Production and Properties of Two NovelExopolysaccharides Synthesized by a ThermophilicBacterium Aeribacillus pallidus 418

Nadja Radchenkova & Spasen Vassilev & Ivan Panchev &

Gianluca Anzelmo & Iva Tomova & Barbara Nicolaus &

Margarita Kuncheva & Kaloyan Petrov &

Margarita Kambourova

Received: 18 March 2013 /Accepted: 17 June 2013 /Published online: 30 June 2013# Springer Science+Business Media New York 2013

Abstract Synthesis of innovative exocellular polysaccharides (EPSs) was reported for fewthermophilic microorganisms as one of the mechanisms for surviving at high temperature.Thermophilic aerobic spore-forming bacteria able to produce exopolysaccharides wereisolated from hydrothermal springs in Bulgaria. They were referred to four species, suchas Aeribacillus pallidus, Geobacillus toebii, Brevibacillus thermoruber, and Anoxybacilluskestanbolensis. The highest production was established for the strain 418, whose phyloge-netic and phenotypic properties referred it to the species A. pallidus. Maltose and NH4Clwere observed to be correspondingly the best carbon and nitrogen sources and productionyield was increased more than twofold in the process of culture condition optimization. Afterpurification of the polymer fraction, a presence of two different EPSs, electroneutral EPS 1and negatively charged EPS 2, in a relative weight ratio 3:2.2 was established. They wereheteropolysaccharides consisting of unusual high variety of sugars (six for EPS 1 and sevenfor EPS 2). Six of the sugars were common for both EPSs. The main sugar in EPS 1 wasmannose (69.3 %); smaller quantities of glucose (11.2 %), galactosamine (6.3 %), glucos-amine (5.4 %), galactose (4.7 %), and ribose (2.9 %) were also identified. The main sugar inEPS 2 was also mannose (33.9 %), followed by galactose (17.9 %), glucose (15.5 %),

Appl Biochem Biotechnol (2013) 171:31–43DOI 10.1007/s12010-013-0348-2

N. Radchenkova : S. Vassilev : I. Tomova :M. Kambourova (*)Institute of Microbiology, Bulgarian Academy of Sciences, Acad G. Bonchev Str. Bl. 26, 1113 Sofia,Bulgariae-mail: margikam@microbio.bas.bg

I. Panchev :M. KunchevaUniversity of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria

G. Anzelmo : B. NicolausInstitute of Biomolecular Chemistry ICB-CNR, Via Campi Flegrei, 34, 80078 Pozzuoli (NA), Italy

K. PetrovInstitute of Chemical Engineering, Acad. G. Bonchev Str. Bl. 103, 1113 Sofia, Bulgaria

galactosamine (11.7 %), glucosamine (8.1 %), ribose (5.3 %), and arabinose (4.9 %). Bothpolymers showed high molecular weight and high thermostability.

Keywords Thermophilic bacilli . Aeribacillus pallidus . EPS synthesis . Microbialheteropolysaccharides . Mannan

Introduction

Over the past two to three decades, a new class of microbial products, such as microbialpolysaccharides, has received significant industrial importance [1, 2]. An increasinginterest towards microbial exopolysaccharides (EPSs) is determined by the wide varietyof their properties as a result of their heterogeneous composition. The major differencebetween bacterial and plant polysaccharides is the lack of extensive branching in themicrobial polymers [3], as well as their predominantly heteropolysaccharide nature. Dueto their many interesting physical and chemical properties, that group of carbohydrateshas found application in many industrial products in which they serve as stabilizers,thickeners, gelling agents, coagulants, suspenders, and film formers [4, 5]. Industriallyscaled microbial EPSs like xanthan, dextran, alginate, gellan gum, and curdlan areproduced by mesophilic microorganisms, but some of them are well-known humanpathogens. Isolation of non-pathogenic producers could enlarge the area of applicationin novel biotechnological processes, such as drug delivery, medical diagnosis, newbiodegradable plastics, etc.

