A new subspecies of Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. with very high ethanol tolerance

Authors


  • Editor: Aharon Oren

Correspondence: Hui Peng, Engineering Technology Research Center of Microorganisms and Biocatalysis, Anhui Province, School of Life Sciences, Anhui University, Hefei 230039, China. Tel.: +86 551 510 7224; fax: +86 551 386 1724; e-mail: pph0259@126.com

Abstract

In a search for thermophilic ethanol-tolerant bacteria, water-sediment samples collected at springs in Yunnan province of China were screened by ethanol enrichment. A novel thermophilic bacterium, strain E13T, was isolated. It exhibits a unique and remarkable ability to preferably grow in the presence of ethanol and is able to tolerate 13% (v/v) ethanol at 60 °C. The isolate is a facultative aerobic, Gram-positive, motile, spore-forming rod that is capable of utilizing a range of carbon sources, such as xylose, arabinose and cellobiose. Phylogenetic analysis based on 16S rRNA gene similarity showed the strain to be affiliated with the species Anoxybacillus flavithermus (99.2% sequence similarity). DNA–DNA hybridization comparisons demonstrated a 64.8% DNA–DNA relatedness between strain E13T and A. flavithermus DSM 2641T. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA hybridization data, it was concluded that the isolate merited classification as a novel subspecies of A. flavithermus, for which the name Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. is proposed. The type strain of this subspecies is E13T (=CCTCC AB2010187T=KCTC 13759T).

Introduction

Organic-solvent-tolerant bacteria are a relatively new subgroup of extremophiles. They are able to overcome the toxic and destructive effects of organic solvents on account of their unique adaptive mechanisms. Ethanol (log Pow=−0.32) (Pow=partitioning coefficient n-octanol/water) is a low toxic compound when compared with extremely toxic solvents with a log Pow value between 1.5 and 4.0. Several mesophilic bacteria capable of tolerating high concentrations of ethanol have been investigated extensively. For example, Lactobacillus heterohiochii (a later heterotypic synonym of Lactobacillus fructivorans) and Zymomonas mobilis exhibited tolerance to ethanol up to 18% (% value is in v/v) (Ingram 1990) and 13% (Liu & Qureshi, 2009), respectively. However, thermophilic bacteria rarely tolerate >2% ethanol (Rani & Seenayya, 1999; Burdette et al., 2002), primarily because the level of ethanol tolerance decreases drastically with increasing temperature (Georgieva et al., 2007). Recently, a mutant strain of Thermoanaerobacter ethanolicus 39E-H8 has been reported to survive and grow weakly in up to 8% ethanol at 60 °C (Burdette et al., 2002). Ethanol tolerance (maintain viability) as high as 10% has been reported in Geobacillus thermoglucosidasius M10EXG (Fong et al., 2006). There is no report of thermophilic bacterial strains capable of active growth in 8% ethanol, or growth in concentrations above 10%.

In the search for new thermophilic ethanol-tolerant bacteria, samples taken from hot springs were screened by ethanol enrichment, resulting in the isolate E13T. It exhibits a unique and remarkable ability to preferably grow in the presence of ethanol (up to 8%) at high temperature and is able to tolerate 13% ethanol at 60 °C. The phylogenetic 16S rRNA gene sequence analysis revealed that strain E13T is affiliated with the recently established genus Anoxybacillus (Pikuta et al., 2000). At present, the genus Anoxybacillus comprises 15 species with validly published names. Only Anoxybacillus kamchatkensis contains two subspecies (Gul-Guven et al., 2008). None of these Anoxybacillus strains is reported to tolerate ethanol. On the basis of phenotypic features as well as molecular studies, we propose to classify the strain E13T as a novel subspecies, Anoxybacillus flavithermus ssp. yunnanensis ssp. nov.

Materials and methods

Isolation of the strain

Cells were isolated from combined water-sediment samples collected from hot springs in Yunnan, China, in rich Luria–Bertani (LB) medium supplemented with 10% ethanol for 12 h at 60 °C without shaking in sealed Balch bottles. After the growth proceeded to opacity, an aliquot was removed and used as an inoculum in a new bottle with fresh medium and an additional 5% ethanol for another 12-h incubation. The cultures were diluted and spread on agar plates without ethanol. Individual colonies, visible after 8–10 h, were subcultured in medium supplemented with 10% ethanol to confirm the ethanol tolerance of the strains. The strains were purified by repeatedly isolating single colonies in agar plates at least five times.

