Bioresource Technology 111 (2012) 328335

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Bioresource Technology
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Biodegradation of rhamnolipids in liquid cultures: Effect of biosurfactant

dissipation on diesel fuel/B20 blend biodegradation efciency and bacterial
community composition
ukasz Chrzanowski a,, Mariusz Dziadas b, ukasz awniczak a, Pawe Cyplik c, Wojciech Biaas c,
Alicja Szulc a, Piotr Lisiecki a, Henryk Jelen b
a
Institute of Chemical Technology and Engineering, Poznan University of Technology, Pl. M. Skodowskiej-Curie 2, 60-965 Poznan , Poland
b
, Poland
Institute of Food Technology of Plant Origin, Poznan University of Life Sciences, Wojska Polskiego 31, 60-624 Poznan
c
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Poznan , Poland

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

Article history: Bacterial utilization of rhamnolipids during biosurfactant-supplemented biodegradation of diesel and
Received 24 November 2011 B20 (20% biodiesel and 80% diesel v/v) fuels was evaluated under conditions with full aeration or with
Received in revised form 30 January 2012 nitrate and nitrite as electron acceptors. Rhamnolipid-induced changes in community dynamics were
Accepted 31 January 2012
assessed by employing real-time PCR and the ddCt method for relative quantication.
Available online 9 February 2012
The experiments with rhamnolipids at 150 mg/l, approx. double critical micelle concentration (CMC)
and diesel oil conrmed that rhamnolipids were readily degraded by a soil-isolated consortium of
Keywords:
hydrocarbon degraders in all samples, under both aerobic and nitrate-reducing conditions. The presence
Biodegradation
Biodiesel
of rhamnolipids increased the dissipation rates for B20 constituents under aerobic conditions, but did not
Diesel inuence the biodegradation rate of pure diesel. No effect was observed under nitrate-reducing condi-
Nitrate-reducing conditions tions. The biodegradation of rhamnolipids did not favor the growth of any specic consortium member,
Rhamnolipids which proved that the employed biosurfactant did not interfere with the microbial equilibrium during
diesel/biodiesel biodegradation.
2012 Elsevier Ltd. All rights reserved.

1. Introduction for soil washing processes, phytoextraction of heavy metals as well

Rhamnolipids are surface-active glycosides of biological origin,
composed of rhamnose and 3-hydroxy fatty acid moieties linked
via an O-glycosidic bond (Dziel et al., 1999). They are among
the most well investigated and comprehensively described biosur-
factants (Chrzanowski et al., 2012). Rhamnolipids have gained
increasing popularity in soil bioremediation as environmentally
friendly and efcient alternatives to synthetic surfactants, due to
their excellent physicochemical properties and considerably lower
toxicity. They can be produced from renewable sources by numer-
ous microorganisms (Nitschke et al., 2005), which contributes to a
large variety of homologs (Abdel-Mawgoud et al., 2010). Their abil-
ity to form emulsions and solubilize water-immiscible compounds
makes them suitable for various industrial and environmental
applications (Costa et al., 2010). Additionally, they can be blended
with other surfactants to obtain the desired characteristics (Urum
et al., 2006).
Rhamnolipids have been used in various processes such as ex
situ bioremediation of hydrophobic contaminants, ushing agents
Corresponding author. Tel.: +48 61 6653716; fax: +48 61 6653649.
E-mail address: lucaschrz@gmx.de (. Chrzanowski).
as oil spill remediation in contaminated aquatic and terrestrial
environments (Maier and Sobern-Chvez, 2000). Most of the
studies carried out with the use of rhamnolipids were mainly
focused on assessing their effect on the biodegradation efciency
of petroleum hydrocarbons (Makkar and Rockne, 2003; Whang
et al., 2008). Although several researchers observed increased
dissipation of target contaminant upon the addition of rhamnoli-
pids (Zhang and Miller, 1992; Maslin and Maier, 2000), a decrease
of biodegradation efciency or no effect caused by rhamnolipid-
supplementation were reported just as frequently (Deschnes et
al., 1996; Owsianiak et al., 2009a). It has been observed, that the
presence of surfactant molecules may induce changes in the micro-
bial community, which in turn corresponds to differences in degra-
dation patterns (Zhu et al., 2010). Many previous studies dealing
with the effect of synthetic surfactants on biodegradation of hydro-
carbons support the fact, that preferential utilization of surfactant
molecules may impact both community dynamics and hydrocar-
bon degradation rates (Colores et al., 2000; Snchez-Peinado
et al., 2010; Wyrwas et al., 2012). Interestingly, even though
rhamnolipids are considered to be biodegradable (Mulligan, 2009),
only few reports demonstrated that they may be co-degraded or
solely utilized as a source of carbon and energy by various