Microbes isolated from extreme environments offer a great diversity in chemical andphysical properties of their EPSs compared to anywhere else in the biosphere [4].Traditionally, extremophilic microorganisms suggest nonpathogenic products, appropriat-ed for application in food industry, pharmacy, and cosmetics. Despite of lower productionrate in comparison with mesophilic processes, thermophilic ones suggest additionallyseveral advantages in EPS production like short fermentation time (lasting from severalhours to a few days), better mass transfer, decreased viscosity of synthesized polymer andculture liquid. Additionally, EPSs synthesized by thermophilic bacteria are suggested tokeep their properties at high temperature, which is a desired feature of the polymersolution. Only few EPS-secreting thermophilic bacteria and archaea have been described[6] and some unusual bacterial EPSs of thermophilic origin were characterized.

Between thermophilic producers of biologically active compounds, Bacillus andrelated genera are of special interest [7, 8] due to their possibility for fast growth ina comparatively simple media with cheap carbon and nitrogen sources, and well-developed vectors for heterological gene expression. Despite of the enormous explora-tion of thermophilic bacilli as sources for thermostable enzymes, the knowledge on theirability to produce EPSs is still in its childhood. EPSs synthesized by thermophilicbacilli were established to express interesting cytotoxic, antiviral, and immunoregulatoryproperties [9–11]. Only two thermophilic genera belonging to the group of Bacillussenso lato [12] are reported up to now as EPSs producers: a genus Bacillus with thespecies Bacillus thermoantarcticus [13] and Bacillus licheniformis [14, 15], and a genusGeobacillus with the species Geobacillus thermodenitrificans [14] and Geobacillustepidamans [11].

In this work, we describe the isolation of a strain-producer referred to another genus,Aeribacillus. The conditions for EPS production were optimized and the chemical compo-sition and other characteristics of isolated two different EPSs were determined.

32 Appl Biochem Biotechnol (2013) 171:31–43

Materials and Methods

Sample Collection and Strain Isolation

Forty-one water, soil, and algobacterial mat samples were collected in summer, 2010, from 19hot springs located in three geographically separated areas in Bulgaria: South-West Bulgaria,Sofia region, and Rodopa Mountain. The temperature of the sites varied from 40 to 100 °C andpH in the range from 6.2 to 9.8. The samples (2 ml) were used as inocula for enrichment in20 ml basal medium containing: (NH4)2HPO4, 0.1 %; MgSO4, 0.01 %; yeast extract, 0.01 %;KCl, 0.02 %; and thiamine, 0.00001 %. Sucrose (0.6 %) was added as a carbon source. The рНwas adjusted to 7.2 and cultures were cultivated at 55 °C, 240 rpm for up to 40 h.

For isolation of spore-forming microorganisms solely, all samples were treated at 80 °Cfor 10 min before inoculation. The mixed cultures were purified by streaking samples onagar medium containing peptone (0.2 %) and yeast extract (0.1 %) for at least three times.Strains with mucoid consistency of their colonies were selected for quantitative analysis ofthe synthesized EPSs. The strain with highest EPS production was selected for further work.Duplicate or triplicate cultures were used for each experiment.

Taxonomic Characterization of the Isolated Bacteria

Genomic DNAs from broth cultures of the strains under investigation were prepared usingGenElute Bacterial Genomic DNA kit (Sigma). The almost full 16S rRNA genes wereamplified from the extracted DNAs using primers specific to bacterial 16S rRNA gene, 8Fand 1492R [16]. Their sequences were determined with Applied Biosystems model 373ADNA sequencer by using the ABI PRISM cycle sequencing kit (Macrogen, South Korea).After BLAST search by using the BLAST network service [17], the sequences were alignedwith sequences from the NCBI database to identify the closest phylogenetic relatives.

The purified strains were cultivated for up to 40 h at 240 rpm, 55 °C, pH of the culture medium7.2. Samples were taken every 5 h and, and after centrifugation (4,000×g for 10min), supernatantswere used as solutions for EPS determination. Growth was determined by measuring of turbidityat 660 nm. A correlation curve reflecting the proportionality between turbidity and dry weight wasdone for the chosen strain. One unit ODwas established to correspond on 1.05 mg/ml dry cells ofa strain 418. Methods described by Smibert and Krieg [18] were used for its physiologicalcharacterization.

After optimization, next medium was used for cultivation of the strain in the fermentor (%):maltose, 0.9; NH4Cl, 0.2; Na2HPO4, 0.89; KH2PO4, 0.63; NaCl, 0.5; MgSO4, 0.02, CaCl2,0.01; yeast extract, 0.04; and thiamine 0.0001. One milliliter of trace element solution contain-ing (mg/l): ZnSO4·7H2O, 440; FeSO4·7H2O, 2300; CuSO4·5H2O, 50; CoSO4·5H2O, 50, wasadded to 1 l medium.

