E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
Turk J Biol 36 (2012) 303-312 © TÜBİTAK doi:10.3906/biy-1102-16
Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
Ebru TEKİN, Mustafa ATEŞ, Özge KAHRAMAN Department of Biology, Basic and Industrial Microbiology Section, Ege University, Bornova, 35100 İzmir - TURKEY
Received: 24.02.2011
Abstract: İzmir Çamaltı Saltern is the biggest seawater-based saltern in Turkey. To date, it has not been investigated extensively for the existence of poly-3-hydroxybutyrate (PHB)-producing species. In this study, 14 extremely halophilic archaea were isolated, purifi ed, and screened for PHB production. One strain was then selected as the best PHB producer, further cultivated in diff erent PHB media, and compared with a positive control, Haloferax mediterranei ATCC 33500. PHB was extracted from cells and measured with a spectrophotometer, and then the amount of PHB was measured through comparison with standard PHB. Th e detected high PHB yield was 6.53% of the dry cell weight in the PHB medium supplemented with acetate. Partial 16S rRNA gene sequence analysis showed 99% similarity to Haloferax alexandrinus strain TMT; therefore, the strain was named Haloferax sp. MA10. Haloferax sp. MA10 and Haloferax alexandrinus TMT have some diff erences in phenotypic and biochemical properties. With this study, the discovery of PHB-producing extreme halophilic archaeon Haloferax sp. MA10 at İzmir’s Çamaltı Saltern is reported for the fi rst time.
Key words: Extremely halophilic archaeon, poly-3-hydroxybutyrate (PHB), PHB extraction and quantifi cation, Haloferax sp. MA10, 16S rRNA gene
İzmir Çamaltı Tuzlası’ndan izole edilen poli-3-hidroksibütirat üreticisi ekstrem halofi lik arkeon: Haloferax sp. MA10
Özet: İzmir Çamaltı Tuzlası, Türkiye’nin deniz suyu kökenli en büyük tuzlası olma özelliğindedir. Günümüze kadar bu bölgede poli-3-hidroksibütirat (PHB) üretici türün varlığı bakımından çok fazla araştırma yapılmamıştır. Bu kapsamda, on dört ekstrem halofi lik arkea izole edilmiş, safl aştırılmış, PHB üretip üretmedikleri saptanmış ve bir suş en iyi PHB üreticisi olarak seçilerek PHB üreticisi Haloferax mediterranei ATCC 33500 pozitif kontrol ile karşılaştırmak suretiyle değişik PHB besiyerlerinde büyütülmüştür. PHB hücrelerden ekstrakte edilmiş, spektrofotometre ile ölçülmüş ve PHB miktarı standart PHB ile karşılaştırılarak tespit edilmiştir. En yüksek PHB veriminin asetat içeren PHB ortamında hücre kuru ağırlığının % 6,53’ü olarak saptanmıştır. Kısmi 16S rRNA gen dizisi analiz sonucu % 99 oranında Haloferax alexandrinus TMT suşuna benzer olduğunu göstermiştir. Bu nedenle suş Haloferax sp. MA10 olarak adlandırılmış olup Haloferax sp. MA10’ın, bazı fenotipik ve biyokimyasal özellikler bakımından Haloferax alexandrinus TMT suşundan farklılıkları olduğu bulunmuştur. Bu çalışma ile İzmir Çamaltı Tuzlası’ndan PHB-üretici ekstrem halofi lik arkeon Haloferax sp. MA10 ilk defa bulunmuştur.