0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2012.01.181
 . Chrzanowski et al. / Bioresource Technology 111 (2012) 328335
monocultures (Mohan et al., 2006; Frank et al., 2010). Furthermore
the exact inuence of rhamnolipid degradation on hydrocarbon
dissipation efciency with respect to microbial community
dynamics has not yet been investigated.
We hypothesize, that rhamnolipids may be preferentially de-
graded by a consortium of hydrocarbon degraders due to structural
similarities between lipid moieties of rhamnolipids and fatty acid
moieties present in biodiesel. This phenomenon will result in dis-
appearance of surface active biosurfactant molecules, which will
further contribute to negative effect on the fuel biodegradation
efciency.
The main objective of the presented study was to assess the bio-
degradation of a rhamnolipids mixture during surfactant-mediated
biodegradation of diesel and diesel/biodiesel (containing 20% v/v
biodiesel in diesel) fuel carried out by a microbial consortium.
The shifts in abundance of specic consortium members were
determined by real-time quantitative PCR. In order to investigate
the possible utilization of rhamnolipids during short-term studies
with electron acceptors other than oxygen, nitrate was chosen, as
this electron acceptor offers a relatively fast reaction rate.
2. Methods
2.1. Fuels
Two different types of fuel were used in the experiments.
Petroleum diesel fuel, produced according to EN 590:2004, was
purchased from a petrol station (PKN Orlen, Poland). Biodiesel, pro-
duced from rapeseed oil according to DIN V 51606, was purchased
from a local supplier in Germany. B20 blend, containing 20% (v/v)
biodiesel in diesel fuel, was prepared by batching and mixing vol-
umetric portions of fuels. The fuels were ltrated (Millex, pore size
of 0.2 lm; Millipore) prior to the experiments.
2.2. Rhamnolipids
Commercial sample of rhamnolipids JBR425 from Jeneil Biosur-
factant Company (US) was used throughout the experiments.
2.3. Microorganisms
Bacterial consortium has been previously isolated from soil pol-
luted with crude oil under aerobic conditions employing diesel fuel
as sole carbon and energy source, identied and maintained as
described by Cyplik et al. (2011). The consortium contained phylo-
types belonging to the following bacterial taxa: Achromobacter sp.,
Alcaligenes sp., Citrobacter sp., Comamonadaceae, Sphingobacterium
sp., Pseudomonas sp., and Variovorax sp.
The consortium has been stored at 80 C in a 30% (v/v) glycerol
stocks. To prepare an inoculum, stock suspension (1 ml) was trans-
ferred to a 300 ml Erlenmeyer ask containing 50 ml of mineral
medium (composition in Owsianiak et al., 2009b) and diesel fuel
(0.5%, v/v), and was cultivated for 24 h at 25 C. Later, 1 ml aliquot
of the cell suspension was transferred to a new ask and the cul-
ture was grown for 3 days in the same conditions. This step was re-
peated three times and cells from the last culture were centrifuged
at 10,000  g, washed twice with 40 ml of mineral medium and
were used to inoculate the experimental samples. In all steps aer-
obic conditions were provided. Polymerase chain reaction denatur-
ing gradient gel electrophoresis (PCRDGGE) revealed that the
community in the culture used for inoculation was the same as
the communities in the parent glycerol stock (data not shown).
The ability of the microbial consortium to produce biosurfactants
under the experimental conditions was investigated by employing
the du Noy ring technique described by Grna et al. (2011).
329
2.4. Biodegradation experiments
Bottle sacrice technique was used for each analysis. Three bot-
tles were sacriced for each analysis.
2.4.1. Full aeration
Loosely closed 250 ml Duran-Schott bottles covered with alu-
minum foil contained 50 ml of mineral medium and fuels at 1.5%
(v/v). Initial inoculum was adjusted to OD600nm 0.1 0.01 by add-
ing approximately 1 ml of dense cell suspension from the precul-
ture grown aerobically on diesel fuel, and cultivation was carried
out at 25 C and 120 rpm. Rhamnolipids (JBR425) were supplied
at 150 mg/l equal to twice their CMC concentration to ask set
up for biosurfactant studies. Each day, a set of six independent rep-
licates run in parallel was sacricially sampled: three for hydrocar-
bon and FAME (Fatty Acid Methyl Esters), three for rhamnolipids
analyses. Cultures lacking biomass served as blanks to account
for abiotic loses.
2.4.2. Nitrate-reducing conditions
Tightly closed 250 ml Duran-Schott bottles contained 50 ml of