Recovery and Purification of EPSs

EPSs were isolated from the culture medium after 20 h of cultivation. The cells wereremoved by centrifugation (4,000 rpm, 20 min) in the late stationary phase. The EPS inthe supernatant was precipitated by an equal volume of cold ethanol added dropwise withstirring in ice bath, held at −18 °C overnight and then centrifuged for 30 min at 13,000 rpm,4 °C. The pellets were washed two times with ethanol and dissolved in hot water, dialyzedagainst distilled water and measured after drying at 105 °C. The samples were tested forcarbohydrate, protein, and nucleic acid content. The EPS fraction collected from strain 418

Appl Biochem Biotechnol (2013) 171:31–43 33

was purified by anion exchange chromatography on DEAE-SepharoseTM CL-6B. Six milli-grams EPS were loaded on a column (1.6×80 cm) and eluted with 360 ml water, followed by200 ml of a linear gradient 0–1 M NaCl at a flow rate of 25 ml/h. Fractions (4 ml each) werecollected and analyzed for carbohydrate, protein, and DNA content. The molecular weightwas determined by gel filtration on a Sepharose CL-6B column using dextrans with differentmolecular weight (200, 500, and 2,000 kDa) as standards.

Chemical Analysis

The total carbohydrate content of the culture supernatant was determined by the method ofDubois et al. [19] with glucose as a standard. Uronic acids determination was performedaccording to the method of Blumenkrantz and Asboe-Hansen [20] with glucuronic acid as astandard. Sugar analysis was carried out after EPS hydrolysis with 2.0 N trifluoroacetic acid at120 °C for 2 h. The sugar components were identified by both, thin layer chromatography(TLC) and GC-MS method. The TLC chromatogram was developed in a solvent systemcontaining butanol/acetic acid/H2O (6:2:2). The molar ratio of monosaccharides was deter-mined by GC-MS method in a two-step derivatization procedure. Firstly, O-methoxyaminehydrochloride (0.5 % w/v in pyridine, 50 μl) was added and the samples were heated in cappedReacti-Vials in a heating block at 70 °C for 60 min. After cooling to room temperature, BSTFA(N,O-bis(trimethylsilyl)trifluoroacetamide, silylation reagent; 50 μl) and pyridine (50 μl) wereadded and the samples were capped and heated again at 70 °C for 60 min, cooled to roomtemperature, and transferred to vials, which were immediately capped. Samples were analyzedusing an Agilent 7890A gas chromatograph fitted with an Agilent 5975C series mass selectivedetector (MSD). For the acquisition of data for multivariate analysis, samples were injected insplit (5:1) mode after 3 min column equilibration onto an Agilent HP-5MS capillary column(0.25 mm film thickness; 30 m×0.25 mm i.d.) subjected to a temperature program of 100 °C for2 min, 15 °/min to 180 °C, hold 1 min, 5 °/min to 300 °C, and hold 10 min (total run time42.33 min). The injector inlet was held at 250 °C and the MSD transfer line at 250 °C; thecarrier gas was helium (1.2 ml/min). Mass spectral data were collected in electron impact mode(70 eV) from 4.0 to 42.33 min between 50.0 and 500.0m/z. The mass spectrometer source wasset at 230 °C and the quadrupole at 150 °C. The MSD tune parameters were all within normalranges. MSD ChemStation software was employed for data analysis. For the determination ofretention time indexes (RI), n-alkanes (C10–C34) mixture was used. The compounds wereidentified as TMSi derivatives comparing their mass spectra andKovats Indexes (RI) with thoseof an online available database (The GolmMetabolome Database) and the National Institute ofStandards and Technology (NIST) mass spectral database library [21].

Differential thermal analyses in combination with thermogravimetric analysis wereperformed by LABSYS TM EVO (SETARAM, France). Ten milligrams of each samplewere heated from 30 to 300 °C at a heating rate of 30 °C/min.

