International Biodeterioration & Biodegradation 65 (2011) 1095e1099
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation
journal homepage: www.elsevier.com/locate/ibiod
Short communication
Phylogenetic characterization of bioemulsifier-producing bacteria
Andrea Franzetti a, *, Isabella Gandolfi a, Valentina Bertolini a, Chiara Raimondi a, Marco Piscitello a,
Maddalena Papacchini b, Giuseppina Bestetti a
a
b
Dept. Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, Milano, Italy
ISPESL, Dept. for Production Premises and Interaction with Environment, via Fontana Candida 1, 00040 Monteporzio Catone (RM), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 November 2010
Received in revised form
27 December 2010
Accepted 12 January 2011
Available online 19 August 2011
Bacteria able to produce biological emulsifiers were isolated from different environments using different
isolation media with the aim of discovering the widest diversity. The phylogenetic diversity of the
isolates was evaluated by 16S rRNA gene analysis. Among 190 isolated strains, 127 released extracellular
emulsifiers able to stabilize oil-water emulsions when grown on low-cost substrates. Among these, the
35 isolates that showed the highest emulsifier production on different substrates were found to belong to
16 different bacterial genera. Overall, this is the first systematic study of the diversity of bioemulsifierproducing bacteria and of their ability to produce bioemulsifiers on low-cost substrates.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Surface-active compound
Biosurfactant
Bioemulsifier
Renewable substrate
Screening
Diversity
1. Introduction
Surface active compounds (SACs) are amphiphilic molecules
containing both hydrophilic and hydrophobic moieties. Neu (1996)
classified SACs into low-molecular-weight SACs, also termed biosurfactants, and high-molecular-weight SACs, including amphiphilic and polyphilic polymers, also termed bioemulsifiers. The
former lower the interfacial tensions of the liquid in which they are
dissolved, whereas the latter are not able to efficiently reduce
interfacial tension; instead, they firmly stabilize oil/water emulsions. A variety of microorganisms produce high-molecular-weight
bioemulsifiers (Satpute et al., 2010a); the best investigated among
them are bioemulsans, which are synthesized by various species of
Acinetobacter. Among these, the first studied compound was RAG-1
emulsan, an amphiphilic polysaccharide produced by Acinetobacter
calcoaceticus, which is also the only commercially available bioemulsifier at present (Rosenberg et al., 1979).
Over the past few years, microbial SACs have received increasing
commercial attention as substitutes for synthetic surfactants owing
to their properties (such as high surfactant and emulsifying activities and stability in extreme physico-chemical conditions) and
advantages (such as lower toxicity and higher biodegradability).
Thanks to these features, microbial SACs are more acceptable,
compared to synthetic surfactants, in various applications, such as
those in the oil industry, microbial-enhanced oil recovery, environmental remediation, oil transportation, tank cleaning, agriculture, medicine, and the cosmetic and food industries (Banat et al.,
2010). The environmental distribution and diversity of lowmolecular-weight SAC-producing bacteria have already been
studied (Bodour et al., 2003; Ruggeri et al., 2009). Moreover, the
recent attention given to the de-emulsifying activity of biological
SACs has led to the publication of studies on the characteristics,
diversity, and distribution of this type of microorganism (Huang
et al., 2009, 2010). Despite the potential for a wide range of applications, the diversity of high-molecular-weight SAC-producing
bacteria has been poorly studied. A systematic study of their
distribution in the environment has not been carried out so far.
The aims of this paper were to study the microbial diversity and
phylogenetic relationships between bioemulsifier-producing
bacteria and the effect of environmental sampling, isolation
media, and low-cost substrates on their isolation.
2. Materials and methods
2.1. Environmental samples
* Corresponding author. Tel.: þ39 02 6448 2927; fax: þ39 02 6448 2996.
E-mail address: andrea.franzetti@unimib.it (A. Franzetti).
0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2011.01.014
Various environmental samples were used to screen for
bioemulsifier-producing bacterial strains. These include a previously
1096
A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e1099
characterized high-quality compost (C) (Gandolfi et al., 2010),
a polycyclic aromatic hydrocarbons (PAHs) e contaminated soil (I), a
diesel-oil-contaminated soil (G), and a heavy-metals-contaminated
soil (M). Soils I and G had previously been characterized by
Gandolfi et al. (2010) and the characteristics of the heavy-metalcontaminated soil were recently reported by Cao et al. (2007).
