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Phylogenetic characterization of bioemulsifier-producing bacteria
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 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: [email protected] (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
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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 kg1. 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 l1, tryptone: 10 g l1, NaCl: 5 g l1) (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 l1. 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
VLAA
VL55
VLX LB NBD NBDD
VL55 LB NB 1:10 NB 1:100
Arabinose 1.5 g l1, xylose 1.5 g l1 glucose 1.8 g l1, galactose 1.8 g l1 Ascorbic acid 1.76 g l1, galacturonic acid 1.12 g l1, glucuronic acid 1.94 g l1 sodium gluconate 2.18 g l1 Sodium acetate 0.8 g l1, sodium lactate 1.12 g l1, methanol 0.32 g l1 Xylane 0.05% w/v
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).
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).
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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%
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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 l1 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
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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: [email protected] (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
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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 kg1. 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 l1, tryptone: 10 g l1, NaCl: 5 g l1) (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 l1. 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
VLAA
VL55
VLX LB NBD NBDD
VL55 LB NB 1:10 NB 1:100
Arabinose 1.5 g l1, xylose 1.5 g l1 glucose 1.8 g l1, galactose 1.8 g l1 Ascorbic acid 1.76 g l1, galacturonic acid 1.12 g l1, glucuronic acid 1.94 g l1 sodium gluconate 2.18 g l1 Sodium acetate 0.8 g l1, sodium lactate 1.12 g l1, methanol 0.32 g l1 Xylane 0.05% w/v
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).
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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).
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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%
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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 l1 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
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