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International Biodeterioration & Biodegradation 65 (2011) 1095e1099

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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

aDept. Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, Milano, Italyb 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

Article history:Received 4 November 2010Received in revised form27 December 2010Accepted 12 January 2011Available online 19 August 2011

Keywords:Surface-active compoundBiosurfactantBioemulsifierRenewable substrateScreeningDiversity

* Corresponding author. Tel.: þ39 02 6448 2927; faE-mail address: andrea.franzetti@unimib.it (A. Fra

0964-8305/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.ibiod.2011.01.014

a b s t r a c t

Bacteria able to produce biological emulsifiers were isolated from different environments using differentisolation media with the aim of discovering the widest diversity. The phylogenetic diversity of theisolates was evaluated by 16S rRNA gene analysis. Among 190 isolated strains, 127 released extracellularemulsifiers able to stabilize oil-water emulsions when grown on low-cost substrates. Among these, the35 isolates that showed the highest emulsifier production on different substrates were found to belong to16 different bacterial genera. Overall, this is the first systematic study of the diversity of bioemulsifier-producing bacteria and of their ability to produce bioemulsifiers on low-cost substrates.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Surface active compounds (SACs) are amphiphilic moleculescontaining both hydrophilic and hydrophobic moieties. Neu (1996)classified SACs into low-molecular-weight SACs, also termed bio-surfactants, and high-molecular-weight SACs, including amphi-philic and polyphilic polymers, also termed bioemulsifiers. Theformer lower the interfacial tensions of the liquid in which they aredissolved, whereas the latter are not able to efficiently reduceinterfacial tension; instead, they firmly stabilize oil/water emul-sions. A variety of microorganisms produce high-molecular-weightbioemulsifiers (Satpute et al., 2010a); the best investigated amongthem are bioemulsans, which are synthesized by various species ofAcinetobacter. Among these, the first studied compound was RAG-1emulsan, an amphiphilic polysaccharide produced by Acinetobactercalcoaceticus, which is also the only commercially available bio-emulsifier at present (Rosenberg et al., 1979).

Over the past few years, microbial SACs have received increasingcommercial attention as substitutes for synthetic surfactants owingto their properties (such as high surfactant and emulsifying activ-ities and stability in extreme physico-chemical conditions) andadvantages (such as lower toxicity and higher biodegradability).

x: þ39 02 6448 2996.nzetti).

All rights reserved.

Thanks to these features, microbial SACs are more acceptable,compared to synthetic surfactants, in various applications, such asthose in the oil industry, microbial-enhanced oil recovery, envi-ronmental remediation, oil transportation, tank cleaning, agricul-ture, medicine, and the cosmetic and food industries (Banat et al.,2010). The environmental distribution and diversity of low-molecular-weight SAC-producing bacteria have already beenstudied (Bodour et al., 2003; Ruggeri et al., 2009). Moreover, therecent attention given to the de-emulsifying activity of biologicalSACs has led to the publication of studies on the characteristics,diversity, and distribution of this type of microorganism (Huanget al., 2009, 2010). Despite the potential for a wide range of appli-cations, the diversity of high-molecular-weight SAC-producingbacteria has been poorly studied. A systematic study of theirdistribution in the environment has not been carried out so far.

The aims of this paper were to study the microbial diversity andphylogenetic relationships between bioemulsifier-producingbacteria and the effect of environmental sampling, isolationmedia, and low-cost substrates on their isolation.

2. Materials and methods

2.1. Environmental samples

Various environmental samples were used to screen forbioemulsifier-producing bacterial strains. These include a previously

A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e10991096

characterized high-quality compost (C) (Gandolfi et al., 2010),a polycyclic aromatic hydrocarbons (PAHs)e contaminated soil (I), adiesel-oil-contaminated soil (G), and a heavy-metals-contaminatedsoil (M). Soils I and G had previously been characterized byGandolfi et al. (2010) and the characteristics of the heavy-metal-contaminated soil were recently reported by Cao et al. (2007).Furthermore, bioemulsifier-producting bacteria were also screenedfrom a laboratory-enriched culture on hydrocarbons (S). Theenrichment culture was prepared from a soil sample collected froma diesel-oil-contaminated site in northern Italy. The concentration ofhydrocarbons (C > 12) in this soil was 3000 mg kg�1. Finally, theisolation of bioemulsifier-producing bacteria was also carried outfrom two commercial preparations that are sold for bioremediationpurposes: ECORPOLL/L1 (M1) and ECORPOLL/L2 (M2), commer-cialized by GIO.ECO srl (Segrate, Italy).

