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Keywords:

  • bioproducts;
  • biotechnology;
  • diversity;
  • environmental;
  • heavy metals tolerance;
  • marine shallow vents

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  To characterize bacilli isolated from shallow hydrothermal vents of Panarea Island (Italy) and evaluate their biotechnological potential.

Methods and Results:  Fifteen isolates were characterized by culture and molecular methods. Eleven isolates were thermophilic, six isolates were alkalophilic and four of them were haloalkalophilic. After 16S rRNA gene sequencing, four strains, exhibiting sequence similarity below 95% with deposited strains, may represent novel species of bacilli. One strain was strictly related to Geobacillus subterraneus, but shared phenotypic characteristics for which it could be considered a new strain of this species. Four strains were affiliated with different Bacillus spp. Most isolates produced gelatinase, lipases and amylase, and some were mercury tolerant. Exopolysaccharides (EPS) production was tested adding different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose, 1% w/v) as a carbon source in a minimal medium. The highest EPS yield (185 mg l−1) was reached by strain 1A70 utilizing ribose as a carbon source.

Conclusions:  Novel strains of Geobacillus and indigenous ribotypes of Bacillus with biotechnological potential inhabit shallow vents of Panarea Island.

Significance and Impact of the Study:  New strains of thermophilic bacilli from Panarea are producers of useful biomolecules for industrial purposes as well as environmental and biotechnological applications.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Extreme environments, such as geothermal areas, terrestrial and marine hot springs, represent sources of novel micro-organisms loving extreme conditions or extremophiles. Hydrothermal systems, characterized by unusual conditions for most organisms (high temperature, high concentrations of H2S, hydrocarbons, heavy metals, etc.), provide a unique opportunity to gain new insights on microbial diversity and to isolate new micro-organisms able to produce compounds with biotechnological potential. Microbial biomolecules, such as enzymes, complex polysaccharides and exopolysaccharides (EPSs) produced in situ, as a strategy for growth and to survive in adverse conditions, represent a rich source of products useful in various industrial processes.

Thermophilic bacilli are widely distributed around the world in geothermal areas, and they have been also isolated from marine extreme environments, from both deep and shallow vents. Thermophiles, showing optimal growth at temperatures ranging from 45 to 70°C (Nazina et al. 2001), are fast growing, and their biomolecules (enzymes and EPSs) may have a great advantage over those from mesophilic or psychrophilic micro-organisms, as they can be produced in large quantities using a relatively simple purification process.

Thermostable enzymes from micro-organisms have received great attention during the last two decades. Enzymes that have optimal activity at extreme temperatures and pH levels are widely used in household detergents and in the food, textile, pulp and paper, leather processing and chemical industries (Podar and Reysenbach 2006).

Bacterial EPS produced in extreme marine habitats, characterized by novel chemical compositions, properties and structures, have been found to possess biotechnological potentialities (Guezennec 2003; Nicolaus et al. 2010; Poli et al. 2010). From deep-sea hydrothermal vents, some bacteria, such as Alteromonas macleodii subsp. fijiensis (Raguénès et al. 1996; Cambon-Bonavita et al. 2002), Vibrio diabolicus strain HE800 (Raguénès et al. 1997a), Pseudoalteromonas strain HYD721 (Rougeaux et al. 1999) and Alteromonas infernus strain 785 (Raguénès et al. 1997b; Colliec Jouault et al. 2001), producers of EPS with original structures, have been isolated. Among hyperthermophiles, the archeon Thermotoga maritima (Van Fossen et al. 2008) and the archeon Thermococcus litoralis (Rinker and Kelly 2000) from shallow springs have been reported as producers of EPS. Micro-organisms producing EPS have also been isolated from hypersaline aquatic environments, including the extremely halophilic marine archeon Haloferax mediterranei (Anton et al. 1988; Parolis et al. 1996), the slightly halophilic marine bacterium Hahella chejuensis (Lee et al. 2001) and the new haloalkalophilic bacterium Halomonas alkaliantarctica, isolated from an Antarctic salt lake (Poli et al. 2007).

Applications of polysaccharides produced by marine bacteria have been demonstrated in the food industry, as viscosity stabilizing, gelling or emulsifying agent, and in the pharmaceutical and medicine industry as antiviral and antitumoral (Umezawa et al. 1983; Okutani 1984; Matsuda et al. 1992; Weiner 1997).

