Barophilic Bacteria Associated with Digestive Tracts of Abyssal Holothurians

Abyssal holothurians and sediment samples were collected at depths of 4,430 to 4,850 m in the Demerara abyssal plain. Bacterial concentrations in progressive sections of the holothurian digestive tract, as well as in surrounding surface sediments, were determined by epifluorescence microscopy. Total bacterial counts in sediments recently ingested by the animals were 1.5- to 3-fold higher than in surrounding sediments at the deepest station. Lowest counts were observed consistently in the foregut, where the digestive processes of the holothurian are believed to occur. In most animals, counts increased 3- to 10-fold in the hindgut. Microbial activity at 3°C and in situ and atmospheric pressure were determined for gut and sediment samples by measuring the utilization of [¹⁴C]glutamic acid, the doubling time of the mixed-population of culturable bacteria, and the percentage of the total bacterial count responsive to yeast extract in the presence of nalidixic acid, using epifluorescence microscopy. A barophilic microbial population, showing elevated activity under deep-sea pressure, was detected by all three methods in sediments removed from the hindgut. Transmission electron micrographs revealed intact bacteria directly associated with the intestinal lining only in the hindgut. The bacteria are believed to be carried as an actively metabolizing, commensal gut flora that transforms organic matter present in abyssal sediments ingested by the holothurian. Using data obtained in this study, it was calculated that sediment containing organic matter altered by microbial activity cleared the holothurian gut every 16 h, suggesting that abyssal holothurians and their associated gut flora are important participants in nutrient cycles of the abyssal benthic ocean.     

Positive Pressure Effect on Manganese Binding by Bacteria in Deep-Sea Hydrothermal Plumes

A positive pressure effect (1.4 to 3.3×) on the binding of Mn²⁺ by a natural population of bacteria in a deep-sea hydrothermal plume was discovered over the intermediate pressure range of 1 to 200 atm (1 to 200 bars; ca. 1.01 × 10² to 2.03 × 10⁴ kPa). The data suggest Mn²⁺ binding is functionally barophilic rather than simply barotolerant.     

Activity and growth of microbial populations in pressurized deep-sea sediment and animal gut samples.

Benthic animals and sediment samples were collected at deep-sea stations in the northwest (3,600-m depth) and southeast (4,300- and 5200-m depths) Atlantic Ocean. Utilization rates of [14C]glutamate (0.67 to 0.74 nmol) in sediment suspensions incubated at in situ temperatures and pressures (3 to 5 degrees C and 360, 430, or 520 atmospheres) were relatively slow, ranging from 0.09 to 0.39 nmol g-1 day-1, whereas rates for pressurized samples of gut suspensions varied widely, ranging from no detectable activity to a rapid rate of 986 nmol g-1 day-1. Gut flora from a holothurian specimen and a fish demonstrated rapid, barophilic substrate utilization, based on relative rates calculated for pressurized samples and samples held at 1 atm (101.325 kPa). Substrate utilization by microbial populations in several sediment samples was not inhibited by in situ pressure. Deep-sea pressures did not restrict growth, measured as doubling time, of culturable bacteria present in a northwest Atlantic sediment sample and in a gut suspension prepared from an abyssal scavenging amphipod. From the results of this study, it was concluded that microbial populations in benthic environments can demonstrate significant metabolic activity under deep-ocean conditions of temperature and pressure. Furthermore, rates of microbial activity in the guts of benthic macrofauna are potentially more rapid than in surrounding deep-sea sediments.     

Pressure and Temperature Effects on Growth and Methane Production of the Extreme Thermophile Methanococcus jannaschii

The marine archaebacterium Methanococcus jannaschii was studied at high temperatures and hyperbaric pressures of helium to investigate the effect of pressure on the behavior of a deep-sea thermophile. Methanogenesis and growth (as measured by protein production) at both 86 and 90°C were accelerated by pressure up to 750 atm (1 atm = 101.29kPa), but growth was not observed above 90°C at either 7.8 or 250 atm. However, growth and methanogenesis were uncoupled above 90°C, and the high-temperature limit for methanogenesis was increased by pressure. Substantial methane formation was evident at 98°C and 250 atm, whereas no methane formation was observed at 94°C and 7.8 atm. In contrast, when argon was substituted for helium as the pressurizing gas at 250 atm, no methane was produced at 86°C. Methanogenesis was also suppressed at 86°C and 250 atm when the culture was pressurized with a 4:1 mix of H₂ and CO₂, although limited methanogenesis did occur when the culture was pressurized with H₂.     