mineral medium, where NH4Cl (1 g/l) was replaced by NaNO3
(1 g/l) and Na2MoO4 (0.5 mg/l). Nitrate-reducing conditions were
achieved by ushing with nitrogen to remove dissolved oxygen
from the medium and the headspace. Cultivation conditions were
the same as in the fully aerobic set up, with cultures lacking bio-
mass serving as blanks to account for abiotic loses. In addition,
an independent set of duplicates run in parallel was set up for ni-
trate and nitrite analyses. All cultures were set up to an OD600 of
0.1 0.01 for each experimental condition.
2.5. Analytical procedures
2.5.1. Analysis of fuels
Residual hydrocarbons and/or FAME were extracted with hex-
ane (ISO, 2000) and analyzed with gas chromatography (GCFID),
as described elsewhere (Owsianiak et al., 2009b).
2.5.2. Analysis of nitrate and nitrite
Residual nitrate and nitrite were determined as described else-
where (Cyplik et al., 2011).
2.5.3. Analysis of rhamnolipids
The cell free cultivation broth (centrifuged at 10,000  g for
10 min.) was rinsed three times with hexane (3  5 ml) to extract
the residual hydrocarbons. The biomass was rinsed three times
with redistilled water (3  5 ml) in order to extract the adsorbed
rhamnolipids and the aliquots were combined with the cultivation
broth. Afterward 10 ml of the cultivation broth containing rhamn-
olipids were collected and lyophilized in vials. The dry matter was
derivatized according to the method described by Schenk et al.
(1995), by dissolving in 1 ml of dry acetonitrile, which contained
p-bromophenacyl bromide and triethylamine in a molar ratio of
1:4:2 (glycolipids: p-bromophenacyl bromide:Et3N). The derivati-
zation reaction was carried out at room temperature for 18 h in a
tightly closed vial with nitrogen as an inert gas. The obtained p-
bromophenacyl rhamnolipid esters were then ltered (0.22 lm)
and analyzed directly by HPLC.
The analysis was carried out using Aglient 1100 liquid chro-
matograph equipped with a DAD detector and an Eclipse Plus
C18 4.6  75 mm, 3.5 lm column. Water (solvent A) and acetoni-
trile (solvent B) was used as the mobile phase: 1 min, A:B, 30:70
(%), 5 min gradient from 30:70 to 0:100 (%), on hold for 5 min, bal-
ancing of the column for 3 min to 30:70. The analysis was carried
out at 25 C with a ow rate of 2 ml per min at 265 and 320 nm.
330 . Chrzanowski et al. / Bioresource Technology 111 (2012) 328335
The separation was carried out on a chromatographic column
lled with silica gel (50 g, 4063 lm) with the use of chloro-
form:methanol:water solvent mixture as the mobile phase
(65:15:2, v/v/v). One gram of the JBR425 rhamnolipids mixture
was frozen, lyophilized and afterward the dry matter was dissolved
in 3 ml of the mobile phase. The obtained fractions were monitored
by TLC (Thin Layer Chromatography) according to the method de-
scribed by Wang et al. (2007). Fractions containing mono-rhamn-
olipids (Rf = 0.69) and di-rhamnolipids (Rf = 0.43) after HPLCMS
studies (Wang et al., 2007) were used as standards for HPLC
analysis.
Analyses of rhamnolipids revealed that JBR425 consist of six
major congeners: 42.0% RhaRhaC10C10, 43.9% RhaC10C10,
11.9% both of RhaRhaC10C12 and RhaRhaC12C10 and 2.2%
both of RhaRhaC10C12:1 and RhaRhaC12:1C10. This corre-
sponds well with the precise description given by Wang et al.
(2007). The same Authors also reported limited separation of
(RhaRhaC10C12/RhaRhaC12C10) and (RhaRhaC10C12:1/
RhaRhaC12:1C10) pairs during HPLC determination. Therefore a
simplication was made and each of those pairs was considered
as a sum of both constituents.
2.6. Bacterial community dynamics
Bacterial community dynamics was studied with relative quan-
titative real-time PCR and the ddCt (delta-delta-Ct algorithm, a
specic approximation method) method (Livak and Schmittgen,
2001) as described elsewhere (Cyplik et al., 2011). To compare
the community dynamics the amount of target rRNA genes in a
culture grown in the presence of rhamnolipids for seven days
was determined by normalizing to the total bacteria number in
the culture of interest without biosurfactant.
2.7. Data processing
The results obtained during the experiment were normalized
with the following equation:
Ct
Y  100 1 to rhamnolipids supplementation.
C t0
where: Y percentage value that describes normalized residual
concentration, its values range <0, 100>, Ct concentration after
the time t [mg/l], Ct=0 concentration at the beginning of the
experiment [mg/l].
Based on the initial experimental data, it was concluded, that
linear regression was most t for further modeling of rhamnolipid