Furthermore, bioemulsifier-producting bacteria were also screened
from a laboratory-enriched culture on hydrocarbons (S). The
enrichment culture was prepared from a soil sample collected from
a diesel-oil-contaminated site in northern Italy. The concentration of
hydrocarbons (C > 12) in this soil was 3000 mg kg 1. Finally, the
isolation of bioemulsifier-producing bacteria was also carried out
from two commercial preparations that are sold for bioremediation
purposes: ECORPOLL/L1 (M1) and ECORPOLL/L2 (M2), commercialized by GIO.ECO srl (Segrate, Italy).
The emulsification assay (EA) was carried out as previously
reported (Franzetti et al., 2008). The height of the emulsion was
measured with respect to the height of the solution, and the degree
of emulsification was classified as follows: 0e10%: 1; 10e20%: 2;
20e50%: 3; >50%: 4; >50% with fine emulsion: 5.
In order to distinguish between bioemulsifiers and lowmolecular-weight SACs, the oil spreading technique (OST) was
carried out as previously reported (Ruggeri et al., 2009). Isolates
with a degree of emulsification greater than the control and
negative in the OST were considered high-molecular-weight SAC
producers (Satpute et al., 2010b). Strains that produced bioemulsifiers on more than one substrate and that showed a difference greater than one between the degrees of emulsification of the
broth and the control were classified as “best producers.”
2.4. 16S rRNA gene sequence analysis
2.2. Isolation procedures
The isolates were obtained by dilution and plating on seven
different isolation media that were solidified with agar. Four media
were based on the oligotrophic VL55 mineral medium (Sait et al.,
2002; Joseph et al., 2003) specifically designed for isolating previously uncultured bacteria. The VL55 medium was separately
amended with either one of the four different mixtures of carbon
sources, as reported in Table 1. Nutrient broth (Biolife, Italy) was
used diluted by 10- or 100-fold (NB10 or NB100); the Luria-Bertani
(yeast extract: 5 g l 1, tryptone: 10 g l 1, NaCl: 5 g l 1) (LB) medium
was used undiluted.
The compost and the contaminated soil samples were plated
onto all of the isolation media while the enrichment culture and
commercial preparations were only plated onto LB media. The agar
plates were incubated at 30 C for 2e10 days to allow colony
growth. Colonies with different morphologies were then picked
and transferred to a fresh agar plate until a pure culture was
obtained. Each isolate was named based on the starting environmental sample, the isolation medium, and the isolation dilution as
follows: acronym of the environmental sample.dilution.acronym of
the isolation medium.ID of the isolate (e.g., D.4.VLAA.8).
2.3. Screening of bioemulsifier-producing strains
Cells for monitoring bioemulsifier production were grown in
either NB10, NB100, or LB broth. They were then washed twice and
resuspended in BH2 mineral medium at an optical density
(600 nm) of 1 (Franzetti et al., 2008). Each different carbon source
(sugar-beet molasses, brewery wastes, ricotta cheese whey, and
glycerol) was filtered, sterilized, and supplied at an initial concentration of 5.0 g l 1. The cultures were incubated at 200 rpm, 30 C,
for six days.
Table 1
The isolation media used in this study.
Isolation medium
Basal medium
Carbon source
VLZ
VL55
VLA
VL55
Arabinose 1.5 g l 1, xylose 1.5 g l 1
glucose 1.8 g l 1, galactose 1.8 g l 1
Ascorbic acid 1.76 g l 1, galacturonic
acid 1.12 g l 1, glucuronic acid 1.94 g l
sodium gluconate 2.18 g l 1
Sodium acetate 0.8 g l 1, sodium
lactate 1.12 g l 1, methanol 0.32 g l 1
Xylane 0.05% w/v
VLAA
VL55
VLX
LB
NBD
NBDD
VL55
LB
NB 1:10
NB 1:100
1
Colony PCR was carried out using Com primers (Schwieger and
Tebbe, 1998) as previously reported (Gandolfi et al., 2010). Taxonomic assignments of sequences were performed using the Ribosomal Database Project (RDP) classifier (Wang et al., 2007). The
nearest relative sequences in GenBank were retrieved using BLAST
(Zhang et al., 2000). A phylogenetic tree was drawn using the
software program MEGA, version 4, by the neighbour-joining
method (Tamura et al., 2007).