Results

Isolation of EPS Producers

A total of 38 aerobic, spore-forming bacterial strains were selected for their ability to formmucoid colonies on solid medium. Twelve of them synthesized more than 20 μg/ml in the basalmineral medium with sucrose as a carbon source (Table 1). The phylogenetic identification

34 Appl Biochem Biotechnol (2013) 171:31–43

revealed their affiliation to four genera, Brevibacillus, Anoxybacillus, Geobacillus, andAeribacillus with similarity to the closest neighbor higher than 97 % suggesting absence ofnovel species. Most of them (nine) were related to the species Brevibacillus thermoruber.Highest EPS production (53 μg/ml) was observed for strain 418, followed by strains 419(identified as Geobacillus toebii) and 425 (identified as B. thermoruber). The strain 418 waschosen for further work.

Characterization of Strain 418

16S rRNA gene sequence of strain 418 comprising 1,446 bp was determined and depositedin the EMBL under accession number HF558447. Phylogenetic analysis using the BLASTprogram showed that the strain 418 belonged to the phylum Firmicutes and that it wasclosely related with other strains from the species Aeribacillus pallidus (99 % sequencesimilarity). The strain was Gram-positive, spore-forming, rod-shaped cells, 0.8 by 2.5 μm insize, neutrophilic, growing in the area 35–72 °C, strict aerobe. Its physiological andbiochemical properties were compared with those of the type strain H12T (DSM 3670T)[22–24]. Both strains were similar in the temperature area for growth, lack of growth atanaerobic conditions, inability to utilize arabinose, ribose, casein, gelatin, good growth onstarch, and carboxymethyl cellulose. They differed in some enzyme activities, ability toutilize some sugars, and the tolerance to NaCl content in the medium. The strain 418 wasneutrophilic (pH area for growth 6.0–8.5), while DSM 3670T was alkaliphilic. Some of thedifferentiating characteristics are reported in Table 2.

EPS Production

Medium composition and physicochemical parameters for EPS production by A. pallidus418 were optimized in shake flasks. To evaluate the effect of the carbon source on EPSproduction, A. pallidus 418 was cultivated in the basal medium in presence of 12 differentsugars as the only carbon source at a concentration of 0.6 %. Growth was not observed in the

Table 1 EPS production by thermophilic strains isolated from Bulgarian hot springs

Strain no. Phylogeneticaffiliation basedon 16S rDNA

Hot spring Тemperatureof isolation(°C)

MaximumEPS production(μg ml−1)

Time for reachingmaximumproduction (h)

418 Aeribacillus pallidus Rupi 55 53 20

419 Geobacillus toebii Rupi 58 50 20

425 Brevibacillus thermoruber Ravno pole 62 49 30

423 Brevibacillus thermoruber Gradeshnitsa 43 41 25

426 Brevibacillus thermoruber Rupi 55 37.4 25

421 Brevibacillus thermoruber Levunovo 85 34 30

416 Brevibacillus thermoruber Rupi 55 33.3 30

422 Brevibacillus thermoruber Rupi 58 32 25

428 Brevibacillus thermoruber DolnoOsenovo

50 30 20

415 Anoxybacillus kestanbolensis Mizinka 85 25.3 15

427 Brevibacillus thermoruber Ravno pole 50 20 25

417 Brevibacillus thermoruber Trebich 50 20 30

Appl Biochem Biotechnol (2013) 171:31–43 35

medium with five sugars tested (arabinose, galactose, lactose, ribose, and raffinose). The finalbiomass and EPS yield after 40 h growth were highest in a medium with maltose (0.75 mg/mland 66 μg/ml, respectively; Fig. 1). Further, the levels of maltose were varied in the range 0.05–1.2 % and a highest polymer production (80 μg/ml) was observed in a concentration of 0.9 %.

The choice of a suitable nitrogen source also influenced polymer production. Amongseven nitrogen sources tested in the basal medium, the highest production was observedwhen ammonium salts were presented in the medium, while organic sources provided bettergrowth (Fig. 2). Growth was poor in the presence of nitrates and EPS was not observed. Thehighest EPS production by A. pallidus 418 was established in a medium containing 0.2 %NH4Cl. Such a way the ratio between carbon and nitrogen source in the optimal medium was4.5:1 (w/w). Na2HPO4 and KH2PO4 in a concentration of 0.89 and 0.63 %, respectively,were able to keep pH 7.0 during the whole process with A. pallidus 418. Addition of smallquantity of yeast extract (0.04 %) was found to increase EPS production of 10 % in weight.As a result of culture condition optimization EPS production was increased up to 95 μg/ml.Among the tested temperatures (between 50 and 70 °C with 5 °C temperature step) and pH(between 6.0 and 8.5 with 0.5 pH steps) for their effect on EPS synthesis, highest polymerproduction was observed at 55 °C and pH 7.0.

