Taxonomic studies of extremely barophilic bacteria isolated from the Mariana Trench and description of Moritella yayanosii sp. nov., a new barophilic bacterial isolate

We have isolated two strains of extremely barophilic bacteria from sediment collected from the world's deepest ocean floor in the Mariana Trench, Challenger Deep, at a depth of 10898m [Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K (1998) Appl Environ Microbiol 64:1510-1513]. One strain, DB21MT-2, was identified as a strain of Shewanella benthica, and the other strain, DB21MT-5, is closely affiliated with members of the genus Moritella on the basis of 16S rDNA sequence analysis. The hybridization values for DNA-DNA relatedness between DB21MT-5 and the Moritella reference strains were significantly lower than that accepted as the phylogenetic definition of a species. Based on this and other taxonomic differences, strain DB21MT-5 appears to represent a novel obligately barophilic deep-sea Moritella species. The name Moritella yaynanosii (JCM 10263) is proposed. This is the first proposed species of obligately barophilic bacteria of the genus Moritella.     

Extremely Barophilic Bacteria Isolated from the Mariana Trench, Challenger Deep, at a Depth of 11,000 Meters

Two strains of obligately barophilic bacteria were isolated from a sample of the world’s deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella. The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment.     

Variability of pressure adaptation in deep sea bacteria

Isolations of pressure-adapted deep sea bacteria from depths of 1,400 to 5,100 m resulted in a variety of psychrophilic barotolerant and barophilic strains. Growth rates determined at different pressures indicated a gradual transition between the two types of pressure-adapted isolates. The presence of barotolerant bacteria in deep water, sustained by sinking particulate matter, causes the nonbarophilic response of natural populations, i.e., increased growth after decompression. With increasing pressure-adaptation in barophilic isolates the maximum growth rates at optimum pressures decrease. Thus, the observed general slow-down of microbial activity in the deep sea takes effect regardless of the common occurrence of psychrophilic and barophilic bacteria. The highest degree of barophilism was observed in isolates from nutrient-rich habitats such as intestinal tracts of deep sea animals or decaying carcasses. Detailed studies with an isolate, growing barophilically on a complex as well as a single-carbon-source medium, showed that (1) culturing at pressures lower than optimal for growth resulted in the formation of cell filaments, (2) growth was unaffected by repeated compression/decompression cycles and (3) no perceptible differences in the distribution of radiolabeled carbon from an amino acid mixture occurred in cells grown at, below and above the pressure optimal for growth.Dedicated to Professor Dr. Hans G. Schlegel on the occasion of his 60th birthday in recognition of his broad microbiological interests and in special appreciation of his lasting support for the Marine Microbiology Course at the Stazione Zoologica (Naples, Italy) now for almost 25 years Non-standard abbreviations. The traditional use of atm as a unit of pressure (=10 m of water column, =1.013 bar, =101.3 kN/m²) is retained here in view of the important relation between water depth and hydrostatic pressure in the present study. Due to the compression of seawater and the geographic variability of gravity, there is a progressive deviation of the actual pressure with depth amounting to +4.9 atm at 5,000 m and a latitude of 30°. EPC, cell counts obtained by epifluorescence microscopy. PY, peptone yeast extract medium     

New Method for Isolating Barophiles from Intestinal Contents of Deep-Sea Fishes Retrieved from the Abyssal Zone

We devised a new method (the dorayaki method) using marine agar under in situ pressures to isolate barophilic bacteria from the intestinal contents of three deep-sea fishes (two Coryphaenoides yaquinae samples and one Ilyophis sp. sample) retrieved from depths of 4,700 to 6,100 m in the Northwest Pacific Ocean. All 10 strains isolated from one sample (C. yaquinae) were obligately barophilic. One of the 10 strains did not grow at atmospheric pressure and 103.4 MPa but did grow well between 20.7 and 82.7 MPa, with optimal growth at 41.4 MPa. This method is useful for isolating psychrophilic and barophilic deep-sea bacteria.     