3. Results and discussion
A total of 190 different isolates were screened: 127 isolates were
positive for bioemulsifier production, while seven strains were
positive for both EA and OST and were considered low-molecularweight SAC-producers.
3.1. Effect of environmental samples, isolation media, and carbon
sources on bioemulsifier production
Notably high percentages of positive isolates were retrieved for
all environmental samples. The commercial mixtures for bioremediation and the enriched culture on hydrocarbons showed the
highest percentages. Approximately 65e70% of the isolates from
the hydrocarbon-contaminated soils (diesel oil and PAHs) were
bioemulsifier producers. These high percentages are in agreement
with the reported role of microbial surface active compounds in
hydrocarbon uptake (Van Hamme et al., 2006). However, these
values are also comparable with the percentage obtained for the
high-biodiversity compost (58%), as expected from the significant
number of hydrocarbon-degrading microorganisms previously
found in the compost (Gandolfi et al., 2010). The lower percentage
of positive isolates retrieved for the metal-contaminated soil (39%)
suggests that the high concentrations of Pb and Zn did not select for
bacteria able to produce chelating bioemulsifiers as a protection
against metal toxicity. This is consistent with the reported dominance of metal-susceptible bacteria in the soil (Cao et al., 2007).
Each isolate was tested for emulsifier production on four
different low-cost substrates (sugar-beet molasses, glycerol,
brewery wastes, ricotta cheese whey). The chosen substrates have
distinct chemical compositions and have been extensively used for
biosurfactant production (Makkar and Cameotra, 2002). Among
them, glycerol emerged as one of the most important potential
feedstocks, available in large quantities as a by-product of the
biodiesel process (Zheng et al., 2008).
All of the isolates were able to grow on at least one of the tested
substrates. All of the substrates allowed the production of bioemulsifiers to a good extent. Glycerol and molasses were the best
substrates (36% and 27%, respectively).
1097
A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e1099
3.2. Taxonomy and phylogeny of the best producers of
bioemulsifiers
Extracellular polymeric substances that can potentially form
stable emulsions between oil and water are produced by many
microorganisms (Satpute et al., 2010a). In order to avoid overclassification of the microorganisms as bioemulsifier producers,
only the isolates with an ability to produce bioemulsifiers on at
least two substrates and with a level of emulsification above
a certain threshold (see Materials and methods section) were
considered.
Table 2 shows the nearest relatives in GenBank, the RDP classification, and the extent of emulsification of the best producers. It
is likely that some of the isolates with the same sequences and from
the same environmental samples were clones. However, they
showed slight differences in emulsification. The use of a typing
technique to distinguish between the strains was beyond the scope
of this work. The presence of 16 genera from the 35 isolates
suggests that there is a wide biodiversity of bioemulsifierproducing bacteria. Bacillus, Acinetobacter, and Rhodococcus are
the best known bacterial groups for biosurfactant and bioemulsifier
production and they were also the most commonly represented
genera in our screening. Bradyrhizobiaceae/Bradyrhizobium were
also dominant bacterial groups found among the “best producers.”
As previously reported, the production of extracellular polymers
has been extensively demonstrated in rhizobia, even though the
surface properties and applicability of these compounds have not
yet been investigated (Skorupska et al., 2006; Ruggeri et al., 2009).
To the best of our knowledge, we are the first to add the following
eight genera to the list of already described bioemulsifierproducing bacteria: Pantoea, Cellulomonas, Luteimonas, Methylobacterium, Micrococcus, Sporosarcina, Georgenia, and Geminicoccus.