Anahtar sözcükler: Ekstrem halofi lik arkeon, poli-3-hidroksibütirat (PHB), PHB ekstraksiyonu ve kantifi kasyonu, Haloferax sp. MA10, 16S rRNA geni
303 Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
Introduction developed PHB copolymer P(3HB-co-3HV), as the Since the Industrial Revolution, the increasingly fraction of the 3-hydroxyvalerate unit increases, the widespread use of petroleum-based plastics has impact strength and elongation to break increase; on the other hand, brittleness, melting point, and Young’s created long-term problems due to the depletion of modulus decrease (2,3). Th us, the polymer becomes oil sources, accumulation of plastic waste in nature, tougher and more fl exible. Due to the decrease in and rising oil prices. Th ese outcomes have directed melting point, the injection molding process of the scientists toward alternative plastic sources that are copolymer occurs without thermal degradation (2). environmentally friendly, biologically degradable Th ere are some useful properties of PHB, such as under appropriate conditions, and obtainable moisture resistance, water insolubility, optical purity, from cheap sources such as waste products, and good oxygen impermeability. Th ese properties microorganisms, or transgenic plants. Th e most are diff erent from those of other biodegradable important consequences of using bioplastics are plastics, which are either water-soluble or moisture- that they can be produced from renewable sources sensitive (2,3). Th e biodegradation of PHB or P(HB- and they are environmentally friendly (1). Th ese so- HV) occurs when microorganisms colonize on called biodegradable polymers or bioplastics were the surface of the polymer and secrete enzymes to fi rst developed as lipid-like intracellular storage degrade the polymer. Th e most important properties granules from Bacillus megaterium (2). of PHB and other polyhydroxyalkanoates are that Microbiologically produced, the most common they do not degrade under the normal conditions of form of bioplastics are polyhydroxyalkanoates storage, and they are stable in air (2). (PHAs). Th ese are a versatile class of polymers with Currently the most important types of bioplastics more than 100 diff erent monomer constituents and suggested by markets are cellulosic esters, starch linear-structured polymers (1,2). Th e most commonly derivatives, PHB, polylactic acids (PLA), and found PHA form produced by microorganisms is polycaprolactone (PCL) (6). Th ese polymers are poly-3-hydroxybutyrate (PHB), which is formed used in areas such as pharmaceutics, biomedical from 3-hydroxybutyric acid monomers by the products, packaging, and agriculture (3,6). Th ere is polymerization reaction of 3 enzymes: 3-ketothiolase, growing interest in the use of bioplastics worldwide. acetoacetyl-CoA reductase, and PHA synthase. Th e total worldwide production of bioplastics was Under stress conditions, microorganisms produce 724,500 t/year in 2010. In 2011, about 1,000,000 t polymers using acetyl-CoA as an initial material, were produced, and it is estimated that the level of and then 3-ketothiolase condenses 2 acetyl-CoAs worldwide bioplastic production will be 1,709,700 to acetoacetyl-CoA. Acetoacetyl reductase reduces t/year by 2015. When compared with conventional acetoacetyl-CoA to (R)-3-hydroxybutynl-CoA and plastics derived from petroleum, however, worldwide PHA synthase polymerizes (R)-3-hydroxybutyryl- consumption levels of bioplastics are small (7). CoA to PHB (2). Th e stress conditions that cause PHA can be produced by several microorganisms, microorganisms to produce these polymers are such as various eubacteria and some haloarchaeal the presence of excess carbon and the absence of strains. Th ere are several advantages to working with an essential nutrient such as oxygen, phosphorus, haloarchaeal strains rather than eubacteria. Due to the nitrogen, or sulfur, or the absence of a trace element high salt concentrations they require in their growth such as magnesium, calcium, or ferrous iron (3-5). medium to maintain cell wall stability, haloarchaeal PHB is a stiff , highly crystalline, and relatively strains do not require strict sterile conditions. Th is brittle thermoplastic. While the melting point of makes cultivation more convenient in simple places PHB is 175 °C, the degradation temperature is 185 such as open ponds. Cell walls can also easily lyse in °C, which makes the injection molding process the absence of salt, especially in distilled water; this diffi cult. Although the mechanical properties of property enables the recovery of PHB or other PHA PHB, such as Young’s modulus (3.5 GPa) and tensile forms from extreme halophiles much more easily and strength (MPa), are similar to those of polypropylene, economically than from eubacteria (8). Investigations the elongation to break of PHB (6%) is signifi cantly of the production of PHB and other PHAs are few. lower than that of polypropylene (400%) (2). In the Studies including some haloarchaeal strains in the