Thermal Inactivation of a Deep-Sea Barophilic Bacterium, Isolate CNPT-3

The barophilic deep-sea bacterium, isolate CNPT-3, was inactivated by exposures to temperatures between 10 and 32°C at atmospheric pressure. Inactivation in samples from warmed cell suspensions was measured as the loss of colonyforming ability (CFA) at 10°C and 587 bars. At atmospheric pressure, there was a slow loss of CFA even at 10°C. The loss of CFA was rapid above 20°C and only slightly affected by high pressures. The first-order rate constants for thermal inactivation fit the Arrhenius equation with an activation energy of 43 kcal (ca. 179.9 kJ)/mol. Light microscopy and scanning transmission electron microscopy revealed morphological changes due to warming of the cells. The changes ensued the loss of CFA. The results supported the hypothesis from an earlier work that indigenous (autochthonous) deep-sea bacteria from cold deep seas are both barophilic and psychrophilic. If ultimately sustained, these characteristics may be useful in designing experiments to assess the relative importance of the autochthonous and allochthonous bacteria in the deep sea. The data were used to evaluate how barophilic bacteria may have been missed in many investigations because of warming of the cells during sample retrieval from the sea or during cultivation in the laboratory. The evaluation revealed the need for temperature and pressure data during retrieval of samples and cultivation in the laboratory. Most deep-ocean microbiology may be possible with thermally insulated equipment for retrieval from the sea and with high-pressure vessels for laboratory incubations.     

Activity and growth of microbial populations in pressurized deep-sea sediment and animal gut samples

Benthic animals and sediment samples were collected at deep-sea stations in the northwest (3,600-m depth) and southeast (4,300- and 5200-m depths) Atlantic Ocean. Utilization rates of [14C]glutamate (0.67 to 0.74 nmol) in sediment suspensions incubated at in situ temperatures and pressures (3 to 5 degrees C and 360, 430, or 520 atmospheres) were relatively slow, ranging from 0.09 to 0.39 nmol g-1 day-1, whereas rates for pressurized samples of gut suspensions varied widely, ranging from no detectable activity to a rapid rate of 986 nmol g-1 day-1. Gut flora from a holothurian specimen and a fish demonstrated rapid, barophilic substrate utilization, based on relative rates calculated for pressurized samples and samples held at 1 atm (101.325 kPa). Substrate utilization by microbial populations in several sediment samples was not inhibited by in situ pressure. Deep-sea pressures did not restrict growth, measured as doubling time, of culturable bacteria present in a northwest Atlantic sediment sample and in a gut suspension prepared from an abyssal scavenging amphipod. From the results of this study, it was concluded that microbial populations in benthic environments can demonstrate significant metabolic activity under deep-ocean conditions of temperature and pressure. Furthermore, rates of microbial activity in the guts of benthic macrofauna are potentially more rapid than in surrounding deep-sea sediments.     

Effects of Hyperbaric Pressure on a Deep-Sea Archaebacterium in Stainless Steel and Glass-Lined Vessels

The effects of hyperbaric helium pressures on the growth and metabolism of the deep-sea isolate ES4 were investigated. In a stainless steel reactor, cell growth was completely inhibited but metabolic gas production was observed. From 85 to 100°C, CO₂ production proceeded two to three times faster at 500 atm (1 atm = 101.29 kPa) than at 8 atm. At 105°C, no CO₂ was produced until the pressure was increased to 500 atm. Hydrogen and H₂S were also produced biotically but were not quantifiable at pressures above 8 atm because of the high concentration of helium. In a glass-lined vessel, growth occurred but the growth rate was not accelerated by pressure. In most cases at temperatures below 100°C, the growth rate was lower at elevated pressures; at 100°C, the growth rates at 8, 250, and 500 atm were nearly identical. Unlike in the stainless steel vessel, CO₂ production was exponential during growth and continued for only a short time after growth. In addition, relatively little H₂ was produced in the glass-lined vessel, and there was no growth or gas production at 105°C at any pressure. The behavior of ES4 as a function of temperature and pressure was thus very sensitive to the experimental conditions.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype.

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Product Inhibition of Butyrate Metabolism by Acetate and Hydrogen in a Thermophilic Coculture

Studies on product inhibition of a thermophilic butyrate-degrading bacterium in syntrophic association with Methanobacterium thermoautotrophicum showed that a gas phase containing more than 2 × 10⁻² atm (2.03 kPa) of hydrogen prevented growth and butyrate consumption, while a lower hydrogen partial pressure of 1 × 10⁻³ to 2 × 10⁻² atm (0.1 to 2.03 kPa) gradually inhibited the butyrate consumption of the coculture. No inhibition of butyrate consumption was found on the addition of 0.75 × 10⁻³ atm (76 Pa) of hydrogen to the gas phase. A slight inhibition of butyrate consumption by the coculture occurred at an acetate concentration of 16.4 mM. Inhibition gradually increased with increasing acetate concentration up to 81.4 mM, when complete inhibition of butyrate consumption occurred. When the culture contained an acetate-utilizing methanogen in addition to M. thermoautotrophicum, the inhibition of the triculture by acetate was gradually reversed as the acetate concentration was lowered by the aceticlastic methanogen. The results show that optimal growth conditions for the thermophilic butyrate-degrading bacterium depend on both hydrogen and acetate removal.     