Studies on shallow hydrothermal vents of the Eolian Archipelago (Italy) in the Mediterranean Sea indicate that these sites are sources of great microbial diversity (Maugeri et al. 2009, 2010a,b). Novel thermotolerant and thermophilic bacteria isolated until now from these sites were spore-forming bacilli, identified as Bacillus and Geobacillus spp. Among the new novel thermophilic species isolated from hot marine vents of Eolian Islands, Geobacillus vulcani (Caccamo et al. 2000), Geobacillus thermodenitrificans strain B3-72 (Nicolaus et al. 2000), Bacillus licheniformis strain B3-15 (Maugeri et al. 2002a) and three novel Geobacillus spp. (Maugeri et al. 2002b) have been described as producing novel EPSs.

EPS of the novel B. licheniformis strain B3-15 and G. thermodenitrificans strain B3-72 have been demonstrated to possess antiviral and immunostimulant effects (Arena et al. 2006, 2009). More recently, thermophilic species of Bacillus and Geobacillus, producers of enzymes potentially useful in biotechnology isolated from the Eolian Islands, have been reported (Lentini et al. 2007).

Here are described fifteen bacilli isolated from marine vents of Panarea Island (Italy) and their potential biotechnological exploitation, such as exopolysaccharide production, heavy metal tolerance and ability to produce biofilm.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Isolation of bacterial strains

Water and sediment samples were collected in June 2006 by SCUBA diving in the immediate vicinity of three vents, Bottaro (latitude 38°38′31″N–longitude 15°06′597″E), Campo 7 (latitude 38°37′59″N–longitude 15°06′360″E) and Black Point (latitude 38°38′23″N–longitude 15°06′28″E), located in front of Panarea Island near Bottaro, Dattilo, Lisca Bianca and Lisca Nera islets. Temperature and conductivity of thermal waters were recorded in situ by a digital thermometer (Hanna Instruments, Milan, Italy). The pH values of thermal waters were determined on board immediately after sampling.

To enrich heterotrophic thermophilic bacteria, 100 ml of thermal water was filtered through 0.2-μm cellulose membrane filters. Each membrane and sediment sample (1 g) was inoculated into Bacto Marine Broth 2216 (Difco Laboratories, Detroit, MI) (100 ml) (MB). Incubation was performed at 60, 70 and 80°C for 3 days under aerobic conditions. Subcultures were made onto Bacto Marine Agar 2216 (Difco) (MA) and incubated for 24 h. Colonies were picked and purified by streaking onto MA plates at least three times.

Phenotypic characterization of isolates

The isolates were observed for cell morphology, spore production and motility, Gram stained and tested for oxidase and catalase activity, H2S production in triple sugar iron (TSI) and glucose fermentation, as reported by Maugeri et al. (2001). Temperature and pH range for growth was determined following incubation of the strains for 3 days at 37°, 40°, 45°, 50°, 55°, 60° and 70°C and pH 5.5, 6, 7,8 and 9 in MB. Halotolerance was tested after incubation in Bacto Nutrient Broth (Difco) (NB) supplemented with 0, 2, 3, 5, 7 and 10% (w/v) NaCl. Optimal growth in MB and NB was evaluated by measuring the increase in turbidity at 600 nm with a spectrophotometer (Ultraspec 3000; Amersham Pharmacia Biotech, Freiburg, Germany). Sensitivity to the following antibiotics polymyxin B (300 U), penicillin G (10 U), novobiocin (30 μg), streptomycin (10 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), bacitracin (10 U) was tested on MA.

Lipase on Tween 20 (0.5%, v/v) and Tween 80 (0.5%, v/v) and hydrolysis of starch and gelatine were tested according to Lentini et al. (2007).

Phenotypic characteristics were compared with those of the following thermophilic reference strains: Geobacillus kaustophilus DSM 7263T, Geobacillus stearothermophilus DSM 22T, G. thermodenitrificans DSM 465T, Geobacillus thermoleovorans DSM 5366T, Bacillus caldolyticus DSM 405, Bacillus caldotenax DSM 406, Bacillus caldovelox DSM 411. B. licheniformis strain B3-15 and G. thermodenitrificans strain B3-72, previously isolated from marine vents of Vulcano Island (Eolian Islands), were also included.

Genotypic characterization of isolates

Amplified ribosomal DNA restriction analysis.