Photobacterium profundum sp. nov., a new, moderately barophilic bacterial species isolated from a deep-sea sediment

A novel, moderately barophilic bacterium was isolated from a sediment sample obtained from the Ryukyu Trench, at a depth of 5110 m. The isolate, designated strain DSJ4, is a Gram-negative rod capable of growth between 4°C and 18°C under atmospheric pressure, with optimum growth displayed at 10°C, and capable of growth at pressures between 0.1 MPa and 70 MPa at 10°C, with optimum growth displayed at 10 MPa. Strain DSJ4 is a moderately barophilic bacterium, and shows no significant change in growth at pressures up to 50 MPa. Phylogenetic analysis of the 16S rRNA sequence of strain DSJ4 places this strain within the Photobacterium subgroup of the family Vibrionaceae, closely related to the strain SS9 that was independently isolated from the Sulu Trough. The temperature and pressure ranges for growth, cellular fatty acid composition, and assorted physiological and biochemical characteristics indicate that these strains differ from other Photobacterium species. Furthermore, both SS9 and DSJ4 displayed a low level of DNA similarity to other Photobacterium type strains. Based on these differences, these strains are proposed to represent a new deep-sea-type species. The name Photobacterium profundum (JCM10084) is proposed. Received June 13, 1997 / Accepted: August 9, 1997     

Molecular analyses of the sediment of the 11000-m deep Mariana Trench

We have obtained sediment samples from the world's deepest sea-bottom, the Mariana Trench challenger point at a depth of 10 898 m, using the new unmanned submersible Kaiko. DNA was extracted from the sediment, and DNA fragments encoding several prokaryotic ribosomal RNA small-subunit sequences and pressure-regulated gene clusters, typically identifed in deep-sea adapted bacteria, were amplifed by the polymerase chain reaction. From the sequencing results, at least two kinds of bacterial 16S rRNAs closely related to those of the genus Pseudomonas and deep-sea adapted marine bacteria, and archaeal 16S rRNAs related to that of a planktonic marine archaeon were identifed. The sequences of the amplifed pressure-regulated clusters were more similar to those of deep-sea barophilic bacteria than those of barotolerant bacteria. These results suggest that deep-sea adapted barophilic bacteria, planktonic marine archaea, and some of the world's most widespread bacteria (the genus Pseudomonas) coexist on the world's deepest sea-bottom. Received: October 10, 1996 / Accepted: March 3, 1997     

The ribonucleotide sequence of 5s rRNA from two strains of deep-sea barophilic bacteria

Deep-sea bacteria were isolated from the digestive tract of animals inhabiting depths of 5900 m in the Puerto Rico Trench and 4300 m near the Walvis Ridge. Growth of two bacterial strains was measured in marine broth and in solid media under a range of pressures and temperatures. Both strains were barophilic at 2 degrees C (+/- 1 degrees C) with an optimal growth rate of 0.22 h-1 at a pressure 30% lower than that encountered in situ. At 1 atm they grew at temperatures ranging from 1.2 to 18.2 degrees C (+/- 0.3 degrees C), while in situ pressures increased the upper temperature limit to 23.3 degrees C. Both strains were identified as members of the genus Vibrio, based on standard taxonomic tests and mol% G + C values (47.0 and 47.1). Ribonucleotide sequences determined for 5S ribosomal RNA from each strain confirmed relationship to the Vibrio-Photobacterium group, as represented by V. harveyi and P. phosphoreum, but the barophiles were clearly distinct from these species. Secondary structure conformed to the established model for eubacterial 5S rRNA.     

Taxonomic studies of deep-sea barophilic Shewanella strains and description of Shewanella violacea sp. nov.

Several barophilic Shewanella species have been isolated from deep-sea sediments at depths of 2,485– 6,499 m. From the results of taxonomic studies, all of these isolates have been identified as strains of Shewanella benthica except for strain DSS12. Strain DSS12 is a member of a novel, moderately barophilic Shewanella species isolated from the Ryukyu Trench at a depth of 5,110 m. On Marine Agar 2216 plates, this organism produced a violet pigment, whereas the colonies of other isolates (S. benthica) were rose-colored. Phylogenetic analysis based on 16 S ribosomal RNA gene sequences showed that strain DSS12 represents a separate lineage within the genus Shewanella that is closely related to S. benthica and particularly to the members of the Shewanella barophiles branch. The temperature range for growth and some of the biochemical characteristics indicate that strain DSS12 differs from other Shewanella species. Furthermore, strain DSS12 displayed a low level of DNA similarity to the Shewanella type strains. Based on these differences, it is proposed that strain DSS12 represents a new deep-sea Shewanella species. The name Shewanella violacea (JCM 10179) is proposed. Received: 15 May 1998 / Accepted: 15 July 1998     

Effects of hydrostatic pressure and temperature on growth and lipid composition of the inner membrane of barotolerant Pseudomonas sp. BT1 isolated from the deep-sea

A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.     