Comparative Physiological Studies on Hyperthermophilic Archaea Isolated from Deep-Sea Hot Vents with Emphasis on Pyrococcus Strain GB-D

Three new sulfur- or non-sulfur-dependent archaeal isolates, including a Pyrococcus strain, from Guaymas Basin hydrothermal vents (Gulf of California; depth, 2,010 m) were characterized and physiologically compared with four known hyperthermophiles, previously isolated from other vent sites, with an emphasis on growth and survival under the conditions particular to the natural habitat. Incubation under in situ pressure (200 atm [1 atm = 101.29 kPa]) did not increase the maximum growth temperature by more than 1°C for any of the organisms but did result in increases in growth rates of up to 15% at optimum growth temperatures. At in situ pressure, temperatures considerably higher than those limiting growth (i.e., > 105°C) were survived best by isolates with the highest maximum growth temperatures, but none of the organisms survived at temperatures of 150°C or higher for 5 min. Free oxygen was toxic to all isolates at growth range temperatures, but at ambient deep-sea temperature (3 to 4°C), the effect varied in different isolates, the non-sulfur-dependent isolate being the most oxygen tolerant. Hyperthermophiles could be isolated from refrigerated and oxygenated samples after 5 years of storage. Cu, Zn, and Pb ions were found to be toxic under nongrowth conditions (absence of organic substrate), with the non-sulfur-dependent isolate again being the most tolerant.     

Biochemical Function and Ecological Significance of Novel Bacterial Lipids in Deep-Sea Procaryotes

The fatty acid composition of the membrane lipids in 11 deep-sea bacterial isolates was determined. The fatty acids observed were typical of marine vibrios except for the presence of large amounts of long-chain polyunsaturated fatty acids (PUFAs). These long-chain PUFAs were previously thought to be absent in procaryotes, with the notable exception of a single marine Flexibacter sp. In three barophilic strains tested at 2°C, there was a general increase in the relative amount of PUFAs as pressure was increased from a low growth pressure towards the optimal growth pressure. In Vibrio marinus MP-1, a psychrophilic strain, PUFAs were found to increase as a function of decreasing temperature at constant atmospheric pressure. These results suggest the involvement of PUFAs in the maintenance of optimal membrane fluidity and function over environmentally relevant temperatures and pressures. Furthermore, since these lipids are essential nutrients for higher taxa and are found in large amounts in the lipids of deep-sea vertebrates and invertebrates, an important, specific role for deep-sea bacteria in abyssal food webs is implicated.     

Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 × 10⁻¹⁰ to 5.3 × 10⁻¹⁰ μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10⁸ to 10¹¹ cells per liter. The muramic acid content in natural marine water decreased from 5.3 × 10⁻¹⁰ to 1.6 × 10⁻¹⁰ μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.     

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     

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.     

Properties of the Glucose Transport System in Some Deep-Sea Bacteria

Many deep-sea bacteria are specifically adapted to flourish under the high hydrostatic pressures which exist in their natural environment. For better understanding of the physiology and biochemistry of these microorganisms, properties of the glucose transport systems in two barophilic isolates (PE-36, CNPT-3) and one psychrophilic marine bacterium (Vibrio marinus MP1) were studied. These bacteria use a phosphoenol-pyruvate:sugar phosphotransferase system (PTS) for glucose transport, similar to that found in many members of the Vibrionaceae and Enterobacteriaceae. The system was highly specific for glucose and its nonmetabolizable analog, methyl alpha-glucoside (a-MG), and exhibited little affinity for other sugars tested. The temperature optimum for glucose phosphorylation in vitro was approximately 20°C. Membrane-bound PTS components of deep-sea bacteria were capable of enzymatically cross-reacting with the soluble PTS enzymes of Salmonella typhimurium, indicating functional similarities between the PTS systems of these organisms. In CNPT-3 and V. marinus, increased pressure had an inhibitory effect on a-MG uptake, to the greatest extent in V. marinus. Relative to atmospheric pressure, increased pressure stimulated sugar uptake in the barophilic isolate PE-36 considerably. Increased hydrostatic pressure inhibited in vitro phosphoenolpyruvate-dependent a-MG phosphorylation catalyzed by crude extracts of V. marinus and PE-36 but enhanced this activity in crude extracts of the barophile CNPT-3. Both of the pressure-adapted barophilic bacteria were capable of a-MG uptake at higher pressures than was the nonbarophilic psychrophile, V. marinus.     