Amplified ribosomal DNA restriction analysis (ARDRA) was used as a prescreen method for clustering the isolates into genetically homogeneous groups before 16S rRNA gene sequencing.

Genomic DNA was extracted from single colonies by using DNeasy tissue kit (Qiagen, Milan, Italy), according to the manufacturer’s directions. The 16S rRNA genes were amplified by PCR using universal bacterial primers 27f and 1525r, and PCRs were performed according to Lentini et al. (2007).

Amplified products were digested with 3 units of AluI and TaqI (Invitrogen, Milano, Italy) restriction enzymes. Approximately, 20–50 ng of amplified 16S rRNA products were cleaved in a total volume of 20 μl by incubating the reaction mixtures at 37°C for 180 min. The restriction enzyme AluI was then inactivated by heating the mixtures at 65°C for 10 min. The reaction products were analysed by agarose gel (2.5% w/v) electrophoresis in TAE buffer (0.04 mol l−1 Trisacetate, 0.001 mol l−1 EDTA) and stained with ethidium bromide.

16S rRNA sequence determination and analysis.

Selected isolates were analysed for 16S rRNA sequencing. The PCR products were purified using the Wizard Genomic DNA Purification kit (Promega, Madison, WI) according to the manufacturer’s protocol and sequenced by BIOFAB (Roma, Italy). A nucleotide BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to obtain sequences with the most significant alignment.

Sequences assigned to isolates and selected reference sequences were used for phylogenetic tree construction generated by the neighbour-joining using the Kimura-2 parameters algorithm. Distance matrix trees were generated by the neighbour-joining (NJ) method with the Felsenstein correction as implemented in the PAUP 4.0B software (Sinauer, Sunderland, MA, USA). The NJ calculation was subjected to bootstrap analysis (1000 replicates).

EPS production

Different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose) in concentration of 1% (w/v) were tested as carbon source in 10 ml of a minimal medium containing 0.01% of yeast extract in seawater (SWY). Cultivation was performed at each strain optimal growth for 3 days, and growth was determined by measuring optical density (OD at 540 nm).

For EPS production, isolates were inoculated into flasks containing 200 ml of the SWY supplemented with each of the seven different sugars before reported and incubated at optimal growth conditions with shaking at 240 rev min−1 for 3 days. Cells were harvested by centrifugation (9800 g, 20 min at +4°C). The supernatant was treated with an equal volume of cold absolute ethanol, added drop wise under stirring in an ice bath, held at −20°C overnight and then centrifuged at 13 000 g for 30 min. The pellets were washed two times with ethanol and dissolved in hot water, dialysed against distilled water, lyophilized and weighed.

EPS were tested for carbohydrate, protein and nucleic acid contents (Manca et al. 1996). EPS production was tested on cell-free cultural broth with the phenol–sulphuric acid method using glucose as standard (Dubois et al. 1956). EPS sugar analysis was performed by hydrolysis of EPS with 0.5 N trifluoroacetic acid (TFA) at 120°C for 2 h. Sugar mixture was identified by HPAE-Pad Dionex (Dionex, Sunnyvale, CA) equipped with Carbopac PA 1 column and was eluted isocratically with 16 mmol l−1 NaOH (Manca et al. 1996).

Adherence test

The ability to adhere on hydrophobic substrates and to form a biofilm was tested according to the method described by Maugeri et al. (2002b). Strains were cultured overnight at each optimal condition, in 15-ml polystyrene tubes (Falcon; Corning, NY) containing MB or SWY plus glucose (1% w/v), mannose (1% w/v), saccharose (1% w/v) or trehalose (1% w/v). The tubes were then carefully emptied of the cultures and stained with safranin. Adherence was assessed by evaluation of a visible slime on the polystyrene tube walls. The presence of only a ring at the liquid–air interface was not considered indicative of slime production. The amount of biofilm production was estimated as weak (+), moderate (++) or strong (+++).

Heavy metals tolerance

To minimize metal complexation, microdilutions were carried out in MB medium containing only 25% of organic substrates concentration. The tested metals were provided as CdCl2 · 5H2O, ZnSO4 · 7H2O, Na2HAsO4 · 7H2O, AgNO3 and HgCl2. Metal-amended media were prepared by adding the following filter-sterilized metal stock solutions: Cd2+ (40, 80, 120 μg ml−1), Zn2+ (50, 100, 150 μg ml−1), As2+ (500, 1000, 1500 μg ml−1), Ag2+ (5, 10, 15 μg ml−1) and Hg2+ (0.5, 1.0, 1.5 μg ml−1).