Stimulation du potentiel de dégradation dans les eaux marines profondes par les bactéries barotolérantes colonisant les fèces du plancton migrateur

At a depth of 1 150 m in the northwest Mediterranean, amino acid uptake and bacterial biomass production rates increased, in a 12-day time interval, 5- and 30-fold, respectively. Simultaneously, bacterial response to pressure changes evolved from barophilic to barotolerant. During the same period, scanning electron microscope observations and in situ observations using Underwater Video Profiler showed a 2-fold increase in particle concentrations, mainly due to faecal pellet production by zooplankton. This input, nutrient rich and largely colonized with bacteria unaffected by pressure variations since they originated from organisms that regularly migrate up and down through the water column, could produce such microbial activity peaking in the deep water masses.     

Species Composition and Barotolerance of Gut Microflora of Deep-Sea Benthic Macrofauna Collected at Various Depths in the Atlantic Ocean

The bacterial flora of marine animals collected at depths of 570 to 2,446 m was examined for population size and generic composition, and the barotolerant characteristics of selected bacterial isolates were determined. Total numbers of culturable, aerobic, heterotrophic bacteria were found to be low in animals collected at the greatest ocean depths sampled in this study. Vibrio spp. were predominant in 10 of 15 samples examined, and Photobacterium spp. and yeasts were the major components of the remainder. Pseudomonas, Achromobacter, and Flavobacterium spp. comprised minor components of the gut flora of deep-sea fish. Forty-six pure cultures isolated from samples of seven animals were tested for growth or viability after incubation for 1 week under pressures ranging from 100 to 750 atm. Strains of bacteria isolated from samples of fish intestine were more barotolerant than those from the stomach (P<0.01). When incubated at a pressure of 600 atm, viability of bacterial cultures originally isolated from fish caught at a depth of 570 m was significantly decreased in comparison with viability of cultures from animals caught at depths of 1,393 and 2,446 m (P<0.01). From results of this study, it is concluded that the gut microflora of animals that dwell in the deeper regions of the ocean are adapted to an increased hydrostatic pressure environment, that is, the gut microflora is less inhibited by elevated hydrostatic pressure with increasing depth from which the host animal was collected.     

Moritella japonica sp. nov., a novel barophilic bacterium isolated from a Japan Trench sediment

Strain DSK1 is a novel moderately barophilic bacterium isolated from the Japan Trench at a depth of 6,356 m. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that strain DSK1 represents a separate lineage with the Shewanella barophiles branch and is closely related to Moritella marina. Comparisons of the temperature and pressure range for growth and some biochemical characteristics indicate that strain DSK1 differs from M. marina and Shewanella barophilic species. Furthermore, strain DSK1 displays a low level of DNA similarity to the Moritella and Shewanella type strains; however this isolate characteristically produces DHA (22:6) as a membrane fatty acids, and the fatty acid profile of this strain is similar to that of M. marina. Because of these differences, strain DSK1 appears to represent a novel deep-sea Moritella species. The name Moritella japonica is proposed. The type strain is JCM 10249.     

A study of deep-sea natural microbial populations and barophilic pure cultures using a high-pressure chemostat

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.     

A Study of Deep-Sea Natural Microbial Populations and Barophilic Pure Cultures Using a High-Pressure Chemostat

Continuous cultures in which a high-pressure chemostat was used were employed to study the growth responses of (i) deep-sea microbial populations with the naturally occurring carbon available in seawater and with limiting concentrations of supplemental organic substrates and (ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea isolates in the presence of limiting carbon concentrations at various pressures, dilution rates, and temperatures. We found that the growth rates of natural populations could not be measured or were extremely low (e.g., a doubling time of 629 h), as determined from the difference between the dilution rate and the washout rate. A low concentration of supplemental carbon (0.33 mg/liter) resulted in positive growth responses in the natural population, which resulted in an increase in the number of cells and eventually a steady population of cells. We found that the growth responses to imposed growth pressure by barophilic and barotolerant pure-culture isolates that were previously isolated and characterized under high-nutrient-concentration conditions were maintained under the low-nutrient-concentration limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.     

Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus spp.

Summary Four strains (W1, W2, W3, and W4) of alkalophilic thermophilic bacteria which produced xylanase were isolated from soils. They were aerobic, spore-forming, Gram-positive, and rod-shaped bacteria and hence identified as the genus Bacillus. The optimal temperatures for growth of the four strains were between 45° C and 50° C and pH optima were between 9.0 and 10.0. No growth occurred below pH 7.0 or above 55° C. The four strains produced xylanases in medium containing xylan or xylose under these conditions. The optimal pH and temperature for activities of the four xylanases ranged from 6.0 to 7.0 and from 65° C to 70° C, respectively. The four xylanases were stable in the wide pH range from 4.5 to 10.5 at 45° C for 1 h. All xylanases split xylan to yield xylose and xylobiose.     

