Wung Yang Shieh

Alterococcus agarolyticus, gen.nov., sp.nov., a halophilic thermophilic bacterium capable of agar degradation

Five strains of facultatively anaerobic moderately thermophilic bacteria were isolated from two hot springs in the intertidal zone of Lutao, Taiwan. They produced extracellular agarase on agar medium, yielding reducing sugars and organic acids as the end products under either aerobic or anaerobic conditions. The growth temperature range was approximately 38-58 degrees C with an optimal temperature of about 48 degrees C. The five strains tolerated a relatively narrow pH range from 7.0 to 8.5. They were Gram-negative halophiles growing optimally at 2.0-2.5% NaCl (ca. 0.34-0.43 M). They were capable of anaerobic growth by fermenting glucose and producing various organic acids such as butyrate, propionate, formate, lactate, and acetate. Cells grown in liquid medium were motile monotrichous cocci, normally 0.8-0.9 micron in diameter. They possessed saturated anteiso-15-carbon acid (anteiso-C15:0) as the most abundant cellular fatty acid (46.0-51.3 mo1%) and had G + C contents ranging from 65.5 to 67.0 mo1%. They are the first thermophiles found to degrade agar and also the first halophilic thermophilic bacteria known to be capable of both aerobic and anaerobic fermentative growth. These bacteria are considered to represent a new genus that we named Alterococcus, and Alterococcus agarolyticus is the type species.

Vibrio aerogenes sp. nov., a facultatively anaerobic marine bacterium that ferments glucose with gas production.

A mesophilic, facultatively anaerobic, marine bacterium, designated strain FG1T, was isolated from a seagrass bed sediment sample collected from Nanwan Bay, Kenting National Park, Taiwan. Cells grown in broth cultures were motile, Gram-negative rods; motility was normally achieved by two sheathed flagella at one pole of the cell. Strain FG1T required Na+ for growth, and exhibited optimal growth at 30-35 degrees C, pH 6-7 and about 4% NaCl. It grew anaerobically by fermenting glucose and other carbohydrates with production of various organic acids, including acetate, lactate, formate, malate, oxaloacetate, propionate, pyruvate and succinate, and the gases CO2 and H2. The strain did not require either vitamins or other organic growth factors for growth. Its DNA G+C content was 45.9 mol%. It contained C12:0 as the most abundant cellular fatty acid. Characterization data, together with the results of a 16S rDNA-based phylogenetic analysis, indicate that strain FG1T represents a new species of the genus Vibrio. Thus, the name Vibrio aerogenes sp. nov. is proposed for this new bacterium. The type strain is FG1T (= ATCC 700797T = CCRC 17041T).

Simiduia agarivorans gen. nov., sp. nov., a marine, agarolytic bacterium isolated from shallow coastal water from Keelung, Taiwan

A Gram-negative, heterotrophic, agarolytic, marine bacterium, designated strain SA1T, was isolated from a seawater sample collected in the shallow coastal region of Keelung, Taiwan. Cells were straight to slightly curved rods. Nearly all of the cells were non-motile and non-flagellated during the exponential phase of growth in broth cultures; a few cells (<1 %) were motile and were considered to have monotrichous flagella. The isolate required NaCl for growth and grew optimally at 30-35 degrees C and 2-3 % (w/v) NaCl. It grew aerobically and was incapable of anaerobic growth by fermentation of glucose or other carbohydrates. However, anaerobic growth could be achieved by reduction of nitrate to nitrite. Polar lipids comprised phosphatidylethanolamine (71.8 %), diphosphatidylglycerol (12.7 %), phosphatidylglycerol (12.2 %) and phosphatidylserine (3.3 %). Isoprenoid quinones consisted of Q-10 (87.5 %), MK-9 (6.6 %) and MK-7 (5.9 %). Major cellular fatty acids were C16 : 1 omega 7c and/or iso-C15 : 0 2-OH (28.6 %), C17 : 1 omega 8c (22.8 %), C16 : 0 (14.5 %), C18 : 1 omega 7c (11.0 %) and C17 : 0 (6.4 %). The DNA G+C content was 55.6 mol%. Phylogeny based on 16S rRNA gene sequence analysis showed that strain SA1T formed a distinct lineage within the class Gammaproteobacteria. Strain SA1T was related most closely to Teredinibacter turnerae, Cellvibrio spp., Saccharophagus degradans, Pseudomonas spp. and Microbulbifer spp., strains of these species sharing <93 % 16S rRNA gene sequence similarity with strain SA1T. The phylogenetic data and those from physiological, morphological and chemotaxonomic characterizations indicated that strain SA1T represents a novel species and genus, for which the name Simiduia agarivorans gen. nov., sp. nov. is proposed. The type strain is SA1T (=BCRC 17597T=JCM 13881T).

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×102 to 4.6×102/ml for seawater, 4.0×104 to 4.3×105/g wet wt for eelgrass-bed sediment, and 2.1 × 105 to 1.2 × 107/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.