However, the bioemulsifiers described in the literature belong to
polysaccharide and lipopolysaccharide families, possibly coupled
with peptide/protein molecules (Ron and Rosenberg, 2001). Some
of the genera that we describe in this work as novel bioemulsifier
producers have previously been described as being able to produce
extracellular polymeric substances (EPS), but without the properties of these compounds being tested for stabilizing oil/water
emulsions. Hallack et al. (2010) isolated an endophytic diazotrophic
Burkholderia kururiensis strain, M130, from rice roots, that produces
two kinds of acetylated acidic exopolysaccharides. The abundance
of EPS-producing bacteria in the rhizosphere is related to the
positive effect of the polymeric substance on the physico-chemical
properties of the soil (Amellal et al., 1998). This is consistent with
the production of emulsifiers by rhizobia, Burkholderia, Agromonas,
and Pantoea. The genus Cellulomonas has already been described by
Nazina et al. (2003) as producing emulsifying agents. However, the
reduction of interfacial tension due to these compounds suggests
that they were low-molecular-weight biosurfactants rather than
high-molecular-weight bioemulsifiers. No information was
retrieved from the literature regarding the ability of Luteimonas sp.
to produce bioemulsifiers or EPS. However, Luteimonas mephitis
was originally described as being close to the Xanthomonas genus
(Finkmann et al., 2000) and Xanthomonas campestris is the
producer of xanthan gum, a commercialized exopolysaccharide
Table 2
Best producers: Nearest relative in GenBank, RDP classification, and results of the EA test for low-cost substrates. M: Sugar-beet molasses, B: brewery wastes, G: glycerol,
W: ricotta cheese whey. *: fine emulsion.
Isolate
C.2.LD.2
C.3.LD.5
C.4.LD.10
C.4.LD.6
C.5.NBDD.11
C.6.LD.1
C.6.VLZ.1
C.8.NBDD.20
G.1.LD.1
G.1.VLZ.7
G.2.LD.10
G.2.NBD.6
G.2.NDB.5
G.2.VLA.4
G.2.VLAA.1
G.2.VLAA.5
G.2.VLAA.8.1
G.2.VLAA.8.2
G.4.LD.1.1
G.4.LD.1.2
G.4.VLAA.7
I.1.LD.3
I.1.LD.4
I.2.VLA.1
I.4.VLA.1
M.5.VLX.3
M.7.VLX.3
M1.5.LD.2
M1.7.LD.3
M1.8.LD.5
M2.5.LD.1
M2.5.LD.3
M2.6.LD.1
S.4.LD.10
S.4.LD.11
Nearest relative in GenBank
RDP Classification (Confidence 80%)
Strain
Accession number
Similarity (%)
Microbacterium sp. M1T8B9
Luteimonas mephiti
Cellulomonas sp. ANA-WS2
Sporosarcina sp. I1
Microbacterium sp. M1T8B9
Cellulomonas sp. ANA-WS2
Rhodococcus sp. 302BRRJ
Microbacterium sp. M1T8B9
Bacillus sp. IHB B 4034
Agromonas sp.S72
Bradyrhizobium sp. DA4
Agromonas sp.S72
Bacillus sp. IHB B 4034
Agromonas sp.S72
Burkholderia caledonica GR24
Methylobacterium radiotolerans P3
Burkholderia caledonica GR24
Burkholderia caledonica GR24
Micrococcus yunnanensis KTH-35
Micrococcus yunnanensis KTH-35
Bradyrhizobium. GASP-WDOW1_E0
Georgenia ferrireducens F6
Rhodococcus sp. 302BRRJ
Uncultured bact gel band 47
Rhodococcus sp. 302BRRJ
Bradyrhizobium sp. GSM-467
Bradyrhizobium liaoningense CCBAU
Bacillus circulans CAIM 245
Pantoea agglomerans 1.224
Pantoea agglomerans strain 1.2244
Uncultured bact. nby323b11c1
Uncultured bact. nby323b11c1
Paenibacillus dendritiformis P411
Rhodococcus erythropolis GT4
Acinetobacter calcoaceticus
GQ246683.1
AB433628.1