Table 2 Differentiating charac-teristics of A. pallidus 418 and A.pallidus DSM 3670T (data fromBanat et al. [4], Logan et al. [19],and Miñana-Galbis et al. [25])

Characteristic A. pallidus 418 A. pallidus DSM 3670T

pH optimum 6.9 8.0–8.5

Sugars used

Maltose + Weak

Sucrose + −Trehalose + Weak

Xylose + −Growth at NaCl 2.5 % 10 %

Catalase − +

Urease activity + −

0

20

40

60

80

mal

tose

gluc

ose

sucr

ose

xylo

serh

amno

se

treha

lose

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(µg

/ml)

0

0,1

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s (m

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l)

Fig. 1 Effect of carbon source on biomass (white bars) and EPS (gray bars) production by A. pallidus 418 ina basal medium supplemented with different sugars. 1 maltose, 2 glucose, 3 sucrose, 4 xylose, 5 rhamnose, 6trehalose, 7 fructose

36 Appl Biochem Biotechnol (2013) 171:31–43

Time course of the growth and EPS production was followed in 1 l fermenter at 600 rpmand aeration 1.2 vvm over a period of 30 h (Fig. 3) in the optimized medium. During thebatch fermentation of the strain 418, EPS production began in the early exponential phaseand continued during the early stationary phase, reaching a value of 130 μg/ml at the 13thhour.

EPS Purification and Characterization

EPS used for purification was recovered after 15 h cultivation in a fermentor. The tests performedwith dry polymer fraction revealed a presence of carbohydrates (80.5 % w/w), protein (19 % w/w),and nucleic acids (0.5 % w/w). The established protein content is probably a result of the observedsynthesis of extracellular enzymes like proteinase, amylase, and cellulase. The measured cellulaseactivity in the supernatant after 15 h of cultivation in a fermentor was 75 U/l. Six milligrams of thisfraction were loaded on a Sepharose DEAECL-6B columnwith a yield of 58% (3.48 mg). Elutionprofile of EPS purification (Fig. 4) revealed a presence of two polysaccharides in the polymerfraction; one of them (EPS 1) was eluted with water and another one was negatively charged and

0

20

40

60

80

NH4Cl

NH4NO3

KNO3

NaNO3

urea

yeas

t extr

act

pepto

ne

EPS

(µg

/ml)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

Bio

mas

s (m

g/m

l)

Fig. 2 Effect of nitrogen source on biomass (white bars) and EPS (gray bars) by A. pallidus 418 in a basalmedium with 0.6 % maltose supplemented with different nitrogen source. 1 NH4Cl, 2 NH4NO3, 3 KNO3, 4NaNO3, 5 urea, 6 yeast extract, 7 peptone

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20 25 30Time (h)

Gro

wth

(m

g/m

l)

0

20

40

60

80

100

120

140

160

EPS

(µg

/ml)

Fig. 3 Time course of growth (diamonds) and EPS production (triangles) by A. pallidus 418 in the optimizedmedium in 1 l fermenter, air flow 1.2 vvm, 600 rpm

Appl Biochem Biotechnol (2013) 171:31–43 37

eluted at 0.4 M NaCl (EPS 2). The ratio between two EPS was 3:2.2 correspondingly. TLCanalysis of EPSs revealed that the polymers synthesized by A. pallidus 418 wereheteropolysaccharides displaying mannose as a main sugar. Furthermore, GC-MS analysis re-vealed that EPS 1 consists of mannose/glucose/galactosamine/glucosamine/galactose/ribose(69.3/11.2/6.3/5.4/4.7/2.9; Fig. 5a). EPS 2 was constituted by similar sugars, however in differentproportions: manose/galactose/glucose/galactosamine/glucosamine/ribose/arabinose(33.9/17.9/15.5/11.7/8.1/5.3/4.9; Fig. 5b). Mannose in both polymers was presentedby two different isomers. The presence of proteins and uronic acids in both EPS wasestablished; the protein content was 3.0 % w/w and 7.0 % w/w for EPS 1 and EPS 2,respectively; uronic acids content was 1.9 % w/w and 6.5 % w/w for EPS 1 and EPS 2,respectively.