biodegradation as well as the studied fuel types. Hence, the depen-
dence of the residual concentration Y on the time of the process t in
the examined system was described with a linear equation:
Y a  x  b
where: a slope (Dx/Dy); b time needed for complete dissipa-
tion (x intercept when y = 0); SE standard error; R2 coefcient
of determination. Calculations were carried out in STATISTICA 6.0
PL software.
3. Results and discussion
3.1. Biodegradation of fuels
3.1.1. Fuels without rhamnolipids
In order to test our hypothesis regarding the similarities
between lipid moieties of rhamnolipids and fatty acid moieties
present in biodiesel, it was essential to distinguish the biodegrada-
tion efciency of both diesel and biodiesel in B20 blends (Fig. 1).
The results obtained for aerobic experiments conrmed that
biodiesel is the preferentially biodegraded fuel type compared to
diesel. Similar results were obtained by Cyplik et al. (2011). In sam-
ples without biosurfactants biodiesel was dissipated at a faster rate
(a = 0.402%/h) compared to pure diesel oil (a = 0.190%/h), while
the removal of diesel oil in B20 blend occurred at a notably slower
rate (a = 0.080%/h).
For both diesel and B20 fuel types reduced degradation rates
were observed under nitrate-reducing conditions. Diesel fuel degra-
dation reached 20% of that noted for aerobic conditions (a =
0.037%/h). In the case of B20 blend biodiesel was also utilized
50% less effectively compared to aerobic degradation within 7 days
(a = 0.212). Interestingly, diesel oil in the B20 blend was biode-
graded at a similar rate (a = 0.066).
3.1.2. Fuels with rhamnolipids
No inuence of rhamnolipids supplementation could be
observed under nitrate-reducing conditions. Both diesel as well
as diesel/biodiesel utilization rates were unaffected by the
presence of the biosurfactant (Fig. 1).
On the other hand, under aerobic conditions, supplementation
with rhamnolipids contributed to an apparent increase in the uti-
lization rate for both biodiesel (a = 0.878%/h) and diesel in B20
(a = 0.145%/h) blend. A complete degradation of B20 biodiesel oc-
curred after approx. 5 days, followed by increased dissipation of
diesel constituents. Overall, the biodegradation rate of B20 (both
diesel and biodiesel) under aerobic conditions was enhanced by
approx 50% after 7 days as a result of biosurfactant amendment.
Interestingly rhamnolipids did not enhance the biodegradation
efciency of pure diesel oil (a = 0.200%/h) as shown in Table 1.
Our observations for pure diesel oil seem to contradict with
many reports that demonstrated stimulating effects of biosurfac-
tants on hydrocarbon degradation (Maier and Sobern-Chvez,
2000; Whang et al., 2008). However contradicting results regarding
the rhamnolipids-induced facilitation or retardation of biodegrada-
tion processes have been previously reported (Mulligan, 2009). For
example such results were presented by Mata-Sandoval et al.
(2001) when they observed limited degradation of pesticides due
The fact that a complete depletion of nitrate occurred only dur-
ing microbial growth on B20 and not during growth on pure diesel
oil is also worth noticing (Fig. 2). This contributes to enhanced deg-
radation of biodiesel/diesel blends, what is usually linked with bio-
diesel content (Mariano et al., 2008). However the depletion of
nitrate and nitrite occurred faster for samples containing rhamnoli-
pids (approx. 5 and 7 days for nitrate and nitrite, accordingly),
compared to samples without the addition of the biosurfactant (ap-
prox. 7 days for nitrate and incomplete depletion of nitrite). This
fact may either be connected with increased biodegradation of fuels
in the presence of the biosurfactants, which was not the case (as
proved by GC analysis), or it might be associated with utilization
of rhamnolipids. Thus it was crucial to determine the rhamnolipids
concentration parallel to diesel/biodiesel degradation.
3.2. Monitoring rhamnolipids concentration during biodegradation of
fuels
Initial experiments were carried in order to nd out whether
the microbial consortium was able to synthesize its own rhamnoli-
pids. No changes in the interfacial tension values were observed
and further HPLC studies conrmed that rhamnolipids were not
produced (data not shown). The assessment of rhamnolipids dur-
ing biodegradation tests demonstrated that under aerobic condi-
tions approx. 90% of analyzed JBR425 components were readily
utilized by microorganisms within 7 days (Fig. 3).
 . Chrzanowski et al. / Bioresource Technology 111 (2012) 328335 331