EU303275.1
HQ111067.1
GQ246683.1
EU303275.1
FJ200396.2
GQ246683.1
HM233998.1
AB531475.1
AJ430822.1
AB531475.1
HM233998.1
AB531475.1
FN796851.1
HM192796.1
FN796851.1
FN796851.1
HM854237.1
HM854237.1
EF075741.1
EU095256.1
FJ200396.2
EU275400.1
FJ200396.2
FN600560.2
HM446270.1
HM583984.1
HM130689.1
HM130689.1
HM816989.1
HM816989.1
HM071942.1
FN796872.1
HM851460.1
99
99
99
99
99
100
100
99
100
98
95
98
100
96
99
100
99
99
100
100
100
100
100
99
100
100
100
100
99
99
100
100
100
100
100
Microbacterium
Luteimonas
Cellulomonas
Sporosarcina
Microbacterium
Cellulomonas
Rhodococcus
Microbacterium
Bacillus
Bradyrhizobiaceae
Agromonas
Bradyrhizobiaceae
Bacillus
Bradyrhizobiaceae
Burkholderia
Methylobacterium
Burkholderia
Burkholderia
Micrococcus
Micrococcus
Bradyrhizobium
Georgenia
Rhodococcus
Geminicoccus
Rhodococcus
Bradyrhizobium
Bradyrhizobium
Bacillus
Pantoea
Pantoea
Acinetobacter
Acinetobacter
Paenibacillus
Rhodococcus
Acinetobacter
EA (%)
M
B
G
W
20e50%
<20%
>50%
20e50%
20e50%
20e50%
<20%
>50%
20e50%
20e50%
>50%*
<20%
>50%
<20%
>50%*
<20%
<20%
<20%
>50%
>50%
>50%*
>50%
>50%
20e50%
20e50%
>50%
>50%
>50%
20e50%
>50%*
>50%
>50%
>50%
>50%
>50%
>50%*
20e50%
>50%*
>50%
>50%*
>50%*
>50%*
>50%*
20e50%
>50%
20e50%
>50%
<20%
>50%
20e50%
>50%
>50%*
>50%*
<20%
>50%
<20%
<20%
>50%*
>50%*
>50%
<20%
<20%
>50%
>50%*
20e50%
>50%*
>50%
>50%*
>50%
>50%
<20%
20e50%
<20%
<20%
>50%*
>50%
>50%*
>50%*
>50%
<20%
20e50%
20e50%
20e50%
>50%*
<20%
>50%
>50%
20e50%
>50%
<20%
>50%*
<20%
<20%
<20%
20e50%
>50%
>50%*
<20%
>50%*
<20%
<20%
>50%*
>50%
20e50%
20e50%
>50%*
>50%
<20%
<20%
<20%
<20%
>50%
<20%
<20%
<20%
<20%
20e50%
<20%
<20%
<20%
>50%
>50%
<20%
<20%
>50%*
<20%
20e50%
>50%*
<20%
<20%
>50%
>50%*
>50%
>50%
>50%
20e50%
20e50%
20e50%
<20%
<20%
1098
A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e1099
Fig. 1. Unrooted phylogenetic tree based on 16S rRNA gene comparisons of the best bioemulsifier producers and microorganisms previously described in the literature as bioemulsifier producers. Bootstrap probability values under 50% were omitted from the figure. The scale bar indicates the substitutions per nucleotide position.
used as a thickener in the food industry. No Methylobacterium
species have previously been described as producing bioemulsifiers, although other Methylobacterium sp. isolates do
produce exopolysaccharides (Ozturk et al., 2008). Although no
Micrococcus sp. have been reported as bioemulsifier producers
(Kilic and Donmez, 2008), a metal-resistant Micrococcus sp. capable
of producing more than 400 mg l 1 of EPS in the presence of Cr(VI)
was recently isolated.
To the best of our knowledge, no information exists in the
literature on the production of emulsifiers or EPS by Sporosarcina
sp., Georgenia sp., or Geminicoccus sp.
Moreover, biodemulsifier-producing microorganisms were
recently isolated (Huang et al., 2010) that belong to genera that
have never before been described as producing these kinds of
surface active compounds. Most of the genera listed in this paper as
biodemulsifier producers are also able to produce emulsifiers,
suggesting an evolutionary relationship between the microorganisms that synthesize these two types of surface active compounds.