304 E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
genera of Haloferax, Haloarcula, Haloquadratum, depth of 10 cm. From each sample, 5 g was transferred Halorubrum, Halobiforma, Halorhabdus, to 50 mL of medium that contained salt solution at Halalkalicoccus, Halobacterium, Natrianema, an approximately 25% fi nal concentration and was Halostagnicola, Natrinema, Natronobacterium, referred to as seawater (SW)-25. Th e composition of
Natronorubrum, Haloterrigena, Halopiger, and the SW-25 medium was (in g/L): NaCl, 202.5; MgCl2,
Halococcus have only been reported from cheap 17.5; MgSO4, 24; CaCl2, 0.9; KCl, 5; NaHCO3, 0.15; carbon sources (8-19). According to detailed analyses, NaBr, 0.065; and yeast extract, 5. Th e pH was adjusted most of the haloarchaeal cells were found to be to 7.2 with 1 M NaOH (22). Th e samples were synthesizing PHB or poly-(3-hydroxybutyrate-co-3- incubated for 7 days at 40 °C in an orbital shaker at hydroxyvalerate) (PHBV) at levels ranging from 0.8% 120 rpm. Aft er the incubation period, isolations were to 22.9% (w/w) of cell dry weight (19). Currently, the carried out using the streak plate method on SW-25 best PHA-producing strain of extremely halophilic agar medium. Plates were incubated at 40 °C for 14 archaea is Haloferax mediterranei, which produces days. Colonies with diff erent colors were picked up PHB-co-PHV up to 60wt% of its dry cell weight with and restreaked several times until pure cultures were starch or glucose as a carbon source under phosphate obtained (23). limitation (8). In continuous cultivation, PHA yields PHB screening increased to a maximum of 87.5wt% of dry cell weight Cells were stained by the Nile blue A staining procedure with diff erent sources such as hydrolyzed whey, (24). Th e type strain Haloferax mediterranei ATCC sodium valerate, and γ-butylolacton (20). Halopiger 33500 was used as a PHB-producing positive control. aswanensis (formerly strain 56) was isolated from Nile blue A staining was performed as follows: broth Egyptian saline soil, and its PHB yield was reported cultures of type strain H. mediterranei ATCC 33500 as 53wt% of dry cell weight (16). Th ese investigations and the isolates were smeared on a glass slide, heat- reveal that cultivation conditions can change the fi xed, and stained with a 1% ethanol solution of Nile production yield. Th erefore, in order to improve blue A for 10 min at 55 °C. Th ey were then washed production yields, there should be further study of with water and 8% aqueous acetic acid for 1 min, haloarchaeal cells and their cultivation conditions. dried with fi lter paper, and covered with a glass Çamaltı Saltern, a salt production site 28 km cover slip. Th e preparation was examined at 600× from the city of İzmir and the largest saltern in magnifi cation using a fl uorescence microscope. Th e Turkey, has water storage ponds, evaporation ponds, procedure was based on measuring at wavelengths and crystallization ponds. Th e salinities of these near 460 nm to detect intracellular PHB granules, ponds are 35%-50%, 50%-150%, and 150%-300%, which produce bright orange fl uorescence at that respectively (21). Our aim was to conduct the fi rst wavelength (24,25). investigation of PHB-producing extremely halophilic Phenotypic and biochemical properties archaea from İzmir’s Çamaltı Saltern, select PHB- SW-25 agar/broth was used as a basal medium, producing isolates via a specifi c PHB staining and incubations were carried out at 40 °C for 14 method, identify isolates both phenotypically and days, unless otherwise indicated. A modifi ed Gram genetically, and determine PHB yields by diff erent reaction was performed according to the method media formulations. We aimed to assess the eff ects of Dussault (26). Motility was checked under a of diff erent media formulations on production yields phase contrast microscope. Growth on nutrient and collect information about the eff ect conditions agar was tested. Antibiotic susceptibility was tested have on improvement of PHB production. by the disk diff usion method using antibiotic disks (Oxoid) of novobiocin (NV, 5 μg), erythromycin (E, Materials and methods 15 μg), streptomycin (S, 10 μg), bacitracin (B, 0.04 U), ampicillin (AM, 10 μg), and tetracycline (TE, 30 Isolation of halophilic archaea μg) (27,28). Th e culture densities were adjusted to