Fig. 1. Biodegradation of diesel starting from 630 mg per sample (a) and diesel/biodiesel blend (B20) presented as biodegradation of diesel fraction starting from 500 mg
per sample (b) and biodiesel fraction starting from 130 mg per sample (c) in liquid cultures: j nitrate-reducing conditions, d nitrate-reducing conditions and
rhamnolipids, h full aeration, s full aeration with rhamnolipids. Results are averages from triplicates.

Table 1
Model parameters describing the dissipation rate of diesel, diesel in B20 and biodiesel in B20.

Fuel type Biodegradation experiments Model parameters R2

aa SE [%/h] bb SE [h]
Diesel O2 0190 0016 523 36,9 0960
O2 + Rhamnolipids 0200 0013 495 27,8 0974
NO3 0037 0004 2677 297,9 0927
NO3 + Rhamnolipids 0046 0006 2165 281,4 0948
Diesel in B20 blend O2 0080 0007 1240 96,0 0960
O2 + Rhamnolipids 0145 0025 665 103,8 0915
NO3 0066 0005 1519 115,6 0963
NO3 + Rhamnolipids 0086 0003 1166 43,0 0995
Biodiesel in B20 blend O2 0402 0054 236 21,7 0903
O2 + Rhamnolipids 0878 0119 117 10,7 0964
NO3 0212 0033 472 60,7 0874
NO3 + Rhamnolipids 0259 0024 385 29,8 0974
a
a slope (Dx/Dy).
b
b time needed for complete dissipation (x intercept when y = 0).

Fig. 2. Depletion of nitrate (a) and production/utilization of nitrite (b) j diesel, h diesel with rhamnolipids, d diesel/biodiesel blend (B20), s diesel/biodiesel blend
(B20) with rhamnolipids. Results are averages from triplicates.

By performing linear regressions for dissipation curves it was
predicted that a complete degradation of all components would
likely occur between the 8th and 10th day. Apparently all compo-
nents were degraded at a similar rate with the exception for Rha
RhaC10C10. Similar biodegradation susceptibility for two major
rhamnolipids constituents (RhaC10C10, RhaRhaC10C10) was
previously reported by Hudak and Cassidy (2004), however under
anaerobic conditions.
However under nitrate-reducing conditions the estimated
complete utilization of biosurfactant varied both for rhamnolipids
species and fuel type used, reaching an average of 35 days for pure
diesel and 27 days for B20 blend (Table 2). Interestingly, faster
degradation occurred exclusively for di-rhamnolipids with either
a saturated or unsaturated C12 3-hydroxy fatty acid moiety
(RhaRhaC10C12 and RhaRhaC12C10 as well as RhaRhaC10
C12:1 and RhaRhaC12:1C10), which together contributed to only
14% of the entire rhamnolipids amount. Both major components
of rhamnolipids (RhaC10C10, RhaRhaC10C10) were dissipated
at a much slower rate compared to RhaRhaC10C12/RhaRha
C12C10 and RhaRhaC10C12:1/RhaRhaC12:1C10 pairs.
The obtained results suggest that during rhamnolipids-supple-
mented biodegradation of diesel fuel in aerobic conditions the bio-
surfactant molecules were dissipated at a faster rate (a = 0.565%/
h) compared to diesel (a = 0.200%/h), which corresponds to
complete degradation after 8 and 21 days, accordingly.
Different observations were made during rhamnolipids-supple-
mented biodegradation of diesel/biodiesel fuel in aerobic
conditions, as biodiesel dissipated more rapidly (a = 0.878%/h)
332 . Chrzanowski et al. / Bioresource Technology 111 (2012) 328335

