Appl Microbiol Biotechnol (2002) 60:347–351 DOI 10.1007/s00253-002-1115-4
SHORT CONTRIBUTION
C. Calvo · F. Martnez-Checa · F. L. Toledo · J. Porcel · E. Quesada
Characteristics of bioemulsifiers synthesised in crude oil media by Halomonas eurihalina and their effectiveness in the isolation of bacteria able to grow in the presence of hydrocarbons Received: 28 May 2002 / Revised: 31 July 2002 / Accepted: 27 August 2002 / Published online: 26 September 2002 Springer-Verlag 2002
Abstract Halomonas eurihalina strains F2–7, H28, H96, H212 and H214 were capable of producing large amounts of exopolysaccharides (EPS) in MY medium with added crude oil. The biopolymers showed lower carbohydrate and protein content than those synthesised in control medium without oil. Nevertheless, the percentages of uronic acids, acetyls and sulphates were higher. The emulsifying activity of biopolymers was measured; crude oil was the substrate most efficiently emulsified. Furthermore, all the EPS tested emulsified higher volumes of crude oil than the commercial surfactants used as controls. We have also proved the effectiveness of both Halomonas eurihalina strains and their EPS to select indigenous bacteria able to grow in the presence of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene and pyrene) from waste crude oil. The majority of isolated strains belonged to the genus Bacillus.
Introduction Over the past few decades, biosurfactants have gained attention because of their biodegradability, low toxicity, ecological acceptability, ability to be produced from renewable resources and functionality under extreme conditions. These compounds have many potential applications, including enhanced oil recovery, as crude oil drilling lubricants, and in surfactant-aided bioremediation of water-insoluble pollutants. (Banat 1995; Banat et al. 2000; Jain et al. 1992).
Petroleum and its residues are some of the most common anthropogenic contaminants of terrestrial and aquatic environments. The bioremediation of such habitats is limited by the poor availability of hydrophobic pollutants to microorganisms. Nevertheless, surfactants can be used to help enhance emulsification and dispersal of immiscible compounds (Atlas and Cerniglia 1995; Zhan and Miller 1992). The growth of microorganisms on hydrocarbons is often associated with the production of compounds that can aid emulsification of these hydrophobic substances in the growth medium. Our previous studies demonstrated that Halomonas eurihalina strains produce exopolysaccharides (EPS) that emulsified different hydrocarbons (Bjar et al. 1998; Calvo et al. 1995; Quesada et al. 1993). The present study was undertaken in order to evaluate the potential application of these EPS in oil pollution bioremediation. We have studied the ability of five strains of H. eurihalina (F2–7, H28, H96, H212 and H214) to produce EPS in MY medium supplemented with crude oil. We have also investigated the chemical composition of the biopolymers and their ability to emulsify hydrocarbons. In addition, we have proved that both the H. eurihalina strains and their EPS enhanced the isolation of indigenous bacteria able to grow in minimal medium supplemented with polycyclic aromatic hydrocarbons (PAH) as sole source of carbon from samples of waste crude oil.
Materials and methods C. Calvo ()) · F.L. Toledo Environmental Microbiology Research Group, Institute of Water Research, University of Granada, C/Ramn y Cajal no. 4. Edificio Fray Luis de Granada, 18071 Granada, Spain e-mail:
[email protected] Fax: +34-958-243094 F. Martnez-Checa · J. Porcel · E. Quesada Microbial Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, University of Granada, 18071 Granada, Spain
Microorganisms The microorganisms used for this study were H. eurihalina strains F2–7, H28, H96, H212 and H214. These are moderately halophilic bacteria, with optimal growth at 7.5% (w/v) salts. All these strains, isolated from hypersaline soils, synthesise EPS (Bjar et al. 1998; Quesada et al. 1990, 1993).