Media were poured in 96-well plates and inoculated with 25 μl of an overnight culture of each strain in MB. Microplates were incubated at 50°C for 3 days, and growth was spectrophotometrically evaluated (600 nm).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Phenotypic and genotypic characteristics of isolates

Temperature, pH and conductivity of thermal waters registered at the studied sites and the origin of isolated strains are reported in Table 1.

Table 1.   Physical and chemical characteristics of thermal waters at the sampling sites of Panarea Island and related isolates
SiteDepth (m)Temperature (°C)pHConductivity (mS cm−1)Isolate
Black point23.01303.3046.20PBP60, SBP3, SBP4
Bottaro8.0555.4242.901A60, 1A70, PB4, PC1, PG1, SB4
Campo 721.3604.9249.20APA, APB, NPA2, PI1, PL2, SC7-3

Isolates, reported in Table 2, were Gram positive, endospore forming, aerobic or facultative anaerobic, oxidase positive and most of them were catalase positive. None produced H2S in TSI. Most isolates (11/15) were thermophilic (optimum temperature ≥ 45°C), and 13/15 were halophilic (Table 2). APB and PG1 strains were strictly thermophilic, unable to grow under 50 and 55°C, respectively. All isolates were able to grow at pH 9. Three strains (NPA2, PG1 and PI1) were moderately acidophilic (optimal pH values 6 and 5.5, respectively). Six isolates were alkalophilic (optimal pH 8) and four of them were haloalkaliphilic (1A60, PL2, SB4 and SBP4) also showing optimal growth at high NaCl concentration (NaCl 3–5%).

Table 2.   Growth characteristics, antibiotic sensitivity and enzymatic production of the fifteen isolates from Panarea Island in comparison with reference strains of Bacillus and Geobacillus spp.
StrainGrowth temperature (°C)Optimum temperature (°C)Growth pHOptimum pHGrowth NaCl (%)Optimum NaCl (%)CatalaseGlucose fermentationHydrolysis ofAntibiotic sensitivity
GelatinStarchTween 20Tween 80BacitracinChloramphenicolNalidixic AcidNovobiocinPenicillin GPolymyxin BStreptomycin
1A6037–60505.5–982–105++++++
1A7037–70607.0–980–22++++++++++
APA37–70455.5–973–73+++++++
APB50–70605.5–970–32+++++++
NPA237–60505.5–962–107++++++
PB437–60405.5–970–53++++++
PBP6037–60505.5–972–32++++++
PC137–70505.5–9700+++++++
PG155–70605.5–960–22++++++++++
PI137–70505.5–95.50–50++++++
PL237–60606.0–982–33++++++
SB437–60406.0–983–55+++++
SBP337–50405.5–982–72++++++
SBP437–60505.5–9855+++++++
SC7-337–60406.0–970–73+++++
Reference
 Bacillus licheniformis strain B3-1525–60455.5–970–72+++++++++
 Bacillus caldolyticus DSM 40537–75705.5–980–20++++++++++
 Bacillus caldotenax DSM 40637–70655.5–95.50–20++++++++++
 Bacillus caldovelox DSM 41137–70705.5–980–20+++++++++
 Geobacillus kaustophilus DSM 7263T37–70655.5–970–22++++++++++
 Geobacillus thermodenitrificans strain B3-7245–70656.0–970–20++++++++
 Geobacillus thermoleovorans DSM 5366T37–70657.0–970–20+++++++++
 Geobacillus stearothermophilus DSM 22T37–75605.5–970–30+++++++++
 G. thermodenitrificans DSM 465T37–70656.0–970–22+++++++++

At optimal growth conditions for each isolate, 12 strains were able to hydrolyse gelatine, five hydrolysed starch, seven were lypolitic on Tween 20 and only one (strain APB) on Tween 80 (Table 2).

All strains were susceptible to novobiocin and chloramphenicol, and 13 of 15 strains were susceptible to bacitracin (Table 2). Only strain 1A70 was sensitive to all antibiotics. Strain PG1 showed the same antibiotic pattern of Bacillus and Geobacillus DSM reference strains. Seven strains (1A60, APA, PBP60, PL2, SB4, SBP4 and SC7-3) possessed the same antibiotic pattern, as they were also sensitive to bacitracin. Strains APB and PB4 were sensitive to penicillin G. Each of the remaining strains (NPA2, PC1, PI1 and SPB3) had a different antibiotic pattern.