Pseudidiomarina marina sp. nov. and Pseudidiomarina tainanensis sp. nov. and reclassification of Idiomarina homiensis and Idiomarina salinarum as Pseudidiomarina homiensis comb. nov. and Pseudidiomarina salinarum comb. nov., respectively.

Two Gram-negative strains of heterotrophic, aerobic, marine bacteria, designated PIM1T and PIN1T, were isolated from seawater samples collected from the shallow coastal region of An-Ping Harbour, Tainan, Taiwan. Cells grown in broth cultures were straight rods and non-motile. The two isolates required NaCl for growth and grew optimally at 30-35 degrees C and 2-5 % NaCl. They grew aerobically and were not capable of anaerobic growth by fermentation of glucose or other carbohydrates. The cellular fatty acids were predominantly iso-branched, with iso-C(15 : 0) (17.0-21.4 %), iso-C(17 : 0) (18.2-21.0 %) and iso-C(17 : 1)omega9c (15.7-16.6 %) as the most abundant components. The predominant isoprenoid quinone was Q-8 (95.2-97.1 %). Strains PIM1T and PIN1T had DNA G+C contents of 46.6 and 46.9 mol%, respectively. Phylogeny based on 16S rRNA gene sequences and DNA-DNA hybridization, together with data from physiological, morphological and chemotaxonomic characterizations, indicated that the two isolates should be classified as representatives of two novel species of the genus Pseudidiomarina of the family Idiomarinaceae, for which the names Pseudidiomarina marina sp. nov. (type strain PIM1T=BCRC 17749T=JCM 15083T) and Pseudidiomarina tainanensis sp. nov. (type strain PIN1T=BCRC 17750T=JCM 15084T) are proposed. In addition, based on the characterization data obtained in this study, it is proposed that Idiomarina homiensis and Idiomarina salinarum should be reclassified as Pseudidiomarina homiensis comb. nov. and Pseudidiomarina salinarum comb. nov., respectively.

Nitrogen Fixation by Marine Agar-degrading Bacteria

Summary: Several strains of agar-degrading bacteria capable of fixing N2 were isolated from seawater and eelgrass-bed sediment in Aburatsubo Inlet, Kanagawa, Japan, during the summer of 1986. All strains were Gram-negative, facultatively anaerobic, and required NaCl for growth. They were straight or slightly curved rods and were motile in liquid medium by means of a single polar flagellum. These characteristics as well as the G+C contents of their DNA (44.7-46.1 mol%) placed them in the family Vibrionaceae. These strains produced extracellular agarase on agar medium, yielding reducing sugars and acids as the end products. They expressed significant nitrogenase (acetylene reduction) activities after a few hours of incubation under anaerobic conditions. They utilized combined nitrogen sources both aerobically and anaerobically, but fixed N2 only under anaerobic conditions. Neither yeast extract nor vitamins were required for N2 fixation. These strains were demonstrated to fix N2 anaerobically using agar as the sole carbon source.

Denitrification in the rhizosphere of the two seagrasses Thalassia hemprichii (Ehrenb.) Aschers and Halodule uninervis (Forsk.) Aschers

Abstract This work examined the denitrification in the rhizosphere (rhizome–root complex with attached sediment) and rhizoplane (rhizome–root complex) of the two seagrass species Thalassia hemprichii and Halodule uninervis , as well as that in the nonrhizosphere sediment and seawater, by the acetylene blockage technique. The samples were collected from a seagrass bed in Nanwan Bay, Kenting National Park, Taiwan. Most of the rhizoplane and rhizosphere samples (25 out of 31 for T. hemprichii and 26 out of 29 for H. uninervis ) exhibited detectable levels of denitrifying (N 2 O-producing) activity within 12 h of aerobic or anaerobic incubation (0.3 to 2.2 μmol N 2 O·g wet wt −1 ·h −1 ); however, this was undetected in the nonrhizosphere sediment and seawater samples. Insufficiency of nitrate or organic matter would be apt to restrict the magnitude of in situ denitrification in the seagrass rhizosphere including rhizoplane. Most-probable-number values of denitrifying bacteria in the rhizosphere and rhizoplane (29 out of 32 counting values between 10 3 and 10 4 cells·g wet wt −1 ) in nearly all cases were significantly greater than those in the nonrhizosphere sediment (seven out of ten counting values lower than 10 3 cells·g wet wt −1 ) and seawater ( 1 cells·ml −1 ). Eighty-five strains of denitrifying bacteria in total were isolated from the rhizosphere, rhizoplane and nonrhizosphere samples. Only 35 strains among them did not lose their denitrifying ability after subculture, the majority of which were motile, slightly to moderately halophilic, Gram-negative rods.

Aliidiomarina taiwanensis gen. nov., sp. nov., isolated from shallow coastal water.