348 Isolation and purification of EPS MY medium, as described by Quesada et al. (1993), was used in this study, substituting crude oil for glucose. Erlenmeyer flasks (500 ml) containing 100 ml medium were inoculated with 1 ml of an overnight culture, grown in the same medium, and incubated at 32C for 8 days without shaking. Cultures were centrifuged at 36,000 g in a Sorvall RC-5B refrigerated centrifuge at 4C for 60 min. EPS in the supernatant was precipitated with three volumes of cold ethanol, dissolved in distilled water and centrifuged at 226,000 g for 60 min in a Beckman L8-M ultracentrifuge. The pellet was dissolved in water, dialysed against distilled water for 24 h, lyophilised and then weighed (Quesada et al. 1993). Analytical procedures Carbohydrates, proteins, uronic acids and acetyl residues were determined by colorimetric assays (Blumenkratz and AsboeHansen 1973; Bradford 1976; Dubois et al. 1956; McComb and McCready 1957). Sulphate content was analysed using an ion liquid chromatography system (Dionex 300) with a conductivity detector. The chromatography conditions were: mobile phase: 3.5 mM Na2CO3/1.0 mM NaHCO3; flow rate: 1.3 ml/min, suppressor: anion self-regeneration suppressor (AMM II model), and an Ionopac column (AS 14, 4-mm model) was used. Emulsification assay Emulsification assays were carried out according to Cooper and Goldenberg (1987). EPS dissolved in 5 ml distilled water (0.5%, w/ v) was mixed with 5 ml of each hydrophobic substrate in test tubes (10515 mm), vortexed to homogeneity, and left to stand for 24 h at 4C. Emulsifying activity was expressed as the percentage of the total height occupied by the emulsion. The hydrophobic substrates tested were xylene, toluene, octane, tetradecane, hexadecane, mineral light oil and mineral heavy oil (Sigma), crude oil and petrol (Repsol YPF, Puertollano, Spain). As controls we used the following chemical surfactants from Sigma: Tween 20, Tween 80 and Triton X-100. Isolation and selection of bacteria from crude oil waste Indigenous bacteria were isolated from solid waste crude oil samples (provided by Delta, Algeciras, Spain) collected from the clean up of oil storage containers. Waste crude samples (10 g) were placed in 500-ml Erlenmeyer flasks and supplemented with 0.5 g glucose, 100 ml distilled water and 10 ml of an overnight culture of the EPS producer strain (108 cells/ml) or with 0.5 g of the
Table 1 Yield and chemical composition of exopolysaccharides (EPS) from Halomonas eurihalina strains
corresponding lyophilised EPS. The flasks were incubated at 32C for 3 months at 150 rpm. Sampling for bacterial isolation was performed every week. Samples (0.1 ml) were plated on BH agar medium (Dziel et al. 1996) with the addition of 1% (w/v) naphthalene, phenanthrene or pyrene. For that purpose, the polycyclic aromatic hydrocarbon (PAH) was dissolved in ether and the solution was then spread onto the surface of the medium. BH is a minimal medium with the following composition (g/l): MgSO4·7H2O, 0.2; K2HPO4, 1; KH2PO4, 1; CaCl2·2H2O, 0.02; NH4NO3·6H2O, 1; FeCl3, 0.05 and agar, 20. Bacterial strains were selected according their ability to grow and produce a clear halo on the surface of the BH medium with added PAH. Identification of bacteria was based on morphological characteristics and on a commercially available diagnostic kit (Api 20NE, Biomerieux, Ghent, Belgium). Growth in the presence of PAH was confirmed using the minimal BH liquid medium (Dziel et al. 1996), to which 0.1% (w/ v) naphthalene, phenanthrene or pyrene was added as sole carbon and energy source. Growth of microorganisms was studied for 72 h at 32C and 100 rpm. Enumeration was performed by viable colony counts.