Fig. 1 shows the phylogenetic tree based on 16S rDNA sequences
of the best producers isolated in this work and some of the
microorganisms previously described in the literature as bioemulsifier producers. The tree was constructed including the 16S
rDNA sequences of biosurfactant-producing strains when available
in the literature or with the sequences of the type strains of the
producing species. The tree shows the wide phylogenetic diversity
of the isolates which are distributed between the divisions of Firmicutes, Actinobacteria, and Proteobacteria. Of the Proteobacteria,
most of the isolates are in the cluster of a-Proteobacteria, as was
found in a previous study (Ruggeri et al., 2009). Of the Actinobacteria, eight isolates, which represent the novel bioemulsifierproducing genera Microbacterium, Cellulosomanas, Georgenia, and
Micrococcus, are clustered with the known bioemulsifier producer
Arthrobacter globifirmis.
Acknowledgements
The authors gratefully acknowledge Romina Fumagalli and
Daniele Terragni for their help with the laboratory analyses. The
commercial bacterial mixtures for bioremediation were provided
by Gio Eco srl. (Segrate, Milan, Italy). The sugar-beet molasses,
ricotta cheese whey, and brewery wastes were provided by Aimex
Zuccheri Srl (Settimo Milanese, Milan, Italy), Società Agricola F.lli
Ponti (Nova Milanese e MB-Italy), and Carlsberg Italia (Induno
Olona e VA- Italy), respectively. This work was partially funded by
A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e1099
ISPESL (project: Biosynthesis and characterization of biosurfactants
for remediation of hydrocarbon contaminated sites).
References
Amellal, N., Burtin, G., Bartoli, F., Heulin, T., 1998. Colonization of wheat roots by an
exopolysaccharide-producing Pantoea agglomerans strain and its effect on
rhizosphere soil aggregation. Applied Environmental Microbiology 64,
3740e3747.
Banat, I., Franzetti, A., Gandolfi, I., Bestetti, G., Martinotti, M., Fracchia, L., Smyth, T.,
Marchant, R., 2010. Microbial biosurfactants production, applications and future
potential. Applied Microbiology and Biotechnology 87, 427e444.
Bodour, A.A., Drees, K.P., Maier, R.M., 2003. Distribution of biosurfactant-producing
bacteria in undisturbed and contaminated arid southwestern soils. Applied
Environmental Microbiology 69, 3280e3287.
Cao, A., Carucci, A., Lai, T., La Colla, P., Tamburini, E., 2007. Effect of biodegradable
chelating agents on heavy metals phytoextraction with Mirabilis jalapa and on
its associated bacteria. European Journal of Soil Biology 43, 200e206.
Finkmann, W., Altendorf, K., Stackebrandt, E., Lipski, A., 2000. Characterization of
N2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas
nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov. International Journal of Systematic and Evolutionary Microbiology 50, 273e282.
Franzetti, A., Bestetti, G., Caredda, P., La Colla, P., Tamburini, E., 2008. Surface-active
compounds and their role in the access to hydrocarbons in Gordonia strains.
Fems Microbiology Ecology 63, 238e248.
Gandolfi, I., Sicolo, M., Franzetti, A., Fontanarosa, E., Santagostino, A., Bestetti, G.,
2010. Influence of compost amendment on microbial community and ecotoxicity of hydrocarbon-contaminated soils. Bioresource Technology 101, 568e575.
Hallack, L.F., Passos, D.S., Mattos, K.A., Agrellos, O.A., Jones, C., MendoncaPreviato, L., Previato, J.O., Todeschini, A.R., 2010. Structural elucidation of the
repeat unit in highly branched acidic exopolysaccharides produced by nitrogen
fixing Burkholderia. Glycobiology 20, 338e347.
Huang, X.F., Liu, J., Lu, L.J., Wen, Y., Xu, J.C., Yang, D.H., Zhou, Q., 2009. Evaluation of
screening methods for demulsifying bacteria and characterization of lipopeptide bio-demulsifier produced by Alcaligenes sp. Bioresource Technology
100, 1358e1365.
