Microbubble assisted polyhydroxybutyrate production in Escherichia coli.

BACKGROUND: One of the potential limitations of large scale aerobic Escherichia coli fermentation is the need for increased dissolved oxygen for culture growth and bioproduct generation. As culture density increases the poor solubility of oxygen in water becomes one of the limiting factors for cell growth and product formation. A potential solution is to use a microbubble dispersion (MBD) generating device to reduce the diameter and increase the surface area of sparged bubbles in the fermentor. In this study, a recombinant E. coli strain was used to produce polyhydroxybutyrate (PHB) under conventional and MBD aerobic fermentation conditions. RESULTS: In conventional fermentation operating at 350 rpm and 0.8 vvm air flow rate, an OD600 of 6.21 and PHB yield of 23 % (dry cell basis) was achieved. MBD fermentation with similar bioreactor operating parameters produced an OD600 of 8.17 and PHB yield of 43 % PHB, which was nearly double that of the conventional fermentation. CONCLUSIONS: This study demonstrated that using a MBD generator can increase oxygen mass transfer into the aqueous phase, increasing E. coli growth and bioproduct generation.

Streptomonospora tuzyakensis sp. nov., a halophilic actinomycete isolated from saline soil.

A novel actinobacterium, designated strain BN506(T), was isolated from soil collected from Tuz (Salt) Lake, Konya, Turkey, and was characterised to determine its taxonomic position. The isolate was found to have morphological and chemotaxonomic properties associated with members of the genus Streptomonospora. The isolate was found to grow optimally at 37 °C and in the presence of 10 % (w/v) NaCl but not in the absence of NaCl. Phylogenetic analyses based on an almost-complete 16S rRNA gene sequences indicated that isolate is closely related to members of the genus Streptomonospora and forms a distinct phyletic line in the Streptomonospora phylogenetic tree. Strain BN506(T) is closely related to Streptomonospora halophila YIM 91355(T) (98.1 % sequence similarity). Sequence similarities with other type strains of the genus Streptomyces were lower than 98.0 %. The cell wall of the novel strain was found to contain meso-diaminopimelic acid. Whole cell hydrolysates were found to contain galactose, glucose and ribose. The predominant menaquinone was identified as MK-10(H8) (57.0 %). The polar lipids detected were identified as diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. The major fatty acids were found to be anteiso-C17:0, iso-C16:0 and 10 methyl C18:0. Based on 16S rRNA gene sequence analysis, DNA-DNA relatedness, phenotypic characteristics and chemotaxonomic data, strain BN506(T) was identified as a member of a novel species of the genus Streptomonospora, for which the name Streptomonospora tuzyakensis sp. nov. (type strain BN506(T) = DSM 45930(T) = KCTC 29210(T)) is proposed.

Brevibacillus gelatini sp. nov., isolated from a hot spring.

Two Gram-stain-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacteria designated PDF4T and PDF10, were isolated from Camkoy hot spring in the provinces of Aydin, Turkey and were characterized in order to determine their phylogenetic position. 16S rRNA gene sequence analysis revealed that the two strains belonged to the genus Brevibacillus. Strain PDF4T showed highest 16S rRNA gene sequence similarity to strain PDF10 (99.5 %), Brevibacillus brevis DSM 30T (98.9 %), Brevibacillus parabrevis DSM 8376T (98.6 %) and Brevibacillus formosus DSM 9885T (98.5 %); similarities to other species of the genus Brevibacillus were less than 98.5 %. The predominant fatty acids of strain PDF4T were anteiso-C15 : 0 (60.0 %) and iso-C15 : 0 (22.3 %). The polar lipids of strain PDF4T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, an unknown phospholipid, two unknown lipid, an unknown aminophospholipid and two unknown aminolipids. MK-7 was detected as a sole respiratory quinone, and the cell wall of strain PDF4T contained meso-diaminopimelic acid. The DNA G+C content of strain PDF4T was 51.7 mol%. DNA-DNA hybridization showed less than 60 % relatedness between strain PDF4T and type strains of the most closely related species given above. Based on these data, the two strains are considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus gelatini sp. nov. is proposed. The type strain is PDF4T ( = NCCB 100559T = DSM 100115T).

Microvirga makkahensis sp. nov., and Microvirga arabica sp. nov., isolated from sandy arid soil.