The type strains of A. flavithermus DSM 2641T, Anoxybacillus pushchinoensis DSM 12423T and Anoxybacillus kestanbolensis NCIMB 13971T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Anoxybacillus eryuanensis KCTC 13720T and Anoxybacillus tengchongensis KCTC 13721T were acquired from the Korean Collection for Type Cultures (KCTC).

Growth in the presence of ethanol

The cells were initially cultured overnight in LB medium with 4% ethanol. The culture was washed twice in unsupplemented medium to remove ethanol and then used for inoculation of medium with ethanol added at concentrations of 0–10%. The cultures were incubated without shaking at temperatures in the range of 45–65 °C for 60 h. Samples for measurement were withdrawn directly from the sealed bottles with sterile syringes before and after incubation. Growth was measured spectrophotometrically at 600 nm. To evaluate whether this organism can metabolize ethanol, ethanol was analyzed by Agilent 7890A GC System equipped with an Agilent 7694E Headspace Sampler (Agilent Technologies).

The microbial biofilm and its formation were observed by light microscopy (Olympus, BX51). Sterile glass slides were placed in LB culture supplemented with 13% ethanol. The culture was incubated without shaking at 60 °C for 24 h. The slides were then taken out of the bottles and washed three times with H2O. The remaining cells were fixed with methanol for 10 min and stained with 2% (w/v) crystal violet for 5 min.

Physiological and biochemical characteristics

Physiological and biochemical tests were carried out without ethanol addition at 60 °C. Conventional biochemical tests were performed according to standard methods (Smibert & Krieg, 1994). Anaerobic growth experiments were carried out in Hungate tubes. The ability to utilize different carbon source was examined in basal medium (Pikuta et al., 2000).

Cellular fatty acids

To minimize the effects of growth temperature and different media on bacterial fatty acid composition, all strains were uniformly incubated at 60 °C for 24 h on agar LB medium. Then, the analysis of cellular fatty acid methyl esters were performed according to the method described in the Sherlock Microbial Identification System manual (version4.0, MIDI). The final extracts were analyzed by GC/MS in scan mode, using an Agilent 7890 GC/5975 MSD system (Agilent Technologies).

16S rRNA gene sequence and phylogenetic analysis

The 16S rRNA gene was amplified by PCR with the following forward and reverse primers: TTAGAGTTTGATCCTGGC and GGTTACCTTGTTACGACT. The sequence was submitted to the GenBank Data Library with the accession number HM016869. The 16S rRNA gene sequence was aligned with equivalent 16S sequences of all closely related strains found in the GenBank database via a blast search and aligned using clustal w. The phylogenetic tree was calculated with the neighbor-joining method in the phylip package (Felsenstein, 2004).

G+C content and DNA–DNA hybridization

The G+C content was determined by the HPLC method (Mesbah et al., 1989). DNA–DNA homology experiments were carried out by the DSMZ Identification Service.

Results and discussion

Growth characteristics of strain E13T in the presence of ethanol

Only one thermophilic isolate that can grow in the presence of 10% ethanol at 60 °C was isolated. The effect of exogenously added ethanol on the growth of strain E13T at the optimum growth temperature of 60 °C is presented in Fig. 1d. The results showed that the strain E13T not only tolerated high concentrations of ethanol, but grew better in the presence of an amount of ethanol. At concentrations below 6%, ethanol stimulated the growth of strain E13T when compared with a control sample incubated without ethanol. The highest growth rates were consistently attained in the presence of 2% and 4% ethanol, and 4% ethanol resulted in the highest cell yield (final OD600 nm at stationary phase). To our knowledge, this is the first report of a wild-type thermophilic bacterium that has a preferable growth in the presence of ethanol. We define this property as ‘ethanol adaptation’, as against ethanol tolerance. In addition, the ability of strain E13T to utilize ethanol was determined by monitoring ethanol concentrations during cell growth. No significant difference in concentrations of ethanol was observed (data not shown). The results showed that the strain E13T was unable to degrade ethanol.

Figure 1.