Soil samples were taken from 2 diff erent parts of an OD520 of 1.0. Cultures were scored as sensitive to İzmir’s Çamaltı Saltern by digging in the soil to a the antibiotic if the inhibition zone extended beyond
305 Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
8 mm (28). Optimal conditions were determined 10 min for protein denaturation and centrifuged at by spotting 10 μL of the selected isolate on the SW- 12,000 rpm for 10 min at 4 °C. Th e supernatant was 25 agar using diff erent NaCl concentrations (0%, taken in a new Eppendorf tube, and 5 μL of RNAse 5%, 8%, 10%, 15%, 20%, 25%, and 30%), diff erent (20 μg/mL) was added and incubated at 37 °C for 30 temperatures (20 °C, 27 °C, 37 °C, 40 °C, and 50 °C), min. Th e purity of the genomic DNA was checked and diff erent pH levels (3, 4, 5, 6, 7, 8, 9, and 10) with 0.8% (w/v) agarose gel in a TBE (0.5×) buff er (27,28). Th e minimal yeast requirement for growth for 1 h at 80 V. Th e 16S rRNA gene was amplifi ed was determined on SW-25 broth with 0%, 0.01%, with domain Archaea-specifi c forward primer 21F 0.1%, and 0.5% yeast extract. Single carbon source (5’-TTCCGGTTGATCCYGCCGGA-3’ where Y = C (20%, w/v) analyses were performed with diff erent or T, but in this study, Y = T) and the universal reverse single carbon sources that were syringe-fi ltered primer 1492R (5’-GGTTACCTTGTTACGACTT-3’) (0.22 μm). Th e carbon sources (citrate, glucose, (33). Polymerase chain reaction (PCR) was performed galactose, maltose, arabinose, fructose, xylose, Na- with a GeneAmp PCR System 9700 thermal cycler acetate, and Na-butyrate) were added to SW-25 agar using the FastStart Taq DNA Polymerase dNTPack medium containing 0.01% yeast extract at a 1% fi nal (Roche, version 2006) for 35 cycles for 5 min at 95 °C concentration, as there was no growth in SW-25 agar for the initial denaturation/enzyme activation. Th is medium containing 0.01% yeast extract. In the growth was followed by 45 s of denaturation at 95 °C, 1 min of test for diff erent single nitrogen sources, the yeast annealing at 50 °C, and 1.4 min of polymerization at extract was omitted from the medium. Instead, yeast 72 °C, with a fi nal extension at 72 °C for 7 min. DNA carbon base medium (Difco) was used according sequencing was carried out by REFGEN/METU to manufacturer’s instructions and supplemented at Middle East Technical University, Turkey. Th e with 1% of a diff erent test nitrogen source (soya sequence result was analyzed in the National Center peptone, ammonium chloride, potassium nitrate, for Biotechnology Information (NCBI) database and meat extract) in the basal medium of SW-25 using the Nucleotide Basic Local Alignment Search salts. Indole production was tested with SW-25 broth Tool (BLAST) (34). Th e reference sequences of 10 supplemented with 1% tryptone (w/v), catalase was closely related taxa were taken from the GenBank tested with 3% H2O2 solution, and gelatin hydrolysis, database. Th e selected taxa and their accession nitrate reduction, and gas production were tested in numbers were submitted to Molecular Evolutionary
SW-25 broth supplemented with 0.1% (w/v) KNO3 Genetics Analysis version 4 (MEGA 4) soft ware (35) (27). Tween 80 hydrolysis was performed according to analyze the evolutionary distances of the taxa. to the methods of Martin et al. (29). Casein hydrolysis Multiple alignments were done using ClustalW (36), was performed according to the method of Sánchez- and evolutionary distances of taxa were computed Porro et al. (30). Starch hydrolyses were tested using a p-distance model before constructing a according to the methods of Ventosa et al. 1998 phylogenetic tree, according to soft ware instructions (31). Aft erwards, 4.2% (w/v) DNase test agar with (37). Evolutionary history was inferred using 0.005% methyl green (Difco) was added to the SW-25 the neighbor-joining method (38). All positions medium to observe clear zones around the colonies containing gaps and missing data were eliminated for DNase activity, according to the manufacturer’s from the dataset with the complete