Leptospirillum-Like Bacteria and Evaluation of Their Role in Pyrite Oxidation

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40°C, respectively. The pH optimum was 2.0–2.3. Bacterial growth and ferrous iron oxidation were inhibited by yeast extract. The pyrite-leaching activity of the Leptospirillum-like bacteria under mesophilic conditions was close to that of Acidithiobacillus ferrooxidans and exceeded by 2.0–2.7 times the activity of these moderately thermophilic bacteria at 37°C. The leaching of pyrite by Leptospirillum-like bacteria increased in the presence of sulfur-oxidizing bacteria, particularly, in their association with a thermotolerant sulfur-oxidizing bacterium.     

Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses and of Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, -amylase and -galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at –5°C), demonstrating the unique properties of these enzymes.     

Extremely thermophilic cellulolytic anaerobes from icelandic hot springs

Anaerobic enrichment cultures with Avicel as substrate and inoculated with biomat samples from Icelandic hot springs were cultured at 70 ° or 78 °C and examined for the presence of microorganisms that produce extracellular cellulolytic and xylanolytic enzymes. From four enrichments grown at 78 °C eighteen strains were isolated. Five of the strains were screened for their substrate utilization, and on the basis of differences in morphology and substrates used, the two most unique strains were selected for further characterization. All cellulolytic cultures were rod-shaped and non-sporeforming. Motility was not observed. Cells stained gram-negative at various stages of the growth phase. During growth on Avicel, most cultures produced acetate as the major fermentation product, with smaller amounts of lactic acid and ethanol. Carbon dioxide and hydrogen were also produced. The phenotypic characteristics of the enrichment cultures and of isolates are described and assessed in relation to temperature and pH in the hot spring environment. A comparison is made between Icelandic strains isolated in our laboratory and strains isolated from hot springs from other parts of the world. The biotechnological potential of this group of bacteria is briefly discussed.     

A Pressurized Chemostat for the Study of Marine Barophilic and Oligotrophic Bacteria

A continuous culture system that allows bacteria to be grown in steady-state populations under pressures of up to 700 atm (71 MPa) was constructed and tested. With readily available or slightly modified high-pressure chromatography equipment, a continuous flow of sterile medium is pressurized and passed through a 500-ml nylon-coated titanium reactor at flow rates of 0.01 to 10 ml min(sup-1). The pressure in the reactor is controlled by a backpressure regulator with greater than 1% accuracy. In test experiments, a culture of a psychro- and barophilic marine isolate from a depth of 4,900 m (strain F1-A, identified as a Shewanella sp.) was grown at 1, 300, and 450 atm (0.1, 30.4, and 40.5 MPa) and dilution rates of 60 and 90% of the organism's maximum growth rate (determined at 1 atm) in the required complex medium at levels of 3.3 and 0.33 mg of dissolved organic carbon per liter in the reservoir. Growth limitation by carbon was assured by an appropriate C/N/P ratio of the medium. The data indicate that barophilic growth characteristics in steady-state cultures of this psychro- and barophilic deep-sea isolate were positively affected by a decreasing growth rate at the higher of two substrate concentrations in the reservoir. After a 10-fold lowering of the substrate concentration, the effect was reversed. Under these conditions, the cell viability increased significantly, especially at the higher of the two pressures tested. The basic design of the system can principally also be used for growth studies on hyperthermophilic bacteria and archaea.     

Population Sizes and Growth Pressure Responses of Intestinal Microfloras of Deep-Sea Fish Retrieved from the Abyssal Zone

The intestinal floras of seven deep-sea fish retrieved at depths of from 3,200 to 5,900 m were examined for population sizes and growth responses to pressure. Large populations of culturable bacteria, ranging from 1.1 x 10(sup6) to 3.6 x 10(sup8) cells per ml of contents, were detected when samples were incubated at conditions characteristic of those of the deep sea. Culturable cell counts at in situ pressures were greater than those at atmospheric pressure in all samples. Most of the strains isolated by the spread-plating method at atmospheric pressure later proved barophilic. Barophilic bacteria were the predominant inhabitants of the abyssal fish intestines.