Fig. 3. Dissipation proles for RhaC10C10 starting from 3.29 mg per sample (a), RhaRhaC10C12:1 and RhaRhaC12:1C10 starting from 0.17 mg per sample (b), RhaRha
C10C10 starting from 3.15 mg per sample (c), RhaRhaC10C12 and RhaRhaC12C10 starting from 0.89 mg per sample (d) and general plot for JBR425 starting from 7.5 mg
per sample (e) in cultures grown on: j diesel, limited aeration with nitrate, d biodiesel/diesel (B20), limited aeration with nitrate, h diesel, full aeration, s biodiesel/
diesel (B20), full aeration.

compared to rhamnolipids (a = 0.456%/h) and diesel oil (a =
0.145%/h). The presence of biodiesel contributed to the decrease
of both rhamnolipids and diesel oil degradation rate. Hydrolysis of
FAME results in the formation of fatty acids, which may be easily
incorporated into cellular processes and structures, therefore bio-
diesel present in B20 blend was preferentially biodegraded com-
pared to diesel oil (as conrmed in Cyplik et al., 2011) and
rhamnolipids. Since the rhamnolipid molecules employed in this
study consisted of two hydroxy fatty acid moieties linked with
rhamnose moieties, their resemblance to fatty acid methyl esters
may be the cause of their preferential degradation compared to die-
sel oil hydrocarbons.
The biodegradation susceptibility order can be established as
follows: biodiesel > rhamnolipids > diesel oil. The complete degra-
dation time for the abovementioned carbon sources was estimated
to 5, 10 and 28 days, accordingly. Similar results were observed for
nitrate-reducing experiments, although the degradation processes
were carried out at a much slower rate. Again the biodegradation
priority was highest for biodiesel, followed by rhamnolipids and -
nally diesel oil.
Structural similarities between rhamnolipids and fatty acid
methyl esters might explain why the introduction of rhamnolipids
into aerobic cultures strongly affected fuel dissipation patterns
for B20 but not for pure diesel oil, thus resulting in enhanced
biodegradation of FAME upon rhamnolipid-supplementation. One
possible explanation involves short-term stimulation of the growth
of bacteria degrading FAME but not degrading other hydrocarbons.
This might have contributed to enhanced biodegradation of
rhamnolipids, owing to the structural similarities mentioned in
the manuscript. Another cause may be explained by possible
co-metabolic transformation of biodiesel and rhamnolipids, which
affected the biodegradation of these compounds in a similar matter
to that observed for biodiesel and diesel (Pasqualino et al., 2006).
In the light of our observations regarding the preferential utili-
zation of rhamnolipids over diesel hydrocarbons, rhamnolipid-
supplementation may have a negative impact on environmental
biodegradation trials. Sole degradation of rhamnolipids was exten-
sively studied by Mohan et al. (2006) and it was concluded that
this biosurfactant was biodegraded rapidly under aerobic condi-
tions whereas under anaerobic conditions with both nitrate and
sulfate as electron acceptors its dissipation was remarkably slower.
Interestingly Mohan et al. (2006) stated that the conducted
research was aimed to show that microorganisms can indeed
degrade the surfactants, however after the biodegradation process.
Similar concept regarding biosurfactant biodegradation following
the dissipation of facilitated hydrocarbon components was previ-
ously reported by Oberbremmer et al. (1990). This approach seems
to be questionable, since surfactant molecules do not act solely as
 . Chrzanowski et al. / Bioresource Technology 111 (2012) 328335 333

Table 2
Model parameters describing the dissipation rate of RhaC10C10, RhaRhaC10C12:1 and RhaRhaC12:1C10, RhaRhaC10C10, RhaRhaC10C12and RhaRhaC12C10 and
summarized data for JBR425.