deletion option. instructions. Th e bootstrap test (1000 replicates) was used to assess 16S rRNA gene analyses confi dence values with resampling. Th e percentage of replicate trees in which the associated taxa clustered Genomic DNA was isolated according to the together in the bootstrap test (1000 replicates) is Halohandbook methods (32) with a modifi cation shown next to the branches (39). of RNase manipulation. An active culture (OD600 of approximately 1) was centrifuged at 12,000 rpm for Growth curve measurements 10 min at 4 °C, and then 400 μL of cold and ultrapure Th e growth curve was established by measuring the sterile distilled water was added to the pellets and OD520 in SW-25 medium at 40 °C. In all measurements, vortexed gently. Th e tube was subjected to 70 °C for active cultures were adjusted to an OD520 of 1.0; the
306 E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
OD520 was measured daily and 2% (v/v) inocula were PHB (poly-[(R)-3-hydroxybutyric acid], natural used. Growth curves were also established to screen origin; CAT: 36.350-2, Sigma Aldrich). PHB yield the eff ect of glucose concentration (1%, 2%) of the was calculated according to the standard curve as PHB medium, the eff ect of 2% glucose-containing mg PHB/mg dry cell weight. All measurements were PHB medium, and the eff ect of 1% acetate- repeated 3 times, and the results were reported as supplemented 2% glucose-containing PHB medium means ± standard deviations. (8). Statistical analysis Diff erent PHB media A 1-way analysis of variance (ANOVA) was carried Th ree diff erent media were used to measure PHB yield. out with the Tukey-Kramer honestly signifi cant Th e PHB medium contained (w/v) 2% glucose, 0.2% diff erence test based on 3 replicates using JMP NH4Cl, 0.03% KH2PO4, 0.0005% FeCl3, and marine soft ware (version 8.00; SAS Institute Inc., Cary, NC, salts at an approximately 25% fi nal concentration USA) in order to establish statistical diff erences (NaCl, 19.4; MgCl2, 1.6; MgSO4, 2.4; CaCl2, 0.1; between diff erent media for the same microorganism. KCl, 0.5; NaHCO , 0.02; NaBr, 0.05) (8). Th e PHB 3 Signifi cance was defi ned at P < 0.05. medium supplemented with acetate contained (w/v)
1% acetate, 2% glucose, 0.2% NH4Cl, 0.03% KH2PO4,
0.0005% FeCl3, and marine salts at an approximately Results and discussion 25% fi nal concentration (NaCl, 19.4; MgCl , 1.6; 2 In the description of new taxa of extremely MgSO , 2.4; CaCl , 0.1; KCl, 0.5; NaHCO , 0.02; 4 2 3 halophilic archaea, there are some recommended NaBr, 0.05) (8). Th e basic medium contained (w/v) minimum standards. Th ese primarily include cell 22.5% NaCl, 0.2% KCl, 0.5% MgSO .7H 0, 0.1% yeast 4 2 morphology, motility, pigmentation, the minimum extract, and 1% glucose at pH 7.2 ± 0.2 (40). Each salt requirement to prevent lysis, optimum NaCl medium (100 mL) was separately inoculated with the and MgCl concentrations and range of salt active cultures of the isolate and the type strain. Th ey 2 concentrations enabling growth, temperature and were then incubated for 6 days at 40 °C in an orbital pH ranges for growth, anaerobic growth in the shaker at 180 rpm (8). presence of nitrate or arginine, acid production from Quantitative analysis a range of carbohydrates, ability to grow on single PHB quantifi cation was performed according to the carbon sources, catalase and oxidase tests, hydrolysis method of Law and Slepecky, with modifi cations of starch, hydrolysis of casein, hydrolysis of Tween (41). Th is method is based on the conversion of 80, sensitivity to diff erent antibiotics, and sensitivity intracellular PHB with concentrated sulfuric acid to to polar lipids. Th e 16S rRNA nucleotide sequence obtain crotonic acid. Crotonic acid was quantifi ed by information and DNA-DNA hybridization data measuring absorbance at 235 nm. At the beginning should be consistent with the descriptions of new of the stationary phase (day 6), 100 mL of the species (23). cultures were harvested by centrifugation at 10,000 Isolation, phenotypic, and biochemical properties rpm at 4 °C for 40 min. Th e pellet was washed with 10% NaCl (w/v), centrifuged, and washed again with Due to the isolation and purifi cation results, a total of 5 mL of sterile distilled water to remove excess salts 14 microorganisms were selected for PHB production from the medium. Th e pellets were then lyophilized screening via a specifi c Nile blue A staining (9). Lyophilized cell pellets were measured as dry cell