Isolation and Diversity of Actinomycetes in the Chesapeake Bay

Chesapeake Bay was investigated as a source of actinomycetes to screen for production of novel bioactive compounds. The presence of relatively large populations of actinoplanetes (chemotype II/D actinomycetes) in Chesapeake Bay sediment samples indicates that it is an eminently suitable ecosystem from which to isolate actinomycetes for screening programs. Actinomycetes were isolated from sediment samples collected in Chesapeake Bay with an isolation medium containing nalidixic acid, which proved to be more effective than heat pretreatment of samples. Actinomycete counts ranged from a high of 1.4 × 10⁵ to a low of 1.8 × 10² CFU/ml of sediment. Actinomycetes constituted 0.15 to 8.63% of the culturable microbial community. The majority of isolates from the eight stations studied were actinoplanetes (i.e., chemotype II/D), and 249 of these isolates were obtained in a total of 298 actinomycete isolates. Antimicrobial activity profiles indicated that diverse populations of actinoplanetes were present at each station. DNA hybridization studies showed considerable diversity among isolates between stations, but indicated that actinoplanete strains making up populations at nearby stations were more similar to each other than to populations sampled at distant stations. The diversity of actinoplanetes and the ease with which these organisms were isolated from Chesapeake Bay sediments make this a useful source of these actinomycetes.     

Anaerobic Methane Oxidation: Occurrence and Ecology

Anoxic sediments and digested sewage sludge anaerobically oxidized methane to carbon dioxide while producing methane. This strictly anaerobic process showed a temperature optimum between 25 and 37°C, indicating an active microbial participation in this reaction. Methane oxidation in these anaerobic habitats was inhibited by oxygen. The rate of the oxidation followed the rate of methane production. The observed anoxic methane oxidation in Lake Mendota and digested sewage sludge was more sensitive to 2-bromoethanesulfonic acid than the simultaneous methane formation. Sulfate diminished methane formation as well as methane oxidation. However, in the presence of iron and sulfate the ratio of methane oxidized to methane formed increased markedly. Manganese dioxide and higher partial pressures of methane also stimulated the oxidation. The rate of methane oxidation in untreated samples was approximately 2% of the CH₄ production rate in Lake Mendota sediments and 8% of that in digested sludge. This percentage could be increased up to 90% in sludge in the presence of 10 mM ferrous sulfate and at a partial pressure of methane of 20 atm (2,027 kPa).     

Higher Abundance of Bacteria than of Viruses in Deep Mediterranean Sediments

The interactions between viral abundance and bacterial density, biomass, and production were investigated along a longitudinal transect consisting of nine deep-sea stations encompassing the entire Mediterranean basin. The numbers of viruses were very low (range, 3.6 × 10⁷ to 12.0 × 10⁷ viruses g⁻¹) and decreased eastward. The virus-to-bacterium ratio was always < 1.0, indicating that the deep-sea sediments of the Mediterranean Sea are the first example of a marine ecosystem not numerically dominated by viruses. The lowest virus numbers were found where the lowest bacterial metabolism and turnover rates and the largest cell size were observed, suggesting that bacterial doubling time might play an important role in benthic virus development.     

Summer abundance and activities of bacteria in the freshwater lakes of Schirmacher Oasis,Antarctica

Bacterial biomass and heterotrophic potential (using ¹⁴C-labeled glucose, glutamic acid and sodium acetate) of water, ice and sediment microbial populations were studied from different lakes of the Schirmacher Oasis, Antarctica. Epifluorescence counts of total bacteria in these lakes were observed to be lower by a factor when compared to some of the ultraoligotrophic Antarctic lakes. Biovolumes of bacteria from different samples did not show significant variations, suggesting that regulatory factors were oligotrophy and low temperatures rather than microzoan grazing. Microbial uptake rates of glutamic acid were generally the fastest, followed by glucose and/or sodium acetate in the lakewater samples. The mean values of V_max cell^–1 for glutamic acid, sodium acetate and glucose were 3.81, 0.91 and 0.71 pgCh^–1. Results of this study are potentially useful in recognizing the relative abundance and activity of limnetic microbial populations in the Schirmacher Oasis during summer — the active period of microbial growth — and for comparing their activities with other ecosystems elsewhere in continental Antarctica.