A Gram-negative, heterotrophic, aerobic, marine bacterium, designated AIT1(T), was isolated from a seawater sample collected in the shallow coastal region of Bitou Harbour, New Taipei City, Taiwan. Cells grown in broth cultures were straight or slightly curved rods that were motile by means of a single polar flagellum. Strain AIT1(T) required NaCl for growth, grew optimally at 30-40°C and with 1.5-5.0% NaCl, and was incapable of anaerobic growth by fermentation of glucose or other carbohydrates. The isoprenoid quinones consisted of Q-8 (95.2%) and Q-9 (4.8%). The major polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The cellular fatty acids were predominantly iso-branched and included iso-C(17:0) (26.5%), summed feature 9 (comprising iso-C(17:1)ω9c and/or 10-methyl C(16:0); 25.9%) and iso-C(15:0) (20.5%). The DNA G+C content was 51.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AIT1(T) formed a distinct lineage within the class Gammaproteobacteria and was most closely related to members of the genus Idiomarina in the family Idiomarinaceae (91.5-93.9% 16S rRNA gene sequence similarity). The phylogenetic data, together with chemotaxonomic, physiological and morphological data, revealed that the isolate should be classified as a representative of a novel species in a new genus in the family Idiomarinaceae, for which the name Aliidiomarina taiwanensis gen. nov., sp. nov. is proposed. The type strain is AIT1(T) (=JCM 16052(T)=BCRC 80035(T)=NCCB 100321(T)).

Prokaryotic assemblages and metagenomes in pelagic zones of the South China Sea

BackgroundProkaryotic microbes, the most abundant organisms in the ocean, are remarkably diverse. Despite numerous studies of marine prokaryotes, the zonation of their communities in pelagic zones has been poorly delineated. By exploiting the persistent stratification of the South China Sea (SCS), we performed a 2-year, large spatial scale (10, 100, 1000, and 3000 m) survey, which included a pilot study in 2006 and comprehensive sampling in 2007, to investigate the biological zonation of bacteria and archaea using 16S rRNA tag and shotgun metagenome sequencing.ResultsAlphaproteobacteria dominated the bacterial community in the surface SCS, where the abundance of Betaproteobacteria was seemingly associated with climatic activity. Gammaproteobacteria thrived in the deep SCS, where a noticeable amount of Cyanobacteria were also detected. Marine Groups II and III Euryarchaeota were predominant in the archaeal communities in the surface and deep SCS, respectively. Bacterial diversity was higher than archaeal diversity at all sampling depths in the SCS, and peaked at mid-depths, agreeing with the diversity pattern found in global water columns. Metagenomic analysis not only showed differential %GC values and genome sizes between the surface and deep SCS, but also demonstrated depth-dependent metabolic potentials, such as cobalamin biosynthesis at 10 m, osmoregulation at 100 m, signal transduction at 1000 m, and plasmid and phage replication at 3000 m. When compared with other oceans, urease at 10 m and both exonuclease and permease at 3000 m were more abundant in the SCS. Finally, enriched genes associated with nutrient assimilation in the sea surface and transposase in the deep-sea metagenomes exemplified the functional zonation in global oceans.ConclusionsProkaryotic communities in the SCS stratified with depth, with maximal bacterial diversity at mid-depth, in accordance with global water columns. The SCS had functional zonation among depths and endemically enriched metabolic potentials at the study site, in contrast to other oceans.

Aliagarivorans marinus gen. nov., sp. nov. and Aliagarivorans taiwanensis sp. nov., facultatively anaerobic marine bacteria capable of agar degradation

Two agarolytic strains of Gram-negative, heterotrophic, facultatively anaerobic, marine bacteria, designated AAM1T and AAT1T, were isolated from seawater samples collected in the shallow coastal region of An-Ping Harbour, Tainan, Taiwan. Cells grown in broth cultures were straight rods that were motile by means of a single polar flagellum. The two isolates required NaCl for growth and grew optimally at about 25-30 degrees C, in 2-4% NaCl and at pH 8. They grew aerobically and could achieve anaerobic growth by fermenting D-glucose or other sugars. The major isoprenoid quinone was Q-8 (79.8-92.0%) and the major cellular fatty acids were summed feature 3 (C16:1omega7c and/or iso-C15:0 2-OH; 26.4-35.6%), C18:1omega7c (27.1-31.4%) and C16:0 (14.8-16.3%) in the two strains. Strains AAM1T and AAT1T had DNA G+C contents of 52.9 and 52.4 mol%, respectively. The two strains had a 16S rRNA gene sequence similarity of 98.6% and shared 84.9-92.4% sequence similarity with the type strains of Agarivorans albus (91.2-92.4%), eight Alteromonas species (84.9-87.1%), two Aestuariibacter species (86.0-87.0%), Bowmanella denitrificans (86.1-86.7%), eight Glaciecola species (85.0-87.9%) and Salinimonas chungwhensis (85.9-86.1%). Despite their high sequence similarity, strains AAM1T and AAT1T had a DNA-DNA relatedness value of only 4.5%. The data obtained from these polyphasic taxonomic studies revealed that the two agarolytic isolates could be classified as representatives of two novel species in a new genus, Aliagarivorans gen. nov., with Aliagarivorans marinus sp. nov. [type strain is AAM1T (=BCRC 17888T=JCM 15522T)] as the type species and Aliagarivorans taiwanensis sp. nov. [type strain is AAT1T (=BCRC 17889T=JCM 15537T)] as a second species.