Results Table 1 shows the yield and chemical composition of heteropolysaccharides V2–7, H28, H96, H212 and H214, extracted from MY medium supplemented with crude oil. EPS were produced with greater yields than in MY with glucose as carbon source (Bjar et al. 1998) and had a lower carbohydrate and protein content, but higher amounts of uronic acids, sulphates and acetyl residues (Bjar et al. 1998). In relation to their emulsifying activity (Table 2), they were active on almost all the hydrocarbons tested (EPS H28 did not emulsify heavy oil). Crude oil was the substrate most efficiently emulsified in all cases; this oil, however, was very poorly emulsified by the chemical surfactants. We analysed whether H. eurihalina strains and/or their polymers were able to enhance the isolation of strains capable of growing in the presence of PAH (naphthalene, phenanthrene and/or pyrene). For this purpose, we isolated all the strains (56 isolates) that grew on BHPAH plates (Table 3): 34 isolates were from samples of solid waste oil that had previously been treated with some of the H. eurihalina strains and 22 isolates were obtained
EPS V2–7 b
Yield production Chemical compositionc Carbohydrates Proteins Uronic acids Acetyls Sulphates a Data b
H28a
H96
H212
H214
0.4€0.1
0.7€0.1
0.6€0.1
0.3€0.1
0.6€0.1
28.3€1.1 2.1€0.2 2.6€0.3 1.1€0.1 20.7€1.0
35.1€0.9 4.1€0.7 2.8€0.2 0.9€0.1 17.1€0.5
20€1.0 7.0€ 0.5 7.0€0.1 0.3€0.1 17.6€1.1
25.2€1 3.2€0.3 2€0.2 0.9€0.1 20.9€0.9
23.1€1.1 3.3€0.3 1.5€0.1 0.7€0.1 15.4€1.2
from EPS H28 has been previously reported (Martnez-Checa et al. 2002) Results are expressed as grams EPS per gram cellular dry weight; values are means of at least three determinations c Results are expressed as percentages of total dry weight of the polymers; values are means of at least three determinations
349 Table 2 Emulsifying activity of EPS from H. eurihalina strains Hydrocarbon
EPS
Chemical surfactants
V2–7 Xylene Toluene Octane Tetradecane Hexadecane Heavy oil Light oil Crude Petrol
b
46€2 25€1 56€2 15€1 32€1 47€2 34€1 76€1 39€0.4
H28
a
42€1 48€1 40€1 39€1 29€1 0 28€1 58€1 34€0.2
H96
H212
H214
Tween 20
Tween 80
Triton-X-100
57€2 15€1 45€1 42 €1 27€1 18€1 19€1 73€2 61€2
25€0.3 21€0.5 19€1 25€1 27€1 9€0.5 31€1 46€1 21€0.8
69€2 50€1 44€1 43€1 56€1 64€2 43€1 49€1 33€0.5
43€2 29€1 49€1 47€2 45€1 51€2 40€1 15€1 45€3
40€1 31€1 43€1 50€2 49€1 37€1 48€1 16€2 41€2
39€0.3 33€1 54€2 54€1 51€0.3 48€1 44€1 12€1 54€2
a Data b
from EPS H28 has been previously reported (Martnez-Checa et al. 2002) Results are expressed as percentages of the total height occupied by the emulsion; values are means of at least three determinations
Table 3 Number of isolates from solid waste oil samples
Sample treatment
Number of isolates
EPS/straina V2–7 H28 H96 H212 H214 Control sample a
13 17 15 6 5 10
Number of isolates able to grow in the presence of polycyclic aromatic hydrocarbons (PAH) Naphthalene
Phenanthrene
Pyrene
13 8 15 3 3 2
2 2 4 1 0 0
3 1 3 1 0 0
EPS or H. eurihalina strain added to the solid waste oil sample
from the samples supplemented with EPS. At the same time, we randomly selected 10 strains (controls) from non-treated samples. Only 2 of the control strains grew on PAH (naphthalene). In contrast, among the selected 56 bacteria, 46 grew with naphthalene, 9 with phenanthrene and 8 in pyrene when they were cultivated in minimal liquid medium. Figure 1 shows, as an example, the growth on PAH of strains 96–7, 27–1 and 28–11 (the first number of each isolate indicates the EPS or the H. eurihalina strain added to the solid waste oil sample). As
can be seen, the number of cfu increased more than 10fold. Bacteria growing in PAH were taxonomically identified as Bacillus (21 strains), Pseudomonas (7 strains), Micrococcus (5 strains), Arthrobacter (4 strains), Aeromonas (4 strains), Xanthomonas (3 strains), Alcaligenes (1 strain), Enterobacter (1 strain), and Citrobacter (1 strain); 19 isolates could not be identified by the methods used. Strains 96–7, 27–1 and 28–11 were Bacillus pumilus (Fig. 1).