method. According to the staining results, the best weights, 10 mL of sulfuric acid (95%-98%) (Merck) PHB-producing strain was selected for further was added to each test tube, and these were incubated identifi cation based on phenotypic and biochemical for 10 min at 100 °C in a water bath. Th e solution was properties, the results of 16S rRNA gene analysis, and cooled, and 10−1-10−4 dilutions were prepared with the eff ects of diff erent media on the production of sulfuric acid. All dilutions were measured against PHB. Th e characteristics of the colony were circular, a sulfuric acid blank at 235 nm. A standard curve convex, entire, translucent, smooth, and orange- was prepared by the same method with commercial red on the SW-25 agar medium. Th e cells of the
307 Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
isolate stained gram-negative. Cell morphology was pleomorphic, especially for short and long rods. Th is K1 K2 K3 10 M strain was motile under a phase-contrast microscope. Growth was negative on nutrient agar medium. Th e strain was susceptible to novobiocin (5 μg) and 5000 bacitracin (0.04 U) but resistant to erythromycin 2000 (15 μg), streptomycin (10 μg), ampicillin (10 μg), and tetracycline (30 μg). Growth was detected in a 850 5%-30% range of NaCl for the defi ned SW medium, and the optimum NaCl concentration was 25%. Th e pH range was detected as pH 5.0-8.0; however, 400 optimum growth was detected at pH 7.0. Growth was positive at 27 °C, 37 °C, 40 °C, and 50 °C and negative at 20 °C. Th e optimum growth temperature was 40 °C. Growth was negative with 0% and 0.01% 100 yeast extract concentrations, but it was positive with 0.1% yeast extract as a carbon and nitrogen source. Th e optimum yeast extract concentration Figure 1. Th e PCR results of Haloferax sp. MA10 as 10, while was 0.5%. Th e results of single carbon source tests K1, K2, and K3 are negative controls. K1 (negative were positive for citrate, glucose, galactose, maltose, control 1): reaction conditions were performed arabinose, fructose, xylose, Na-acetate, and Na- without reverse primer; K2 (negative control 2): butyrate; growth was negative on lactose. Th e results reaction conditions were performed without forward of single nitrogen source tests were positive for soya primer; K3 (negative control 3): reaction conditions were performed without DNA; 10: main reaction, peptone, ammonium chloride, potassium nitrate, consisting of full reaction conditions. M = marker; and meat extract. Indole from tryptone was negative. Fast RulerTM Middle Range DNA Ladder; 100-5000 bp. Nitrate reduction or gas production was negative. Th e catalase test was positive. Gelatin hydrolysis, Tween 80, starch, and casein hydrolysis results were strain under the existing conditions, the strain is negative. DNase activity was positive with clear zones proposed as Haloferax sp. MA10 until further DNA- around the colonies. DNA hybridization data results are obtained. Th e 16S rRNA gene analyses nucleotide sequence of the partial 16S rRNA gene Genomic DNA isolation was easier than the of the strain Haloferax sp. MA10 was submitted conventional procedures. On the other hand, there to GenBank and assigned the accession number was a problem with impurities on the gel aft er HQ908091. Th e constructed phylogenetic tree is running the agarose gel electrophoresis of genomic given in Figure 2. DNA. Th ese impurities were prevented by the PHB production addition of RNase. Th e results of PCR of a partial Due to the results of the specifi c PHB staining 16S rRNA gene on agarose gel are given in Figure method, Haloferax sp. MA10 was selected as the 1. Due to the sequence analysis of PCR results, a best PHB producer from 14 isolates. Th e stationary 1392-bp length of a partial 16S rRNA gene sequence phase of Haloferax sp. MA10 was determined on day was obtained. Th e results of the partial 16S rRNA 6 by measuring the OD of the culture daily with a gene sequence of the isolate showed 99% similarity 520 to the H. alexandrinus type strain according to spectrophotometer (Figure 3). the NCBI/BLAST database. For this reason, the Th e eff ect of the glucose concentration (1%, 2%) existing properties of the strain and the type of the PHB medium on Haloferax sp. MA10 and H. strain H. alexandrinus strain TMT were compared mediterranei ATCC 33500 is shown in Figure 4. Th e (Table 1). Due to the diff erent properties of each eff ect of 1% acetate on the growth of Haloferax sp.