The analysis of ARDRA profiles allowed us to group the fifteen isolates into four clusters. Strains 1A60, APA and APB were grouped into the first cluster and showed the same pattern profile of G. stearothermophilus DSM 22T. Strains 1A70, PB4, PC1 and PG1 were grouped into the second cluster together with B. caldolyticus DSM 405 and B. caldotenax DSM 406. The third cluster was composed by strains PI1, PL2, SBP4, SB4, SC7-3, PBP60 and B. caldovelox DSM 411. Strain NPA2 showed the same pattern of B. licheniformis strain B3-15. Strain SBP3 showed an unique restriction pattern, as well as the remaining thermophilic reference strains.

Nine isolates representative of each ARDRA group or showing unique ARDRA profile were selected for the partial 16S rRNA gene sequence analysis (Table 3).

Table 3.   Amplified ribosomal DNA restriction analysis (ARDRA) groups and restriction pattern profiles of the fifteen strains from Panarea Island in comparison with reference strains (in bold, strains selected for 16S rRNA gene sequence analysis)
ARDRA groupSpeciesRestriction pattern of 16S rRNA genes digested withStrain
Alu ITaq I
1Geobacillus stearothermophilus DSM 22TDA1A60, APA, APB
2Bacillus caldolyticus DSM 405 Bacillus caldotenax DSM 406DD1A70, PB4, PC1, PG1
3Bacillus caldovelox DSM 411EDPI1, PL2, SBP4, SB4, SC7-3, PBP60
4Bacillus licheniformis strain B3-15BANPA2
FDSBP3
Geobacillus thermoleovorans DSM 5366TAA 
Geobacillus kaustophilus DSM 7263TBC 
Geobacillus thermodenitrificans strain B3-72CA 
G. thermodenitrificans DSM 465TEC 

Phylogenetic analysis revealed that all sequences from the nine strains were similar to those from different species of Bacillus and Geobacillus genera (Table 4). Strains APA and 1A60 exhibit sequence similarity below 95% with GenBank deposited strains and may represent a novel species of Geobacillus genus. Strain SBP3 was strictly related (99% similarity) to Geobacillus subterraneus strain R-35641. Four strains (PG1, 1A70, SB4 and NPA2), sharing high sequence similarity level (from 97 to 99%) were affiliated with different Bacillus species. Two strains (SBP4 and PI1) showed DNA sequences ≤89% similarity with respect to the other previously described bacilli. In Fig. 1 is shown the phylogenetic tree based on the 16S rRNA sequences of the nine isolates and related species of Bacillus and Geobacillus genera.

Table 4.   Similarity percentage (%) based on the partial 16S rRNA gene sequences of isolates from Panarea Island to their closest bacteria present in the NCBI database
StrainAffiliationSequence similarity (%)GenBank accession
1A60Geobacillus stearothermophilus strain mt-292EU652074
1A70Bacillus oceanisediminis strain m3498JF411236
APAGeobacillus stearothermophilus strain mt-794EU652066
NPA2Bacillus pichinotyi strain ra-2499EU652096
PI1Bacillus alcaliinulinus strain AM3170AB018595
PG1Bacillus firmus strain IARI-J-2898JN411422
SB4Bacillus sporothermodurans strain DSMZ 1059994U49079
SBP3Geobacillus subterraneus strain R-3564199FN428689
SBP4Bacillus licheniformis strain CICC 1033289GQ375242
image

Figure 1.  Phylogenetic neighbour-joining tree obtained with the 16S rRNA sequences of the nine strains isolated from Panarea Island and related validated Bacillus and Geobacillus spp. Neighbour-joining tree was constructed from each data set using the Kimura 2-parameters model in MEGA 4.0 (Biodesign Institute, AZ, USA). Bootstrap tree was constructed using 1000 replicates.