Discussion
Fig. 1 Growth of Bacillus pumilus strains in BH liquid medium supplemented with 0.1% (w/v) polycyclic aromatic hydrocarbons (PAH). Open circles Strain 96–7, pyrene; asterisks strain 27–1, naphthalene; open triangles strain 28–11, phenanthrene
Microorganisms have been shown to produce bioemulsifiers on a wide variety of hydrophobic substrates, such as n-paraffin, C12 n-alkanes and olive oil mill effluent (AbuRuwaida et al. 1991; Robert et al. 1989). H. eurihalina is a moderately Gram-negative bacterium, which produces EPS with interesting properties such as the increase of its solution viscosity at low pH values and the ability to emulsify hydrophobic substrates (Bjar et al. 1996, 1998; Calvo et al. 1995, 1998; MartnezCheca et al. 2002; Quesada et al. 1993). We are currently focusing our attention on the bioemulsifiers synthesised by this microorganism with a view to their application in bioremediation processes. The results of this work indicate that H. eurihalina strains grew in the presence of crude oil and produced increased quantities of EPS (Table 1). This fact suggests
350
that hydrophobic substrates enhance synthesis of EPS with emulsifying activities in H. eurihalina, as happens in other microorganisms that synthesise surfactant and/or emulsifiers. EPS chemical composition (Table 1) varied with respect to previously published data (Bjar et al. 1998). This is a common phenomenon described for other EPS, since culture conditions can modify quantity and quality of the biopolymers, although their basic structure is maintained (Sutherland 1990, 2001). As already mentioned, crude oil was the substrate most effectively emulsified by our polymers (Table 2). This result is in agreement with the higher amounts of EPS produced in the MY-crude oil medium (Table 1). Moreover, this fact suggested that H. eurihalina may modify its polymer to confer a specific activity according to the hydrophobic substrate used in the medium as carbon source. We also point out that H. eurihalina EPS were more active on crude oil than chemical surfactants (Table 2). PAH are organic compounds that constitute an important fraction of petroleum hydrocarbons and are widely distributed in diverse environments. Bacterial strains capable of degrading PAH have been isolated from these contaminated sites, although biodegradation of hydrocarbons seems to be limited by the low availability of hydrophobic pollutants to microorganisms. Nevertheless, it has been demonstrated that emulsifying agents enhance the degradation process due to better solubilisation of hydrocarbons prior to microbial degradation. (Dziel et al. 1996; Lin 1996). Addition of compounds able to emulsify hydrocarbons into microscopic droplets increases the surface area exposed to bacteria. This produces a readily available food source that bacteria can assimilate quickly. At the same time, the toxic effect of hydrocarbons increases and the sensitive microbial population begins to disappear. Bearing in mind these facts, we inoculated waste crude oil samples with either the bioemulsifying agents or the producer strains to isolate hydrocarbon-degrading bacteria. Our results indicated that the presence of either EPS or the producer strain allowed the isolation of major numbers of strains with the potential ability to degrade hydrocarbons (Table 3; Fig. 1). Abu-Ruwaida et al. (1991) and Banat (1995) reported similar results in relation to some Pseudomonas, Acinetobacter or Rhodococcus strains. Inoculation with these bacteria stimulated the isolation of indigenous microorganisms able to grow in the presence of hydrocarbons. We are currently carrying out a comprehensive study of those isolates that grew better in the presence of PAH. The majority of these bacteria belonged to the genus Bacillus, a microorganism usually found in soil and frequently involved in bioremediation processes (Doddamani and Ninnekar 2000; Stapleton et al. 2000). Some of these strains produce biosurfactants, which have been identified as lipopeptides, and have great emulsifying activity on hydrocarbons and other hydrophobic substrates (results not shown).
Acknowledgements Financial support was provided by grants from the Spanish Ministerio de Educacin y Cultura (BIO 98– 0897-C02–01) and from the Junta de Andaluca.
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