The polysaccharides from A. pallidus 418 displayed high molecular weight with values of700 kDa for EPS1 and over 1,000 kDa for EPS 2, estimated from a calibration curve of standarddextrans obtained by gel filtration on Sepharose CL-6B.

The results received from the thermogravimetric analysis showed a weight loss due to waterpresence, centered at 50–80 °C for EPS 1 and 50–60 °C for EPS 2. From this temperature to160 °C EPSs were stable and a transition maximum of the decomposition process was observedat 176 °C for EPS 1 and 226 °C for EPS 2.

Discussion

EPS producers belonging to four thermophilic bacterial species were isolated in the current study.The highest EPS production was observed for the strain 418, phylogenetically affiliated to thespecies A. pallidus.Up to now only two thermophilic genera from the bacilli group are known asEPS producer, current investigation is the first announcement for EPS producer belonging to thegenus Anoxybacillus. The species A. pallidus was originally described as Bacillus pallidus [25]and further reclassified as Geobacillus pallidus [22] and A. pallidus [24]. Still the biosyntheticcapacity of this species is not well investigated. Recently, A. pallidus YM-1 was reported toproduce a novel bioemulsifier [26] consisting of lipids (47.6 %), carbohydrates (41.1 %), and

0

10

20

30

40

50

60

70

80

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100

0 12 24 36 48 60 72 84 96 108 120Fractions

EPS

(µg

/ml)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

NaC

l (M

)

Fig. 4 Purification of EPS on a Sepharose DEAE CL-6B column. Elution was performed with water from0 to 36 fraction and 200 ml 0–1 M gradient NaCl from 37 to 90 fraction. Eluted polymers: EPS (solid line),protein (dashed line), and nucleic acids (dotted line)

38 Appl Biochem Biotechnol (2013) 171:31–43

proteins (11.3 %). Carbohydrate fraction consisted of glucose (36.6 %), altrose (30.9 %),mannose (24.4 %), and galactose (8.1 %).

Optimization of the medium composition revealed some specific requirements of the strain.Although glucose was reported as a preferred carbon substrate for EPS production [27], thepolymer production was about twofold lower in comparison with maltose in the case of A.pallidus 418. Highest polymer production was established in a presence of ammonium salts,

Fig. 5 GC chromatogram of a EPS 1 (1, ribose, 2 and 2′ two mannose isomers, 3 glucose, 4 glucosamine, 5galactosamine, 6 galactose, 7 trehalose) and b EPS 2 (1 arabinose, 2 ribose, 3 and 3′ two mannose isomers, 4glucose, 5 glucosamine, 6 galactosamine, 7 galactose)

Appl Biochem Biotechnol (2013) 171:31–43 39

while organic nitrogen provided good growth but low EPS production. Growth was poor in thepresence of nitrates and EPS was not observed. Contrary, a gellan production fromPropionibacterium acidi-propionici was higher in the presence of nitrates in comparison toammonia [28]. Utilization of inorganic nitrogen source for EPS production by A. pallidus 418is an important advantage for further industrial design of cost-effective medium. The ratiobetween carbon and nitrogen source was 4.5:1 (w/w). The increased production in abundanceof carbon source and minimal nitrogen was reported by several authors [3, 29, 30]. The additionof extra nitrogen favors the biomass production but diminishes EPS production.

Investigation of the influence of different buffer systems on EPS production revealed10 % enhancement of EPS in presence of phosphate buffer in the medium. This result is inan agreement with the lower xanthan production from Xanthomonas campestris [31] in thepresence of Tris with regard to phosphate buffer. They ascribed this effect to phosphatedeficiency of the medium and buffering properties of the phosphate system, reducing the pHfluctuation of the culture. A positive effect on EPS production by A. pallidus 418 wasobserved in a presence of a small quantity of yeast extract. Similar influence of complexcompounds in low concentration was established by other authors [32]. There are only fewreports regarding the study of the effect of trace metals on EPS production, and a positiveeffect of Mg and Mn was observed [30].