308 E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
Table 1. Comparison of Haloferax sp. MA10 and Haloferax alexandrinus strain TMT.
Haloferax sp. MA10 Haloferax alexandrinus strain TMT* Color Orange-red Red Cell shape Pleomorphic Pleomorphic Motility + - Colony diameter 0.5-1 mm, circular 0.5-1 mm, circular Lysis in distilled water + + NaCl range 5%-30% 10%-32% NaCl optimum 25% 25% pH, optimum pH 5.0-8.0, optimum 7.0 5.5-7.5, optimum 7.2 Temperature optimum 40 °C 37 °C Temperature range 27-50 °C 20-55 °C Nitrate reduction (aerobic) - + Nitrite reduction (aerobic) - + Catalase + + Gas formation from nitrate - - Hydrolysis of starch - - Hydrolysis of casein - - Hydrolysis of gelatin - + Hydrolysis of Tween 80 - + Sensitivity to antibiotics Novobiocin, bacitracin Novobiocin, bacitracin
*Properties of Haloferax alexandrinus strain TMT were taken from the literature (42). +: Positive growth; -: negative growth.
50 Haloferax alexandrinus AB037474 42 Haloferax sp. MA10 HQ908091 55 Haloferax volcanii AY425724 44 Haloferax denitrificans D14128 93 Haloferax gibbonsii D13378 35 Haloferax lucentensis AB081732 84 Haloferax prahovense AB258305 Haloferax mucosum DQ860980 Haloferax sulfurifontis AY458601 Haloferax elongans DQ860977 100 Haloferax larsenii AY838278
0.001 Figure 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the position of strain Haloferax sp. MA10. Bootstrap values are shown as percentages of 1000 replicates. Th ere were a total of 1353 positions in the fi nal dataset. Scale bar (0.001) indicates the number of nucleotide substitutions per site.
MA10 and H. mediterranei ATCC 33500 is shown in sp. MA10 and H. mediterranei ATCC 33500 in Figure 5. diff erent PHB production media, there was a slightly positive eff ect of 1% acetate on the PHB production Th e results of PHB yield are given in Table 2. of Haloferax sp. MA10 (from 5.34% to 6.53% yield). According to the PHB yield results from Haloferax On the other hand, according to the basic medium
309 Poly-3-hydroxybutyrate-producing extreme halophilic archaeon: Haloferax sp. MA10 isolated from Çamaltı Saltern, İzmir
3.5 3 3 2.5 2.5 2 520
520 2 1.5 1.5 OD OD 1 1 0.5 0.5 0 0 1 2 3 4 5 6 7 8 9 10111213141516 123456 days Days 2% glucose,1% acetate, H. mediterranei ATCC 33500 Figure 3. Th e optic density measurement of Haloferax sp. MA10. 2% glucose, H. mediterranei ATCC 33500 2% glucose, 1% acetate, Haloferax sp. MA10 2% glucose, Haloferax sp. MA10 1.6 1.4 Figure 5. Th e eff ect of 1% acetate in 2% glucose-containing PHB medium on the growth of Haloferax sp. MA10 and H. 1.2 mediterranei ATCC 33500. 1 520 0.8
OD signifi cant yield, however, was acquired with the 0.6 basic medium (54.26%). PHB production yield was 0.4 measured with diff erent medium formulations to 0.2 determine whether there were diff erences in yield 0 results. Acetate produced a slightly positive eff ect 123456 Days on PHB production in both microorganisms. As 2% glucose, H. mediterranei ATCC 33500 seen in Table 2, the basic medium was much more 1% glucose, H. mediterranei ATCC 33500 2% glucose, Haloferax sp. MA10 eff ective than acetate-containing or acetate-lacking 1% glucose, Haloferax sp. MA10 PHB medium for H. mediterranei ATCC 33500. Figure 4. Th e eff ect of 1% and 2% glucose concentrations of We used acetate because it is the initial material for PHB medium on the growth of Haloferax sp. MA10 PHB production and has been used before (8). Th e and H. mediterranei ATCC 33500. limiting condition of the basic medium is much greater than that of the PHB media, so the PHB yield production yield, PHB was the lowest (4.48% yield) should have been much greater than in the other 2 in Haloferax sp. MA10. Th e results of PHB yield for media. It is interesting that the eff ect of acetate on the type strain H. mediterranei ATCC 33500 also PHB production yield in Haloferax sp. MA10 was showed a slight positive eff ect of acetate on PHB slightly higher than in the basic medium. Th e reason production (from 38.59% to 40.30%). Th e most why 2 microorganisms belonging to the same genus
Table 2. Th e PHB yield (%) in diff erent medium conditions of Haloferax sp. MA10 and H. mediterranei ATCC 33500.