The taxonomic positions of two Gram-negative strains, SV1470(T) and SV2184P(T), isolated from arid soil samples, were determined using a polyphasic approach. Analysis of the 16S rRNA gene and the concatenated sequences of three housekeeping gene loci (dnaK, rpoB and gyrB) confirmed that the strains belong to the genus Microvirga. Strain SV1470(T) was found to be closely related to Microvirga vignae BR3299(T) (98.8 %), Microvirga flocculans TFB(T) (98.3 %) and Microvirga lupini Lut6(T) (98.2 %), whilst similarity to other type strains of the genus ranged from 97.8 to 96.3 %; strain SV2184P(T) was found to be closely related to Microvirga aerilata 5420S-16(T) (98.0 %), Microvirga zambiensis WSM3693(T) (97.8 %) and M. flocculans ATCC BAA-817(T) (97.4 %), whilst similarity to other type strains of the genus ranged from 97.2 to 95.9 %. The G + C content of the genomic DNA was determined to be 61.5 mol % for strain SV1470(T) and 62.1 mol % for strain SV2184P(T). Both strains were found to have the same quinone system, with Q-10 as the major ubiquinone. The polar lipid profile of strain SV1470(T) was found to consist of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and one unidentified aminolipid, while that of strain SV2184P(T) consisted of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, one unidentified aminolipid, one unidentified aminophospholipid and two unidentified phospholipids. DNA-DNA relatedness studies showed that the two strains belong to different genomic species. The strains were also distinguished using a combination of phenotypic properties. Based on the genotypic and phenotypic data, the novel species Microvirga makkahensis sp. nov. (type strain SV1470(T) = DSM 25394(T) = KCTC 23863(T) = NRRL-B 24875(T)) and Microvirga arabica sp. nov. (type strain SV2184P(T) = DSM 25393(T) = KCTC 23864(T) = NRRL-B 24874(T)) are proposed.

Thermus anatoliensis sp. nov., a thermophilic bacterium from geothermal waters of Buharkent, Turkey.

A Gram-stain-negative, lack of motility, catalase- and oxidase- positive bacterium (strain MT1(T)) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45-80 °C, pH 5.5-10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1(T) was able to utilize d-mannitol and l-arabinose, not able to utilize d-cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1(T) detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK-8. The DNA G+C content of MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1(T) (=NCCB 100425(T) =LMG 26880(T)).

Algoriphagus trabzonensis sp. nov., isolated from freshwater, and emended description of Algoriphagus alkaliphilus.

A Gram-staining-negative, non-motile, catalase- and oxidase-positive strain, designated MS7(T), was isolated from freshwater of a river near Trabzon, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain grew optimally at 28 °C and pH 7.5 and in the presence of 2.0% NaCl. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Algoriphagus; strain MS7(T) showed highest sequence similarity to the type strains of Algoriphagus alkaliphilus (97.3%), Algoriphagus terrigena (96.8%), Algoriphagus jejuensis (96.2%), Algoriphagus boritolerans (96.1%) and Algoriphagus aquatilis (95.8%). The major fatty acids of strain MS7(T) were iso-C15 : 0 (30.14%) and summed future 9 (10-methyl C16 : 0 and/or iso-C17 : 1ω9c 18.75%). Polar lipid analysis revealed phosphatidylethanolamine, a variety of unidentified lipids, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified aminolipid. The major isoprenoid quinone was MK-7.The DNA G+C content of MS7(T) was 41.6 mol%, a value consistent with that of members of the genus Algoriphagus. The level of DNA-DNA relatedness between strain MS7(T) and A. alkaliphilus LMG 22694(T) was 41%, which is clearly below the 70% threshold accepted for species delineation. Thus, our results support the placement of strain MS7(T) within a separate and previously unrecognized species. On the basis of these data, the strain is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus trabzonensis sp. nov. is proposed. The type strain is MS7(T) ( = NCCB 100372(T) = LMG 26290(T)). An emended description of A. alkaliphilus is also provided.

Flavobacterium anatoliense sp. nov., isolated from fresh water, and emended description of Flavobacterium ceti.

A Gram-staining-negative, catalase- and oxidase-positive, strictly aerobic, rod-shaped bacterial strain isolated from fresh water in Trabzon, Turkey and designated MK3(T) was characterized by phenotypic and molecular methods in order to determine its phylogenetic position. On the basis of 16S rRNA gene sequence similarity, strain MK3(T) was shown to belong to the genus Flavobacterium, being most closely related to Flavobacterium ceti CECT 7184(T) (93.6%). Sequence similarity with other species of the genus Flavobacterium with validly published names was less than 91.6%. Phenotypic and chemotaxonomic data supported the affiliation of strain MK3(T) to the genus Flavobacterium. The only menaquinone was MK-6; the major fatty acids were iso-C15:0 (45.2%), summed feature 9 (C16:0 10-methyl and/or iso-C17:1ω9c; 20.4%) and summed feature 3 (C16:1ω7c and/or C16:1ω6c; 13.3%) and the major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and two unidentified phospholipids. The G+C content of the genomic DNA was 38.6 mol%. The results of physiological and biochemical tests allowed strain MK3(T) to be distinguished phenotypically from Flavobacterium ceti CECT 7184(T). Strain MK3(T), therefore, represents a novel species of the genus Flavobacterium, for which the name Flavobacterium anatoliense sp. nov. is proposed. The type strain is MK3(T) (=LMG 26441(T)=NCCB 100384(T)). An emended description of Flavobacterium ceti is also proposed.

Anoxybacillus kaynarcensis sp. nov., a moderately thermophilic, xylanase producing bacterium.