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Exopolysaccharide production

Growth of isolates utilizing different carbohydrates as carbon sources is shown in Fig. 2(a). Although several strains were able to use different carbohydrates, sucrose, ribose and glucose were the most efficient carbon source for growth, followed by trehalose, xylose and fructose. Growth of strains 1A60, APA, PC1 and PG1 was strongly supported by sucrose. Strains 1A70, NPA2, PB4 and SBP3 grew well with ribose; PI1, PL2 and SC7-3 with glucose; SBP4 and PBP60 with trehalose. Strains APB and SB4 reached the best growth with xylose and fructose, respectively.

image

Figure 2.  Growth (a) and exopolysaccharides production (≥10 mg l−1) (b) of the fifteen strains from Panarea Island with different carbohydrates as carbon sources after 3 days at each optimal growth. OD, Optical density. (inline image) Suc; (inline image) Tre; (inline image) Glu; (inline image) Man; (inline image); Xyl (inline image) Fru; (inline image) Rib; (inline image) Xylose; (inline image) Trehalose; (inline image) Sucrose; (inline image) Fructose; (inline image) Mannose; (inline image) Risbose and (inline image) Glucose.

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The EPS production, using different carbohydrates in SWY medium, for each isolate is reported in Fig. 2(b). Most strains (10/15) utilized glucose as carbon source to produce great amounts of EPS ranging from 60 to 90 mg l−1, the remaining strains produced EPS in a small amount (≤10 mg l−1).

The 1A60 EPS was chosen among other EPS for a better characterization because it showed the highest rate (70%) of carbohydrate content. The EPS also contained protein (0.8%) and nucleic acid (0.6%). 1A60 EPS contained mannose/galactose/galactosamine/fucose and glucose in a relative proportion of 1 : 0.69 : 0.65 : 0.59 : 0.35.

Adherence test

Results of the adherence test are reported in Table 5. The greatest amount of biofilm on polystyrene tubes walls was produced by strain PL2 and SBP3 after growing in MB plus glucose and trehalose, respectively. Strain 1A70 and SBP4 showed a moderate biofilm production in MB plus sucrose and trehalose, respectively. Most strains (7/15) produced a weak biofilm in MB plus glucose, and four strains in MB plus trehalose. Only strains APB and SC7-3 produced a weak biofilm in SWY plus mannose and trehalose, respectively.

Table 5.   Adherence test of isolates from Panarea Island (MB, marine broth; SWY, seawater plus yeast extract 0.01%)
StrainBiofilm production on polystyrene tubes with:
MB + glucoseSWY + glucoseMB + mannoseSWY + mannoseMB + sucroseSWY + sucroseMB + trehaloseSWY + trehalos
1A60+
1A70+++
APA+
APB++
NPA2+
PB4+
PBP60+++
PC1+
PG1
PI1+
PL2+++
SB4+
SBP3+++
SBP4++
SC7-3+++

Heavy metals tolerance

Strains 1A60, APA, APB, NPA2, PB4, PBP60, PL2 and SB4 displayed tolerance to the highest concentration of mercury used (1.5 μg ml−1), and all strains were sensitive to the other heavy metals.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Shallow hydrothermal system of Panarea Island, the most active of the Eolian Islands, characterized by fluid temperatures reaching 130°C and gas emissions dominated by CO2, provides a natural experimental site to examine the way in which organisms respond to the unusual, extreme and environmental conditions. Microbial diversity in the submarine hydrothermal vents of the Eolian Islands has recently been explored (Maugeri et al. 2009, 2010a,b).

Here are described fifteen bacilli isolated from three marine vents of Panarea Island (Italy). Most new isolates were thermophilic and halophilic, some of them were haloalkaliphilic and others were acidophilic, indicating a great physiological versatility that allows them to adapt to the severe environmental conditions. Compared with other groups of extremophiles, especially thermophilic, biotechnological applications of halophilic micro-organisms are until now underestimated, probably due to the fact that few species of Bacillus able to grow with high NaCl concentrations have been described (Romano et al. 2005; Oren 2010). Several Bacillus and Geobacillus species are considered useful bacteria in industrial processes, as they are able to produce proteases, amylases and lipases (Lentini et al. 2007). New haloalkaliphilic isolates (strains 1A60, PL2, SB4 and SBP4) could also have important industrial applications because of their ability to produce protease resisting to high pH and high temperature conditions.