Residual rhamnolipids (%) Biodegradation experiments Model parameters R2

a b
a SE [%/h] b SE [h]
RhaC10C10 DO2 0670 0086 166,66 12,80 0909
DNO3 0101 0010 970,44 85,11 0948
B20O2 0595 0090 194,68 18,66 0880
B20NO3 0072 0014 1350,11 253,57 0806
RhaRhaC10C12:1 RhaRhaC12:1C10 DO2 0590 0059 167,23 11,41 0961
DNO3 0450 0065 214,62 20,63 0887
B20O2 0480 0064 195,68 16,55 0904
B20NO3 0438 0029 240,66 10,90 0975
RhaRhaC10C10 DO2 0478 0066 231,89 21,95 0896
DNO3 0058 0010 1718,64 278,19 0852
B20O2 0292 0099 376,32 100,35 0954
B20NO3 0129 0023 769,22 120,70 0844
RhaRhaC10C12 RhaRhaC12C10 DO2 0561 0057 182,87 11,42 0942
DNO3 0297 0034 315,23 27,02 0928
B20O2 0495 0043 210,15 12,03 0956
B20NO3 0313 0044 336,28 35,83 0912
Rhamnolipids all DO2 0565 0050 194,08 10,86 0955
DNO3 0119 0011 829,46 69,96 0950
B20O2 0456 0061 242,73 22,53 0903
B20NO3 0154 0001 649,58 5,34 0995
a
a slope (Dx/Dy).
b
b time needed for complete dissipation (x intercept when y = 0).