A novel moderately thermophilic, Gram-positive, endospore-forming, rod-shaped, motile bacteria and alkaline active xylanase producing strain D1021(T) , was isolated from Kaynarca hot spring in the province of Izmir, Turkey and was characterized in order to determine its phylogenetic position. Growth was observed at 35-70 °C (optimum 60 °C), at pH 6.0-10.0 (optimum pH 7.0). The strain D1021(T) grew on a wide range of carbon sources including ribose, xylose, glucose, maltose, sucrose, arabinose, and melibiose. The major isoprenoid quinone was MK-7. 16S rRNA gene sequence analysis showed that strain D1021(T) is related to members of genus Anoxybacillus, with similarities to Anoxybacillus spp. varying from 94.7 to 98.7. The major fatty acids of strain D1021(T) were iso-C15:0 (57.46%) and iso-C17:0 (13.98%). The DNA G + C content was 42.9 mol %. DNA-DNA relatedness values between Anoxybacillus spp. and D1021(T) were lower than 70%. On the basis of phenotypic characteristics, rpoB analysis, phylogenetic and DNA-DNA hybridization data, it was proposed that the strain D1021(T) (= DSM 21706(T) = LMG 25303(T) ) should be placed in the genus Anoxybacillus as the type strain of a novel species, Anoxybacillus kaynarcensis sp. nov. The GenBank accession number for the 16S rRNA sequence is EU926955.

Cloning, purification and characterization of an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71.

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al(3+) and Cu(2+) but strongly inhibited by Hg(2+). The enzyme follows Michaelis-Menten kinetics, with K(m) and V(max) values of 0.425 mg xylan/ml and 500 µmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.

Molecular analysis of the genus Anoxybacillus based on sequence similarity of the genes recN, flaA, and ftsY.

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550-600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3-42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.

Brevibacillus aydinogluensis sp. nov., a moderately thermophilic bacterium isolated from Karakoc hot spring.

Two Gram-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacteria, designated PDF25T and PDF30, were isolated from Karakoc hot spring in the province of Izmir, Turkey, and were characterized in order to determine their phylogenetic positions. 16S rRNA gene sequence analysis revealed that the two strains belonged to the genus Brevibacillus; strain PDF25T showed highest sequence similarity to strain PDF30 (99.4 %) and Brevibacillus thermoruber DSM 7064T (98.5 %). The major fatty acids of strain PDF25T were iso-C15:0 (39.30 %), anteiso-C15:0 (26.10 %) and iso-C16:0 (14.75 %). Polar lipid analysis revealed diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, and a variety of unidentified aminophospholipids, phospholipids and aminolipids. The major isoprenoid quinone was MK-7. The G+C content of the genomic DNA was 56.09 mol%. DNA-DNA hybridization experiments revealed 58 % relatedness between strain PDF25T and B. thermoruber DSM 7064T. Based on these data, the two strains are considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus aydinogluensis sp. nov. is proposed. The type strain is PDF25T (=DSM 24395T=LMG 26289T).

Use of rpoB sequences and rep-PCR for phylogenetic study of Anoxybacillus species.

This study was conducted to investigate the applicability of rpoB, which encodes the ß subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.

Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1.

The gene encoding a carboxylesterase from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21, under the control of isopropyl-ß-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature range (25-90 °C), and it had an optimum pH of 8.0 and an optimum temperature of 60 °C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 °C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K(m), V(max), and K(cat) values of 0.348 ± 0.030 mM, 3725.8 U/mg, and 1500 ± 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity.

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey.

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)(5), the (GTG)(5) and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)(5) and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.

Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari.

The gene, AbfAC26Sari, encoding an alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterized. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45-85 degrees C) and it has an optimum pH of 5.5 and an optimum temperature of 65 degrees C. Kinetic experiment at 65 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax and Km values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu2+ and Hg2+. The recombinant arabinofuranosidase released L-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.

Evaluation of arabinofuranosidase and xylanase activities of Geobacillus spp. isolated from some hot springs in Turkey.

Some hot springs located in the west of Turkey were investigated with respect to the presence of thermophilic microorganisms. Based on phenotyping characteristics and 16S rRNA gene sequence analysis, 16 of the isolates belonged to the genus Geobacillus and grew optimally at about 60 degrees C on nutrient agar. 16S rRNA gene sequence analysis showed that these isolates resembled Geobacillus species by > or = 97%, but SDS-PAGE profiles of these 16 isolates differ from some of the other species of the genus Geobacillus. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. It is proposed that recN sequence comparisons could accurately measure genome similarities for the Geobacillus genus. Based on recN sequence analysis, isolates 11, IT3, and 12 are strains of G stearothermophilus; isolate 14.3 is a strain of G thermodenitrificans; isolates 9.1, IT4.1, and 4.5 are uncertain and it is required to make further analysis. The presence of xylanase and arabinofuranosidase activities, and their optimum temperature and pH were also investigated. These results showed that 7 of the strains have both xylanase and arabinofuranosidase activities, 4 of them has only xylanase, and the remaning 5 strains have neither of these activities. The isolates 9.1, 7.1, and 3.3 have the highest temperature optima (80 degrees C), and 7.2, 9.1, AO4, 9.2, and AO17 have the highest pH optima (pH 8) of xylanase. Isolates 7.2, AO4, AC15, and 12 have optimum arabinofuranosidase activities at 75 degrees C, and only isolate AC 15 has the lowest pH of 5.5.