2.2. Isolation procedures

The isolates were obtained by dilution and plating on sevendifferent isolation media that were solidified with agar. Four mediawere based on the oligotrophic VL55 mineral medium (Sait et al.,2002; Joseph et al., 2003) specifically designed for isolating previ-ously uncultured bacteria. The VL55 medium was separatelyamended with either one of the four different mixtures of carbonsources, as reported in Table 1. Nutrient broth (Biolife, Italy) wasused 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) mediumwas used undiluted.

The compost and the contaminated soil samples were platedonto all of the isolation media while the enrichment culture andcommercial preparations were only plated onto LB media. The agarplates were incubated at 30 �C for 2e10 days to allow colonygrowth. Colonies with different morphologies were then pickedand transferred to a fresh agar plate until a pure culture wasobtained. Each isolate was named based on the starting environ-mental sample, the isolation medium, and the isolation dilution asfollows: acronym of the environmental sample.dilution.acronym ofthe 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 ineither NB10, NB100, or LB broth. They were then washed twice andresuspended 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, andglycerol) was filtered, sterilized, and supplied at an initial concen-tration of 5.0 g l�1. The cultures were incubated at 200 rpm, 30 �C,for six days.

Table 1The isolation media used in this study.

Isolation medium Basal medium Carbon source

VLZ 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

VLA VL55 Ascorbic acid 1.76 g l�1, galacturonicacid 1.12 g l�1, glucuronic acid 1.94 g l�1

sodium gluconate 2.18 g l�1

VLAA VL55 Sodium acetate 0.8 g l�1, sodiumlactate 1.12 g l�1, methanol 0.32 g l�1

VLX VL55 Xylane 0.05% w/vLB LBNBD NB 1:10NBDD NB 1:100

The emulsification assay (EA) was carried out as previouslyreported (Franzetti et al., 2008). The height of the emulsion wasmeasured with respect to the height of the solution, and the degreeof 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 low-molecular-weight SACs, the oil spreading technique (OST) wascarried out as previously reported (Ruggeri et al., 2009). Isolateswith a degree of emulsification greater than the control andnegative in the OST were considered high-molecular-weight SACproducers (Satpute et al., 2010b). Strains that produced bio-emulsifiers on more than one substrate and that showed a differ-ence greater than one between the degrees of emulsification of thebroth and the control were classified as “best producers.”

2.4. 16S rRNA gene sequence analysis

Colony PCR was carried out using Com primers (Schwieger andTebbe, 1998) as previously reported (Gandolfi et al., 2010). Taxo-nomic assignments of sequences were performed using the Ribo-somal Database Project (RDP) classifier (Wang et al., 2007). Thenearest relative sequences in GenBank were retrieved using BLAST(Zhang et al., 2000). A phylogenetic tree was drawn using thesoftware program MEGA, version 4, by the neighbour-joiningmethod (Tamura et al., 2007).

3. Results and discussion

A total of 190 different isolates were screened: 127 isolates werepositive for bioemulsifier production, while seven strains werepositive for both EA and OST and were considered low-molecular-weight SAC-producers.

3.1. Effect of environmental samples, isolation media, and carbonsources on bioemulsifier production

Notably high percentages of positive isolates were retrieved forall environmental samples. The commercial mixtures for biore-mediation and the enriched culture on hydrocarbons showed thehighest percentages. Approximately 65e70% of the isolates fromthe hydrocarbon-contaminated soils (diesel oil and PAHs) werebioemulsifier producers. These high percentages are in agreementwith the reported role of microbial surface active compounds inhydrocarbon uptake (Van Hamme et al., 2006). However, thesevalues are also comparable with the percentage obtained for thehigh-biodiversity compost (58%), as expected from the significantnumber of hydrocarbon-degrading microorganisms previouslyfound in the compost (Gandolfi et al., 2010). The lower percentageof positive isolates retrieved for the metal-contaminated soil (39%)suggests that the high concentrations of Pb and Zn did not select forbacteria able to produce chelating bioemulsifiers as a protectionagainst metal toxicity. This is consistent with the reported domi-nance of metal-susceptible bacteria in the soil (Cao et al., 2007).