 Effect of exogenously added ethanol (v/v) on the growth of strain E13T at different temperatures. The cells were grown to early exponential phase (OD600 nm≈0.25) in sealed Balch bottles at 60°C, and various concentrations of ethanol from 0% to 10% was added. Subsequently, the cultures were incubated at temperatures in the range of 45–65°C for 60 h. (a) 45°C; (b) 50°C; (c) 55°C; (d) 60°C; (e) 65°C. (○ 0%, • 2%, □ 4%, ▪ 6%, ◊ 8% and ♦ 10%). All data were based on at least three independent replicates, and each replicate experiment had three parallel cultures.

Comparison of the growth of strain E13T at different temperatures showed that the ethanol adaptation was temperature dependent (Fig. 1). The growth rates remained relatively high up to 8% ethanol at 45 and 50 °C (Fig. 1a and b), but in 8% ethanol at 55 °C, the growth rate decreased significantly although the cell yield reached under this condition was still much higher than that reached in the control sample (Fig. 1c). The addition of 8% ethanol repressed the microbial growth, causing a decrease in the achieved cell yield at 60 °C (Fig. 1d), while no increase in OD600 nm readings was observed for the ethanol concentration of 8% at 65 °C (Fig. 1e). The results indicated that ethanol adaptation increased to 8% ethanol with decreasing temperature, which was similar to previous investigations of ethanol tolerance reported in the literature (Bascaran et al., 1995; Georgieva et al., 2007). In the case of Thermoanaerobacter A10, Georgieva and colleagues demonstrated that a temperature increase of 15 °C, from 50 to 65 °C, resulted in a decrease in the critical inhibitory ethanol concentration from 6.1% to 5.5%.

Anoxybacillus flavithermus DSM 2641T did not exhibit significant tolerance to ethanol (up to 3%), nor did it show any ethanol adaptation under the same culture conditions. The same happened in the cases of A. pushchinoensis, A. kestanbolensis, A. eryuanensis and A. tengchongensis.

Biofilm formation

Although no discernible increase in turbidity (OD600 nm) was measured at concentrations of ethanol in the media above 10%, a biofilm consisting of bacterial cells enclosed in an extracellular polysaccharide matrix actively growing on a surface (Hamon & Lazazzera, 2001) was observed on the glass surface of the bottles after a 24-h incubation. Moreover, multilayer biofilms were clearly seen even though the ethanol concentration eventually reached 13% at 60 °C (Fig. 2a). The freely suspended cells of strain E13T incubated with 8% ethanol showed a tendency to aggregate. Some cells adhered to each other and formed tree-like structures, which might be important for its initial attachment to a surface (Fig. 2b). Biofilm formation is an important strategy for bacterial accumulation in natural aquatic habitats. Biofilms have been proposed to constitute an environmental refuge for a number of bacteria and to provide bacteria with an adaptive advantage promoting their environmental persistence (Matz et al., 2005). In many bacteria, especially strains of pathogenic genus, ethanol stress has been reported to lead to induction of biofilm formation (Knobloch et al., 2006; Mukherjee et al., 2006). Therefore, we suggest that the biofilm formation by strain E13T has an important contribution to the adaptive advantage of growth under high ethanol stress conditions.

Figure 2.

 Light microscope images showing the biofilm formation of strain E13T. (a) A multilayer biofilm growth on the glass slide surface after 24-h incubation with 13% (v/v) ethanol at 60°C; (b) the freely suspended cells after 24-h incubation with 8% (v/v) ethanol at 60°C. Arrow points to the cells forming tree-like structures.

The ability of A. flavithermus CM to produce biofilms has been investigated (Burgess et al., 2009). The biofilm of A. flavithermus DSM 2641T was also observed in LB medium without ethanol after 12 h of incubation at 60 °C.

Morphological and biochemical analysis

The cells of strain E13T appeared as Gram-staining-positive, motile, spore-forming rods. At stationary phase, the cells were 0.4–0.7 μm in width and 1.2–7.0 μm in length. The temperature growth range was from 30 to 66 °C with an optimal growth at 60 °C. The pH growth range was from 5.5 to 10.0 with an optimum growth at 7.0–7.5. The strain E13T was catalase positive while it was negative for gelatin hydrolysis, starch hydrolysis, nitrate reduction, indole production and phenylalanine deaminase. Growth of strain E13T was inhibited in the presence of NaCl concentration above 3.5% (w/v) and the optimal NaCl concentration for growth was 0.3% (w/v). The isolate E13T utilized a wide range of carbon sources including arabinose, cellobiose, galactose, gluconate, glucose, maltose, mannitol, sucrose, trehalose and xylose. The following carbon sources did not support growth: ethanol, fructose, lactose, mannose, rhamnose and ribose. The differentiating phenotypic features between the new isolate and phylogenetically as well as phenotypically related species are indicated in Table 1. The major distinctions include substrate specificities with particularly good growth on arabinose and xylose and the lack of growth on mannose.