mediators between hydrocarbon molecules and microbial cells,

that otherwise stay intact during the biodegradation process
(Deschnes et al., 1996). The results obtained by Maslin and Maier
(2000) conrmed that rhamnolipids may serve as a preferred car-
bon source. Our results, particularly for aerobic degradation of pure
diesel and B20 diesel fuels in the presence of rhamnolipids,
strongly support the idea of biosurfactants being degraded prior
to the utilization of diesel hydrocarbons. Many reports revealed
that bioavailability of organic compounds solubilized in micelles
to microbial cells is negligible. This was proven for nonionic surfac-
tants, like Triton X-100 (Dai et al., 2010), and also for rhamnolipids
(Chrzanowski et al., 2009). Therefore it may be plausible that
primary degradation of rhamnolipids forming micelles and aggre-
gates is a key process for further utilization of fuel substrates under
aerobic conditions.
The available data concerning the applications of surfactants
under anaerobic conditions is limited and even fewer studies deal
with rhamnolipids. Bregnard et al. (1998), reported that no stimu-
lation of weathered diesel fuel biodegradation occurred under
anaerobic conditions after addition of either rhamnolipids or Triton
X-100 at above CMC concentrations. Similar results were observed
during our studies, however under nitrate-reducing conditions.
The use of rhamnolipids seemed to have very limited inuence
on both diesel and B20 blend utilization rate. It was concluded that
the denitrifying biodegradation of weathered diesel fuel was pre-
sumably not limited by bioavailability of hydrocarbons but by
the slower reaction rate under nitrate-reducing conditions.
Nevertheless, the fact that biodegradation of rhamnolipids and
no enhancement of fuel dissipation rates were observed under ni-
trate-reducing conditions may be a crucial factor inuencing their
applicability during actual bioremediation processes, where low
concentration of oxygen is a common issue.
For most of the experiments dealing with degradation of hydro-
carbons, rhamnolipids were commonly applied at concentrations
similar to the one employed in this study i.e. Whang et al. (2008)
used rhamnolipid concentrations ranging between 80 and
160 mg/l, while Zhang and Miller (1992) employed rhamnolipids
at 100300 mg/l. Faster decrease of nitrate and nitrite concentra-
tions observed in this study for rhamnolipid-supplemented
samples, which was most likely associated with the degradation
of rhamnolipids, may lead to the depletion of electron acceptors,
thus contributing to a decreased degradation rate of target pollu-
tants. However it should be emphasized that other electron accep-
tors, such as ferric iron, sulfate or carbonate, are more relevant
during long-term eld studies. Therefore it is plausible that differ-
ent results may be observed in the presence of other electron
acceptors.
3.3. Bacterial community dynamics
In order to investigate how the utilization of rhamnolipids dur-
ing the biosurfactant-supplemented biodegradation of diesel and
B20 fuels inuenced the structure of the microbial consortium,
the changes in the abundance of specic consortium members
were studied (Fig. 4).
The observed changes in the number of rRNA genes among con-
sortium members with respect to different fuel types (either diesel
or biodiesel) were mostly within one order of magnitude. This is
also true when comparing aerobically grown cultures with those
grown under nitrate-reducing conditions. This fact suggests that
the community structure was not notably altered under all studied
experimental conditions and that the consortium remained stable.
Overall, the depletion of rhamnolipids was not associated with
shifts in the abundance of specic species. It is plausible, that
due to structural similarities between lipid moieties of rhamnoli-
pids and FAME the microbial consortium members were able to
shift their metabolism from biodiesel to rhamnolipids, what would
explain why no substantial changes in community dynamics could
be observed.
In contrast, several studies presented that the addition of syn-
thetic surface active agents may have a profound impact on micro-
bial community dynamics. For example Snchez-Peinado et al.
(2010) reported that the structure of the Alphaproteobacteria
community was signicantly changed while evaluating the effects
of linear alkyl sulfonates (LAS) on the community structure of
Alphaproteobacteria, Actinobacteria and Acidobacteria in an agricul-
tural soil. Similar effects were observed during surfactant-
mediated biodegradation trials, where changes in the community
structure were connected with decreased biodegradation ef-
334 . Chrzanowski et al. / Bioresource Technology 111 (2012) 328335
Fig. 4. Changes in bacterial community monitored by relative quantitative (ddCt) real-time PCR during biodegradation of fuels under conditions with full aeration (left) or
limited aeration with nitrate (right). Biomass from cultures grown on appropriate fuel type collected at day 7th served as reference for cultures grown with rhamnolipids.
Achromobacter sp. (AchrP); Alcaligenes sp. (AlcP); Citrobacter sp. (CKK); Comamonadaceae (ComP); Sphingobacterium sp. (SphiP); Pseudomonas sp. (PseuP); Variovorax sp.
(VariP). Standard deviations of the relative quantity for bacterial taxa in the reference culture (RQ = 1) are given in brackets: AchrP (0.5384.159); AlcP (0.0740.654); CKK
(0.3774.898); ComP (0.0170.544); PseuP (0.0460.294); SphiP (0.1970.745); VariP (0.1610.559).
ciency of target pollutants (Chang et al., 2008; Zhu et al., 2010).
Such results indicate that addition of synthetic surfactants may
have substantial, differential effects on populations of organisms
responsible for degradation of contaminants within a microbial
community, whereas the use of rhamnolipids did not inuence
the composition of the microbial consortium. The use of rhamnoli-
pids seems to be an environmentally friendly solution, however it
should be considered that the presented experiments were carried
out under articial laboratory conditions, which differ from actual
environmental conditions. Although no notable shifts in bacterial
community dynamics occurred, it is plausible that different results
may be observed for other microbial consortia. Moreover, the use
of monocultures or mixed cultures may result in other effects.
The potential environmental implications of this study may be
presented in a form of a single question: is the addition of rhamn-
olipids in order to stimulate the biodegradation of target pollutants
a valid strategy? We believe that the answer is unequivocal, since
the problem is strongly related to the conditions of the biodegrada-
tion process. The obtained results suggest that the biosurfactant
was preferentially degraded and no enhancement of diesel oil
was observed, hence the addition of rhamnolipids seems to be
unjustied. On the other hand it should be emphasized that nota-
bly different results may be obtained under actual environmental
conditions. The introduction of rhamnolipids into soil containing
weathered hydrocarbons bound with the soil matrix may prove
to be a promising approach, since enhanced biodegradation may
occur due to solubilization and increased bioavailability of other-
wise inaccessible compounds. Future studies should be focused
on evaluating the inuence of rhamnolipids on biodegradation of
diesel/biodiesel fuels in soil.
4. Conclusions
The addition of rhamnolipids into samples containing either
diesel or biodiesel fuel did not enhance the biodegradation
efciency of petroleum hydrocarbons. On the other hand rhamn-
olipids were readily degraded by a soil-isolated consortium of
hydrocarbon degraders in all samples, under both aerobic and
nitrate-reducing conditions. The biodegradation susceptibility or-
der can be established as follows: biodiesel > rhamnolipids > diesel
oil. Since the biodegradation of rhamnolipids seems to be preferred
while no stimulation of diesel degradation rate was observed, this
process may potentially cause a decrease of electron acceptors in
the contaminated environment, which could otherwise be utilized
during degradation of petroleum pollutants. These ndings may be
of importance for all those interested in using rhamnolipids for
enhanced biodegradation of diesel oil. The obtained results suggest
that rhamnolipids-mediated biodegradation is not a universal
strategy and numerous environmental and microbial factors
should still be considered. The validation of such factors and their
impact on biodegradation of diesel oil during actual environmental
clean-up attempts is an important challenge for future research.
Acknowledgements
A.S. and .C. were supported by the Polish Ministry of Science
and Higher Education through Grant No. N N305 035434, years
2008-2010. A.S., P.L and .C. were supported by Cooperation grant
PUT-PULS 32-087/09-BW and by Ict 32-194-11 DS-MK. Dr. Iren-
eusz Miesiac (PUT) is gratefully acknowledged for constant supply
with biodiesel.
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