Each isolate was tested for emulsifier production on fourdifferent low-cost substrates (sugar-beet molasses, glycerol,brewery wastes, ricotta cheese whey). The chosen substrates havedistinct chemical compositions and have been extensively used forbiosurfactant production (Makkar and Cameotra, 2002). Amongthem, glycerol emerged as one of the most important potentialfeedstocks, available in large quantities as a by-product of thebiodiesel process (Zheng et al., 2008).

All of the isolates were able to grow on at least one of the testedsubstrates. All of the substrates allowed the production of bio-emulsifiers to a good extent. Glycerol and molasses were the bestsubstrates (36% and 27%, respectively).

A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e1099 1097

3.2. Taxonomy and phylogeny of the best producers ofbioemulsifiers

Extracellular polymeric substances that can potentially formstable emulsions between oil and water are produced by manymicroorganisms (Satpute et al., 2010a). In order to avoid over-classification of the microorganisms as bioemulsifier producers,only the isolates with an ability to produce bioemulsifiers on atleast two substrates and with a level of emulsification abovea certain threshold (see Materials and methods section) wereconsidered.

Table 2 shows the nearest relatives in GenBank, the RDP clas-sification, and the extent of emulsification of the best producers. Itis likely that some of the isolates with the same sequences and fromthe same environmental samples were clones. However, theyshowed slight differences in emulsification. The use of a typingtechnique to distinguish between the strains was beyond the scopeof this work. The presence of 16 genera from the 35 isolatessuggests that there is a wide biodiversity of bioemulsifier-producing bacteria. Bacillus, Acinetobacter, and Rhodococcus arethe best known bacterial groups for biosurfactant and bioemulsifierproduction and they were also the most commonly representedgenera in our screening. Bradyrhizobiaceae/Bradyrhizobium werealso dominant bacterial groups found among the “best producers.”As previously reported, the production of extracellular polymershas been extensively demonstrated in rhizobia, even though thesurface properties and applicability of these compounds have notyet been investigated (Skorupska et al., 2006; Ruggeri et al., 2009).

Table 2Best producers: Nearest relative in GenBank, RDP classification, and results of the EA teW: ricotta cheese whey. *: fine emulsion.