Table 1.   Differential characteristics of strain E13T and its closest relatives
Characteristic1234567
Motility++++++
  1. 1, Strain E13T; 2, strain PGDY12; 3, Anoxybacillus flavithermus ssp. flavithermus DSM 2641T; 4, Anoxybacillus eryuanensis KCTC 13720T; 5, Anoxybacillus tengchongensis KCTC 13721T; 6, Anoxybacillus kestanbolensis NCIMB 13971T; 7, Anoxybacillus pushchinoensis DSM 12423T.

  2. +, positive; −, negative. The tests for the reference strains were also performed in the present study.

Temperature range for growth (optimum) (°C)30–66 (60)30–65 (55)30–72 (60–65)35–70 (55)30–75 (50)40–70 (50–55)37–66 (62)
pH range for growth (optimum)5.5–10.0 (7.0–7.5)5.0–10.0 (7.0)5.5–9.0 (7.0)7.0–11.0 (8.0)7.0–11.0 (8.5)6.0–10.5 (7.5–8.5)8.0–10.5 (9.5–9.7)
Tolerance to NaCl (%)3.53.52.53.04.04.03.0
Substrates utilized
 l-Arabinose++
 d-Xylose++
 d-Mannose++++
 Starch++++
Nitrate reduction++++
Hydrolysis of gelatine++
Catalase++++++
DNA G+C content (mol %)42.344.541.842.641.15042.2

Fatty acid profile

The results from the fatty acid analysis revealed that the strain E13T synthesized nine kinds of saturated fatty acids, similar to thermophilic Anoxybacillus species (Table 2). However, the cellular fatty acid compositions of strain E13T differed remarkably from that of the known members of the genus Anoxybacillus. The major fatty acid of the strain E13T was a straight-chain C16 : 0 (33.4%). For the members of the genus Anoxybacillus, the most abundant was a branched-chain iso-C15 : 0 (average value 58.9%) whose value for strain E13T was only 14.5%. Therefore, the known Anoxybacillus species contain branched-chain fatty acids as the major component, but the strain E13T differs by having straight-chain fatty acids (63.7% in total) as the major component. The proportional relationship between straight-chain fatty acids and branched-chain fatty acids plays an essential role in membrane fluidity (Nielsen et al., 2005; Giotis et al., 2007). The alteration of the membrane fatty acid composition has been reported to be an important mechanism of organic solvent tolerance in bacteria (Ramos et al., 2002). Ethanol tolerance has been strongly correlated with adaptive changes in plasma membrane composition and membrane fluidity, with a few studies of thermophilic bacteria suggesting the role for long-chain (C30) fatty acids (Burdette et al., 2002). We hypothesize that the unusual ethanol adaptation may be one reason for the fatty acid compositions of strain E13T.

Table 2.   Cellular fatty acid composition (% w/w) of strain E13T and its closest relatives
Fatty acid1234567
  1. 1, Strain E13T; 2, strain PGDY12; 3, Anoxybacillus flavithermus ssp. flavithermus DSM 2641T; 4, Anoxybacillus eryuanensis KCTC 13720T; 5, Anoxybacillus tengchongensis KCTC 13721T; 6, Anoxybacillus kestanbolensis NCIMB 13971T; 7, Anoxybacillus pushchinoensis DSM 12423T.