Time course of growth and EPS synthesis revealed that polymer production is growthassociated. Similar observation for growth-associated EPS synthesis was reported for glu-can, xanthan, and scleroglucan production [11, 31, 33] and differed from the reports for otherproducers synthesizing EPS during the whole stationary phase [34, 35]. Typically forthermophilic process, maximum EPS production by A. pallidus 418 was determined forsignificantly shorter period than for mesophilic processes.

The established EPS production by A. pallidus 418 is comparable with those reported forother thermophilic producers (55–165 μg ml−1) [11, 13, 14].

A presence of uronic acids was established for both EPSs. The presence of uronic acids in themolecule of EPS is desirable for cosmetics as they are good hydrating agents [3]. The increasedcontent of uronic acids in EPS 2 is in coincidence with its anionic nature. The presence of uronicacids or ketal-linked pyruvate determines polyanionic character of the majority of microbial EPS[6]. Additionally, a presence of glucosamine and galactosamine was established for both EPSs.Small quantities of proteins, uronic acids, and hexosamines attached to the sugar residues andimparting unique characteristics of EPS were also reported by other authors [36].

In contrast to plant polysaccharides, accumulated knowledge on the structural properties ofbacterial exopolysaccharides is scarce and the information concerning thermophilic EPS is ex-tremely limited. According to Sutherland [37] and Bhaskar and Bhosle [38], majority of microbialEPS have generally heteropolymeric composition with two to four to five sugar components. GC-MS analysis revealed a heterogeneous sugar content for EPS 1 and EPS 2. Each of them wascomposed of unusual high number of monosaccharides—six for EPS 1 and seven for EPS 2—sixof thembeing common, however presented in different proportions.Mannose represented 69.3% inEPS 1 and 33.9 % in EPS 2. Glucose and mannose formed the molecule of EPS from B.thermoantarcticus [13]. EPS synthesized byG. tepidamans is glucan, in which glucose represented88% of its content; galactose, fucose, and fructose represented together about 12 % [11]. Similarly,more than 90 % was glucose in the polymer produced from Thermotoga maritima, howeveradditional sugars were ribose and mannose [39]. The polymer from B. licheniformis is a mannan[40]. EPSs isolated from two Geobacillus sp. strains contained as main sugars glucose, galactose,and mannose in different proportions and the third strain from the same genus contained glucos-amine and arabinose together with galactose and mannose [14]. According to Sutherland [41],microbial EPSs are rich in hexoses like glucose and galactose whereas relatively higher content of

40 Appl Biochem Biotechnol (2013) 171:31–43

rhamnose, mannose, and xylose are typical for phytoplankton [42]. However, concerning thermo-philic EPSs, mannose seems to be often a component of the molecule together with glucose andgalactose. Corsaro et al. [43] accept that the type of the produced EPS is specific for themicroorganism and bacteria derived from the same species produce EPS consisting of the sametype of monosaccharides. Isolation of new EPS producer could suggest identification of new EPS.Indeed, an isolation of a species A. pallidus as new EPS producer resulted in isolation of two EPSswhose high number monosaccharide components have not been reported previously.

Another feature of thermophilic EPSs seems to be their high molecular weight. Themolecular weight was determined to be about 700 kDa for EPS 1 and higher than 1,000 kDafor EPS 2. According to Bhaskar and Bholse [38], the molecular weight of the microbialEPSs varied in the range 10 to 30 kDa. However, a molecular weight of 380, 400, 600, and1,000 kDa was reported for EPSs from thermophilic bacilli [14]. EPS from another thermo-phile, B. thermoantarcticus had a molecular weight approximately 300 kDa [13]. Themolecular weight of the polymer from G. tepidamans V264 was higher than 1,000 kDa [11].

Like other EPSs from thermophilic bacteria, the isolated EPSs from A. pallidus 418 werethermostable, especially EPS 2 which decomposition process started at 226 °C. The highestthermostability was reported for the polymers from G. tepidamans V264 (280 °C) [11], G.thermodenitrificans strain B3-72 (240 °C) [9] and B. licheniformis (240 °C) [44].

As a conclusion, a new EPS producer, A. pallidus 418, synthesizes novel EPSs withinteresting properties and high thermostability revealing its perspective for industrial explo-ration in new processes performed in more severe conditions.

Acknowledgments The authors are grateful to the National Fund for Scientific Research, Bulgaria forfinancial support of this work (Contract DTK 02/46).

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