Isolate Nearest relative in GenBank

Strain Accession number Similarity (%

C.2.LD.2 Microbacterium sp. M1T8B9 GQ246683.1 99C.3.LD.5 Luteimonas mephiti AB433628.1 99C.4.LD.10 Cellulomonas sp. ANA-WS2 EU303275.1 99C.4.LD.6 Sporosarcina sp. I1 HQ111067.1 99C.5.NBDD.11 Microbacterium sp. M1T8B9 GQ246683.1 99C.6.LD.1 Cellulomonas sp. ANA-WS2 EU303275.1 100C.6.VLZ.1 Rhodococcus sp. 302BRRJ FJ200396.2 100C.8.NBDD.20 Microbacterium sp. M1T8B9 GQ246683.1 99G.1.LD.1 Bacillus sp. IHB B 4034 HM233998.1 100G.1.VLZ.7 Agromonas sp.S72 AB531475.1 98G.2.LD.10 Bradyrhizobium sp. DA4 AJ430822.1 95G.2.NBD.6 Agromonas sp.S72 AB531475.1 98G.2.NDB.5 Bacillus sp. IHB B 4034 HM233998.1 100G.2.VLA.4 Agromonas sp.S72 AB531475.1 96G.2.VLAA.1 Burkholderia caledonica GR24 FN796851.1 99G.2.VLAA.5 Methylobacterium radiotolerans P3 HM192796.1 100G.2.VLAA.8.1 Burkholderia caledonica GR24 FN796851.1 99G.2.VLAA.8.2 Burkholderia caledonica GR24 FN796851.1 99G.4.LD.1.1 Micrococcus yunnanensis KTH-35 HM854237.1 100G.4.LD.1.2 Micrococcus yunnanensis KTH-35 HM854237.1 100G.4.VLAA.7 Bradyrhizobium. GASP-WDOW1_E0 EF075741.1 100I.1.LD.3 Georgenia ferrireducens F6 EU095256.1 100I.1.LD.4 Rhodococcus sp. 302BRRJ FJ200396.2 100I.2.VLA.1 Uncultured bact gel band 47 EU275400.1 99I.4.VLA.1 Rhodococcus sp. 302BRRJ FJ200396.2 100M.5.VLX.3 Bradyrhizobium sp. GSM-467 FN600560.2 100M.7.VLX.3 Bradyrhizobium liaoningense CCBAU HM446270.1 100M1.5.LD.2 Bacillus circulans CAIM 245 HM583984.1 100M1.7.LD.3 Pantoea agglomerans 1.224 HM130689.1 99M1.8.LD.5 Pantoea agglomerans strain 1.2244 HM130689.1 99M2.5.LD.1 Uncultured bact. nby323b11c1 HM816989.1 100M2.5.LD.3 Uncultured bact. nby323b11c1 HM816989.1 100M2.6.LD.1 Paenibacillus dendritiformis P411 HM071942.1 100S.4.LD.10 Rhodococcus erythropolis GT4 FN796872.1 100S.4.LD.11 Acinetobacter calcoaceticus HM851460.1 100

To the best of our knowledge, we are the first to add the followingeight genera to the list of already described bioemulsifier-producing bacteria: Pantoea, Cellulomonas, Luteimonas, Methyl-obacterium,Micrococcus, Sporosarcina, Georgenia, and Geminicoccus.However, the bioemulsifiers described in the literature belong topolysaccharide and lipopolysaccharide families, possibly coupledwith peptide/protein molecules (Ron and Rosenberg, 2001). Someof the genera that we describe in this work as novel bioemulsifierproducers have previously been described as being able to produceextracellular polymeric substances (EPS), but without the proper-ties of these compounds being tested for stabilizing oil/wateremulsions. Hallack et al. (2010) isolated an endophytic diazotrophicBurkholderia kururiensis strain, M130, from rice roots, that producestwo kinds of acetylated acidic exopolysaccharides. The abundanceof EPS-producing bacteria in the rhizosphere is related to thepositive effect of the polymeric substance on the physico-chemicalproperties of the soil (Amellal et al., 1998). This is consistent withthe production of emulsifiers by rhizobia, Burkholderia, Agromonas,and Pantoea. The genus Cellulomonas has already been described byNazina et al. (2003) as producing emulsifying agents. However, thereduction of interfacial tension due to these compounds suggeststhat they were low-molecular-weight biosurfactants rather thanhigh-molecular-weight bioemulsifiers. No information wasretrieved from the literature regarding the ability of Luteimonas sp.to produce bioemulsifiers or EPS. However, Luteimonas mephitiswas originally described as being close to the Xanthomonas genus(Finkmann et al., 2000) and Xanthomonas campestris is theproducer of xanthan gum, a commercialized exopolysaccharide

st for low-cost substrates. M: Sugar-beet molasses, B: brewery wastes, G: glycerol,

RDP Classification (Confidence 80%) EA (%)