Iso-C14:01.30.71.51.00.60.40.5
C14:09.26.41.60.70.90.950.5
Iso-C15:014.512.852.259.565.363.654.3
Anteiso-C15:09.57.52.96.79.36.85.6
C15:014.013.70.91.51.52.10.7
Iso-C16:08.111.23.66.94.38.44.9
C16:033.437.824.16.96.65.28.6
Iso-C17:02.93.39.611.96.812.518.8
C17:07.16.73.65.04.66.1

Phylogenetic analysis and DNA–DNA hybridization studies

On the basis of 16S rRNA gene sequence analysis, the strain E13T (1449 bp) showed high 16S rRNA gene sequence similarity to members of the genus Anoxybacillus. Although there are obvious differences in biochemical characters, the results of molecular identification show that the strain E13T is closely related to the species of A. flavithermus. Based on its 16S rRNA gene sequence, strain E13T is closely related to A. flavithermus DSM 2641T (99.2% sequence similarity, see Supporting Information, Fig. S1). The genomic G+C contents of strain E13T was 42.3 mol%, which was also close to that of A. flavithermus DSM 2641T (41.6 mol%). As only DNA–DNA hybridization could provide definite identification at the species level (Fox et al., 1992), hybridizations between the strain E13T and A. flavithermus DSM 2641T were performed repeatedly. The average value was 64.8%. DNA–DNA similarity of >70% is used to place bacteria into the same species while bacteria with DNA–DNA similarity of <60% should be considered as genetically independent (Wayne et al., 1987; Stackebrandt & Goebel, 1994). The value of 64.8% was the borderline with the recommended threshold values. Therefore, more evidence, such as carbon sources, fatty acid analysis and the property of ethanol adaptation, was required to establish the strain E13T as a new subspecies of A. flavithermus.

Only strain E13T was isolated in the 10% ethanol enrichment. Previously, we had isolated the strain PGDY12 using the same samples by toluene enrichment (Gao et al., 2011). This strain exhibited a similar solvent (benzene and p-xylene) adaptation, but could not adaptively thrive at high concentrations of ethanol. Analysis of the 16S rRNA gene sequence revealed that this strain showed 99.7% similarity to strain E13T. The phenotypic characteristics and the fatty acid profiles of strain PGDY12 are indicated in Tables 1 2, respectively. We propose that strains E13T and PGDY12 represent a new subspecies of A. flavithermus, for which we offer the name Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. According to Rule 40b of the Bacteriological Code, the creation of this subspecies automatically creates the subspecies A. flavithermus ssp. flavithermus ssp. nov.

Description of A. flavithermus ssp. yunnanensis ssp. nov

Anoxybacillus flavithermus ssp. yunnanensis (yun.nan.en′sis. N.L. masc. adj. yunnanensis pertaining to the Yunnan site, southern China, where the type strain was isolated). Cells are Gram-positive, rod (0.4–0.7 μm width and 1.2–7.0 μm length), motile, occurring in single, pairs or sometimes in long chains with terminal ellipsoidal endospores. The colonies of the strain with round edges are 1–2.0 mm in diameter, usually cream and smooth. It is a facultative aerobic microorganism. The temperature growth range is from 30 to 66 °C with an optimal growth at 60 °C. The pH growth range is from 5.5 to 10.0 with an optimum growth at 7.0–7.5. Cells preferably grow in the presence of solvent and tolerate solvents. The NaCl tolerance range is 0–3.5% (w/v) and the optimal NaCl concentration for growth is 0.3% (w/v). It is able to utilize arabinose, cellobiose, galactose, gluconate, glucose, maltose, mannitol, sucrose, trehalose and xylose. Negative reactions for ethanol, fructose, lactose, mannose, rhamnose and ribose as carbon sources were obtained. It is positive for catalase, and negative with respect to gelatin hydrolysis, starch hydrolysis, nitrate reduction, indole production and phenylalanine deaminase. The major cellular fatty acid is C16 : 0, followed by iso-C15 : 0, C15 : 0, anteiso-C15 : 0, C14 : 0, iso-C16 : 0, C17 : 0, iso-C17 : 0 and iso-C14 : 0. The G+C content of the DNA of the type strain is 42.3 mol%. The type strain E13T was isolated from a geothermal spring in Yunnan Province of China. It was deposited at the China Center for Type Culture Collection (CCTCC, AB2010187T) and the KCTC (13759T). The strain PGDY12 (=DSM 23293) is an additional strain of this subspecies.

Description of A. flavithermus ssp. flavithermus ssp. nov

The creation of A. flavithermus ssp. yunnanensis automatically creates the subspecies A. flavithermus ssp. flavithermus. The description is the same as that given for A. flavithermus by Pikuta et al. (2000). The type strain is strain DSM 2641T.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (30800010).

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