Huang, X., Guan, W., Liu, J., Lu, L., Xu, J., Zhou, Q., 2010. Characterization and
phylogenetic analysis of biodemulsifier-producing bacteria. Bioresource Technology 97, 317e323.
Joseph, S.J., Hugenholtz, P., Sangwan, P., Osborne, C.A., Janssen, P.H., 2003. Laboratory cultivation of widespread and previously uncultured soil bacteria. Applied
Environmental Microbiology 69, 7210e7215.
Kilic, N.K., Donmez, G., 2008. Environmental conditions affecting exopolysaccharide
production by Pseudomonas aeruginosa, Micrococcus sp., and Ochrobactrum sp.
Journal of Hazardous Material 154, 1019e1024.
1099
Makkar, R., Cameotra, S., 2002. An update on the use of unconventional substrates
for biosurfactant production and their new applications. Applied Microbiology
and Biotechnology 58, 428e434.
Nazina, T.N., Sokolova, D.S., Grigor’yan, A.A., Xue, Y.F., Belyaev, S.S., Ivanov, M.V.,
2003. Production of oil-releasing compounds by microorganisms from the
Daqing oil field, China. Microbiology 72, 173e178.
Neu, T.R., 1996. Significance of bacterial surface-active compounds in interaction of
bacteria with interfaces. Microbiology Review 60, 151e166.
Ozturk, S., Aslim, B., Ugur, A., 2008. Chromium(VI) resistance and extracellular
polysaccharide (EPS) Synthesis by Pseudomonas, Stenotrophomonas and Methylobacterium strains. Isij International 48, 1654e1658.
Ron, E.Z., Rosenberg, E., 2001. Natural roles of biosurfactants. Environmental
Microbiology 3, 229e236.
Rosenberg, E., Zuckerberg, A., Rubinovitz, C., Gutnick, D., 1979. Emulsifier of
Arthrobacter RAG-1-Isolation and emulsifying properties. Applied and Environmental Microbiology 37, 402e408.
Ruggeri, C., Franzetti, A., Bestetti, G., Caredda, P., La Colla, P., Pintus, M., Sergi, S.,
Tamburini, E., 2009. Isolation and characterisation of surface active compoundproducing bacteria from hydrocarbon-contaminated environments. International Biodeterioration and Biodegradation 63, 936e942.
Sait, M., Hugenholtz, P., Janssen, P.H., 2002. Cultivation of globally distributed soil
bacteria from phylogenetic lineages previously only detected in cultivationindependent surveys. Environmental Microbiology 4, 654e666.
Satpute, S.K., Banat, I.M., Dhakephalkar, P.K., Banpurkar, A.G., Chopade, B.A., 2010a.
Biosurfactants, bioemulsifiers and exopolysaccharides from marine microorganisms. Biotechnology Advances 28, 436e450.
Satpute, S.K., Banpurkar, A.G., Dhakephalkar, P.K., Banat, I.M., Chopade, B.A., 2010b.
Methods for investigating biosurfactants and bioemulsifiers: a review. Critical
Reviews in Biotechnology 30, 127e144.
Schwieger, F., Tebbe, C.C., 1998. A new approach to utilize PCR-single-strandconformation polymorphism for 16s rRNA gene-based microbial community
analysis. Applied Environmental Microbiology 64, 4870e4876.
Skorupska, A., Janczarek, M., Marczak, M., Mazur, A., Król, J., 2006. Rhizobial exopolysaccharides: genetic control and symbiotic functions. Microbial Cell
Factories, 5e7.
Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary
genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution
24, 1596e1599.
Van Hamme, J.D., Singh, A., Ward, O.P., 2006. Physiological aspects. Part 1 in a series
of papers devoted to surfactants in microbiology and biotechnology. Biotechnology Advances 24, 604e620.
Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for
rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied
and Environmental Microbiology 73, 5261e5267.
Zhang, Z., Schwartz, S., Wagner, L., Miller, W., 2000. A greedy algorithm for aligning
DNA sequences. Journal of Computational Biology 7, 203e214.
Zheng, Y., Chen, X., Shen, Y., 2008. Commodity chemicals derived from glycerol, an
important biorefinery feedstock. Chemical Reviews 108, 5253e5277.