) M B G W

Microbacterium 20e50% >50%* <20% >50%*Luteimonas <20% 20e50% 20e50% >50%Cellulomonas >50% >50%* <20% <20%Sporosarcina 20e50% >50% <20% <20%Microbacterium 20e50% >50%* >50%* <20%Cellulomonas 20e50% >50%* >50% <20%Rhodococcus <20% >50%* >50%* >50%Microbacterium >50% >50%* >50%* <20%Bacillus 20e50% 20e50% >50% <20%Bradyrhizobiaceae 20e50% >50% <20% <20%Agromonas >50%* 20e50% 20e50% <20%Bradyrhizobiaceae <20% >50% 20e50% 20e50%Bacillus >50% <20% 20e50% <20%Bradyrhizobiaceae <20% >50% >50%* <20%Burkholderia >50%* 20e50% <20% <20%Methylobacterium <20% >50% >50% >50%Burkholderia <20% >50%* >50% >50%Burkholderia <20% >50%* 20e50% <20%Micrococcus >50% <20% >50% <20%Micrococcus >50% >50% <20% >50%*Bradyrhizobium >50%* <20% >50%* <20%Georgenia >50% <20% <20% 20e50%Rhodococcus >50% >50%* <20% >50%*Geminicoccus 20e50% >50%* <20% <20%Rhodococcus 20e50% >50% 20e50% <20%Bradyrhizobium >50% <20% >50% >50%Bradyrhizobium >50% <20% >50%* >50%*Bacillus >50% >50% <20% >50%Pantoea 20e50% >50%* >50%* >50%Pantoea >50%* 20e50% <20% >50%Acinetobacter >50% >50%* <20% 20e50%Acinetobacter >50% >50% >50%* 20e50%Paenibacillus >50% >50%* >50% 20e50%Rhodococcus >50% >50% 20e50% <20%Acinetobacter >50% >50% 20e50% <20%

Fig. 1. Unrooted phylogenetic tree based on 16S rRNA gene comparisons of the best bioemulsifier producers and microorganisms previously described in the literature as bio-emulsifier producers. Bootstrap probability values under 50% were omitted from the figure. The scale bar indicates the substitutions per nucleotide position.

A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e10991098

used as a thickener in the food industry. No Methylobacteriumspecies have previously been described as producing bio-emulsifiers, although other Methylobacterium sp. isolates doproduce exopolysaccharides (Ozturk et al., 2008). Although noMicrococcus sp. have been reported as bioemulsifier producers(Kilic and Donmez, 2008), a metal-resistantMicrococcus sp. capableof 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 theliterature on the production of emulsifiers or EPS by Sporosarcinasp., Georgenia sp., or Geminicoccus sp.

Moreover, biodemulsifier-producing microorganisms wererecently isolated (Huang et al., 2010) that belong to genera thathave never before been described as producing these kinds ofsurface active compounds. Most of the genera listed in this paper asbiodemulsifier producers are also able to produce emulsifiers,suggesting an evolutionary relationship between the microorgan-isms that synthesize these two types of surface active compounds.

Fig. 1 shows the phylogenetic tree based on 16S rDNA sequencesof the best producers isolated in this work and some of themicroorganisms previously described in the literature as bio-emulsifier producers. The tree was constructed including the 16S

rDNA sequences of biosurfactant-producing strains when availablein the literature or with the sequences of the type strains of theproducing species. The tree shows the wide phylogenetic diversityof the isolates which are distributed between the divisions of Fir-micutes, Actinobacteria, and Proteobacteria. Of the Proteobacteria,most of the isolates are in the cluster of a-Proteobacteria, as wasfound in a previous study (Ruggeri et al., 2009). Of the Actino-bacteria, eight isolates, which represent the novel bioemulsifier-producing genera Microbacterium, Cellulosomanas, Georgenia, andMicrococcus, are clustered with the known bioemulsifier producerArthrobacter globifirmis.

Acknowledgements

The authors gratefully acknowledge Romina Fumagalli andDaniele Terragni for their help with the laboratory analyses. Thecommercial bacterial mixtures for bioremediation were providedby Gio Eco srl. (Segrate, Milan, Italy). The sugar-beet molasses,ricotta cheese whey, and brewery wastes were provided by AimexZuccheri Srl (Settimo Milanese, Milan, Italy), Società Agricola F.lliPonti (Nova Milanese e MB-Italy), and Carlsberg Italia (IndunoOlona e VA- Italy), respectively. This work was partially funded by

A. Franzetti et al. / International Biodeterioration & Biodegradation 65 (2011) 1095e1099 1099

ISPESL (project: Biosynthesis and characterization of biosurfactantsfor remediation of hydrocarbon contaminated sites).

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