; Pasqualino et al., 2006). In contrast, DeMello et al. (2007)
more complex microbial degradation process. We observed that observed that the degradation of fossil fuel components in the
in diesel blend-contaminated soils, biodiesel seemed to facilitate marine environment was unaffected by the presence of biodiesel.
Analysis of the biodegradability of different types of fatty acid
Table 1 esters showed that non-saturated fatty acids, i.e., linoleic C18:2 and
Percentage of degradation of fatty acid esters (Et), diesel (D) and the sum of these linolenic C18:3 acids were more degradable than saturated fatty
components in blends B5, B20 and B50 after 60 days of incubation. acids (Table 2). The fatty acid ester C18:3 was the only biodiesel
Components Degradation (%) component that was 100% degraded in all samples. The esters com-
posed of stearate (C18:0) and oleate (C18:1) were significantly less
B5 B20 B50 B100 D100
biodegradable in the B50 and B100 blends (Table 2) than were the
Et 100 99 67 84 – fatty acids C18:2 and C18:3 (P ≤ 0.05). Palmitate (C16:0) had a lower
D 29 39 57 – 35
percentage of degradation only in the B50 blend.
Et + D 51 80 62 – –
The B50 blend exhibited lower rates of degradation of its fatty
The calculation was performed using the following formula: %degrada- acid esters. In blends B5 and B20, all the fatty acid esters were
tion = [(control peak-areas − test peak-areas)/control peak-areas] × 100. The
nearly 100% degraded. This may be related to differences between
peak areas in the mixtures correspond to the sums of peak areas of fatty acid esters
(Et) and peak areas of the components of diesel (D). Et + D corresponds to the total a highly complex mixture (diesel) and a simpler one (biodiesel).
degradation of blends. The B5 and B20 blends have a high concentration of diesel, which
34 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35

Fig. 9. Chromatogram of sample D100, showing the extent of degradation after 0, 30 and 60 days of incubation.

likely interfered with the degradation of the biodiesel constituents, DeMello et al. (2007), also in water, fatty acid methyl esters (FAMEs)
including the five constituent fatty acid esters of biodiesel. were degraded at approximately the same rate as the n-alkenes.
A high degree of unsaturation renders biodiesel chemically less Within seven days, the biodegradation in one of the three replicates
stable, so oxidation and subsequent degradation is more favorable was almost complete, and only traces of hydrocarbons (1–2% of the
(Parente, 2003; Mauro, 2007). However, it is not known if there initial amount) were detected in samples incubated for 31 days.
is a direct relationship between the degree of unsaturation of the The biodegradation of B20 in experiments by Prince et al. (2008)
fatty acid esters and the degradation of biodiesel. DeMello et al. was essentially complete and extremely rapid. The biodegradation
(2007) showed that the C16 fatty acid methyl ester was degraded of the fatty acid methyl esters and diesel hydrocarbon in B20 was
faster than the C18 fatty acid methyl ester and that the degrada- almost complete, as detected by GC, in contrast with the study by
tion rate between different C18 fatty acid methyl esters did not Penet et al. (2006) on the biodegradation of diesel in soil. This
correspond to their degree of unsaturation. In contrast, Miller and study found that only one out of 19 soil samples demonstrated
Mudge (1997), in experiments to determine the effectiveness of nearly complete biodegradation in 28 days. A possible explana-
bioremediation of biodiesel spills, found that several methyl esters tion of these contradictory results is that diesel hydrocarbons were
of C18 unsaturated fatty acids were degraded more rapidly than the aggregated to the organic matter of the soil and were thus less
methyl esters of saturated C16 fatty acids.Experiments by Prince bioavailable. This interpretation gives the soil composition a deci-
et al. (2008) with biodiesel-contaminated water showed substan- sive role in the biodegradation of diesel/biodiesel. As in Penet et al.
tial biodegradation within two days, and according to findings by (2006), we found that the rate of biodegradation for both pure diesel
and diesel/biodiesel blends was low. While B100 was almost com-
Table 2 pletely degraded (84%) after 30 days, D100 was only degraded 13.6%
Percentage of degradation of the fatty acid esters present in the blends B5, B20 and (Figs. 8 and 9). For the B5 and B20 blends, degradation of diesel was
B50 and pure biodiesel (B100) after 60 days of incubation. also low compared with pure biodiesel. After 30 days of incubation
Esters of fatty acids Degradation (%) only B100 was almost completely biodegraded; this was not the
case for all biodiesel blends and pure diesel. Our results thus con-
B5 B20 B50 B100
firm that the biodegradation of diesel and biodiesel in soil is much
Palmitate (C16:0) 100 100 59 86 slower than in water.
Stearate (C18:0) 100 91 23 31
Oleic (C18:1) 100 99 34 62
Linoleic (C18:2) 100 100 80 100 4. Conclusions
Linolenic (C18:3) 100 100 100 100

Percentage of degradation was calculated by as follows: %degradation = [(control Our results show that biodiesel is more biodegradable than
peak-areas − test peak-areas)/control peak-areas] × 100. diesel in contaminated soil and that a higher concentration of
 G.S. Silva et al. / Applied Soil Ecology 55 (2012) 27–35
biodiesel in blends favors the biological breakdown of diesel. B20
and B100 were degraded particularly well by soil microbes; B50,
however, while better degraded than pure diesel, was inferior
to the other two blends. Despite being degraded, pure biodiesel
adversely affected microbial community diversity, both in het-
erotrophic count and the number of bands observed in DGGE. The
effect of different mixtures on soil microbial diversity is unclear
and requires further investigation. Similarly, a deeper understand-
ing of the degradation process of biodiesel is needed for to properly
implement bioremediation strategies for biodiesel-contaminated
soils.
Acknowledgments
Research supported by Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq-Process no. 558272/2009-6) and
the Universidade Estadual de Santa Cruz (UESC). We thank Charles
Greer for reviewing the manuscript. The financial support granted
to Gislaine Santos Silva was provided by CNPq (Process no.
564757/2008-0).
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