The combination of ARDRA profiles, obtained using AluI and TaqI restriction enzymes, provided a satisfactory tool in clustering our isolates into genetically homogeneous groups before 16S rRNA gene sequencing, as well as to distinguish from the well-known thermophilic reference strains, which possess high similarity levels of their 16S rRNA gene sequences. The identification of the new isolates was obtained by using 16S rRNA gene sequencing. In details, strains APA and 1A60, exhibiting sequence similarity below 95% with GenBank deposited strains, may represent novel species of Geobacillus genus. Strain SBP3, strictly related (99% similarity) to a strain of G. subterraneus, could be considered a new strain of this species, differing from the type strain G. subterraneus DSM 13552T in that strain SBP3 is less thermophilic, more halophilic and able to hydrolyse gelatine. Four strains (PG1, 1A70, SB4 and NPA2), sharing similarity levels ≥97%, were affiliated with different Bacillus species. Two strains (SBP4 and PI1) showed DNA sequences similar ≤89% with respect to previously described bacilli, suggesting the presence of indigenous bacterial ribotypes at shallow hydrothermal vents of Panarea Island. These findings confirm that members of Bacillus and Geobacillus genera are widespread in the shallow hydrothermal system of the Eolian Islands (Maugeri et al. 2001, 2009, 2010a) and revealed a high taxonomic diversity among bacilli isolated from vents of Panarea Island. Heterotrophic bacilli play an active role in the biogeochemical cycles at hydrothermal vents (Maugeri et al. 2009, 2010a). As they live in both hot and salty environments with great fluctuations of physico-chemical conditions, it is expected that their EPS represent a cellular protection from stressful conditions of the surrounding environment or against possible predators. EPS could serve in the biofilm production to closely adhere and colonize the substrata and to contrast the floating induced by the gas emissions. Adherence test to hydrophobic surface was tested as indicative for the production of biofilm, as well as for the potential affinity to other hydrophobic substances, such as hydrocarbons (Rosenberg 1984). It is known that on the basis of their structural characteristics, some EPS are employed as biosurfactants in detoxification mechanisms of oil-polluted area (Poli et al. 2010).

Exploration of antibiotic pattern and resistance to heavy metals are also indicative for the potential of our isolates in responding to stressful conditions, such as those involved in various applicative processes. Most isolates were tolerant to mercury and possessed an unusual characteristic among bacilli from the Eolian Islands until now described (Maugeri et al. 2002a,b). Although resistance to the mercuric ion is a widely distributed trait among bacteria, very few works reported its tolerance among extremophilic micro-organisms (Glendinning et al. 2005). We can suppose that bacterial EPS also protect the cells in situ, by binding heavy metals released from vents. The important role of EPS in the removal of heavy metals from the environment is because of their involvement in flocculation and their ability to bind metal ions from solutions (Nicolaus et al. 2010). Biosorption of toxic heavy metals constitute an attractive alternative to commonly used physicochemical remediation processes. As surface-active agents, bacterial EPS could be used for heavy metal removal in the treatment of metal-contaminated wastewaters, in mining wastes and effluents from metallurgic industries (Pagnanelli et al. 2000).

When compared with other bacilli producers of EPS previously isolated from other Eolian sites, the yield of EPS by the new isolates from Panarea Island, using glucose as carbon source, was higher than that produced by G. thermodenitrificans strain B3-72 (70 mg l−1) (Nicolaus et al. 2000), but was lower than that obtained by B. licheniformis strain B3-15 (165 mg l−1) (Maugeri et al. 2002a). The highest EPS yield (185 mg l−1) was produced by Bacillus strain 1A70, growing with ribose as carbon source, increasing the yield more than twofolds with respect to the glucose. This amount was lower than that produced by B. thermoantarcticus (400 mg l−1) using mannose (Manca et al. 1996), but was higher than those produced by bacilli recently isolated from hot springs, such as Geobacillus tepidamans V264 (111.4 mg l−1, with sucrose) (Kambourova et al. 2009), Geobacillus strain 4004 (65 mg l−1, with sucrose) and Geobacillus strain 4001 (55 mg l−1, with sucrose) (Nicolaus et al. 2002). These results confirm that members of the genus Bacillus are more EPS productive than those of genus Geobacillus described until now.

Even though shallow hydrothermal vents of Eolian Islands offer a great microbial diversity, at the moment, micro-organisms thriving in this ecosystem represent an almost unexploited resource of biomolecules. Bacterial EPS from shallow hydrothermal vents of Panarea Island are a promising biotechnological source in responding to the increasing demand for natural polymers in biotechnological applications. Characteristics of the new bacilli, such as production of bioactive molecules (enzymes and EPSs) and tolerance to heavy metals, may be useful for industrial purposes and for environmental applications in bioremediation.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study was supported by research grants (2002 funds) from the University of Messina, Italy.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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