Microorganism PHB medium (100 mL) Dried cell weight (mg) PHB amount (mg) Yield (%) Haloferax sp. MA10 2% glucose 20.6 ± 0.01[c] 1.10 ± 0.01[c] 5.34 ± 0.01[b] Haloferax sp. MA10 1% acetate, 2% glucose 148.0 ± 0.01[a] 9.67 ± 0.01[a] 6.53 ± 0.01[a] Haloferax sp. MA10 Basic medium 56.8 ± 0.01[b] 2.55 ± 0.01[b] 4.48 ± 0.01[c] H. mediterranei ATCC 33500 2% glucose 29.2 ± 0.01[c] 11.27 ± 0.01[c] 38.59 ± 0.01[c] H. mediterranei ATCC 33500 1% acetate, 2% glucose 184.8 ± 0.01[a] 74.49 ± 0.01[a] 40.30 ± 0.01[b] H. mediterranei ATCC 33500 Basic medium 60.0 ± 0.01[b] 32.56 ± 0.01[b] 54.26 ± 0.01[a]
Letters in square brackets refer to statistical analysis performed within each column between diff erent media for the same microorganism. Diff erent letters refer to signifi cantly diff erent values (ANOVA, P < 0.05).
310 E. TEKİN, M. ATEŞ, Ö. KAHRAMAN
had diff erent PHB production yields may be related strain, it could be enhanced by gene transfer using to the generation of their PHB-producing enzymes recombinant DNA technology for future studies. or other factors. Th ere are several studies on increasing the yield of Based on the comparison of some biochemical PHB in bacterial species by mutagenesis studies (43), properties, PHB production; aerobic nitrate and and these manipulations can also be used to increase nitrite reduction; hydrolysis of gelatin, Tween PHB yield in haloarchaeal cells. 80, casein, and starch; indole production; growth temperature; NaCl range; color; and motility Acknowledgments properties were diff erent for each strain. In this study, we present the fi rst report of Haloferax sp. Th is work was supported by the Scientifi c Research MA10 from İzmir’s Çamaltı Saltern. Th ere should Project Council of Ege University, Project No. FEN- be more detailed analyses, such as DNA-DNA 045, 2006. We thank the Scientifi c Research Project hybridization, to elucidate the strain from the genus Council of Ege University. We also thank Dr Aziz level to species level. We aimed to assess the eff ects Akın Denizci, Assoc Prof Dr Birgül Özcan, and Assoc of diff erent media formulations on the production Prof Dr Kemal Sami Korkmaz. yield and to obtain information about the eff ect of conditions on the improvement of PHB production. Corresponding author: We found that acetate has a positive eff ect on PHB yield; however, in future studies, conditions should Ebru TEKİN be much more controlled. For example, continuous Department of Biology, fermentation may elucidate other factors that Basic and Industrial Microbiology Section, enhance the production yield. In addition, the eff ect of cheap or waste carbon sources on PHB production Bornova, 35100 İzmir - TURKEY should be examined. Although the yield is low in our E-mail: [email protected]
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