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.     

Morphological survey of microbial mats near deep-sea thermal vents

[This corrects the article on p. 531 in vol. 41.].     

A new screening method for investigating microbial interactions

Interactions among Candida albicans, Staphylococcus aureus and Escherichia coli were investigated using a screening system in which test micro-organisms were incorporated in agar discs and effector micro-organisms in fluid growth media. Total as well as partial inhibition of test micro-organisms was observed in agar discs when these were incubated in broths containing effector micro-organisms. The ratio of numbers of test to effector micro-organisms was found to be of importance in the inhibition effect. The technique was found to be cheap, simple and versatile.     

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.     

A model for real thermal control in high-pressure treatment of foods

A model for the simulation of thermal exchanges in a complete high-pressure equipment was developed. Good agreement between simulated and experimental time-temperature profiles was found during different processes of pressurization and depressurization. The model allows study of the effect of different variables to improve thermal control in the treatments performed. This work involved an important advance in optimization and regulation of high-pressure processes in the food industry.     

A submerged-coil heating apparatus for investigating thermal inactivation of micro-organisms

A novel apparatus has been designed for the investigation of the thermal inactivation of micro-organisms over the temperature range 20–90C. It comprises a stainless steel coil fully submerged in a thermostatically-controlled water bath which allows very rapid temperature equilibration of microbial suspensions. Automated repeat sampling is possible at short time intervals (variable between 6 and 999 s) by means of an electronically controlled displacement mechanism. Validation of the apparatus performance and typical death curves are described.     

A microassay system for measuring esterase activity and protein concentration in small samples and in high-pressure liquid chromatography eluate fractions

A microtiter plate spectrophotometric system has been used together with the Bradford, Ellman, and van Asperen assays to measure protein concentration (to 0.5 μg) and the activities of acetylcholinesterase (to 10^?3 units) and carboxylesterase (to 30 μg β-napthol reaction product) in small samples such as high-performance liquid chromatographic eluate fractions. For 100-μl samples, at least 300 Ellman acetylcholinesterase or Bradford protein assays can be conducted and read in less than 30 min, and a like number of van Asperen nonspecific esterase assays (including 1-h incubation) run in less than 90 min. The eluate from a single 20-min high-performance liquid chromatographic run of a biological sample can be collected as up to 300 fractions directly into microtiter plate wells, and the three assays run on all fractions in less than 90 min.     

Soil microbial community structure across a thermal gradient following a geothermal heating event

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.     

A diffusion gradient chamber for studying microbial behavior and separating microorganisms

The natural habitats of most microbes are dynamic and include spatial gradients of growth substrates, electron acceptors, pH, salts, and inhibitory compounds. To mimic this diffusive aspect of nature, we developed an analytical diffusion gradient chamber (DGC) that can be used to separate, enrich for, isolate, and study the behavior of microorganisms. The chamber is a polycarbonate box containing an arena (5 by 5 by 2 cm) into which is cast a semisolid growth medium. Continuously replenished solute reservoirs positioned on each side of the arena but separated from it by a porous membrane enable the formation throughout the gel of multiple, intersecting gradients of solutes in two dimensions. With glucose as the solute, a gradient which spanned a 100-fold range in concentration was established across the arena in about 4 days. The shape of the glucose gradient was accurately predicted by a mathematical model based on Fickian diffusion. The growth and migratory behavior of Escherichia coli in response to imposed gradients of attractants (aspartate, alpha-methyl aspartate, and serine) and a repellent (valine) were examined. Cells responded in predictable ways to such gradients by forming distinctive growth and migration patterns in the DGC. This was true for wild-type E. coli as well as specific chemotaxis and motility mutants. The patterns yielded information about the threshold concentration of chemoeffectors needed to elicit a response as well as their saturating concentration. It was also evident that the metabolism of attractants significantly affected the gradients and, hence, the movement of cells. Finally, it was possible to separate E. coli and Pseudomonas fluorescens in the DGC on the basis of their differential responses to gradients of various chemoeffectors.     

Hydrolytic enzymatic activity in deep-sea sediments

Abstract Hydrolytic activities of five enzymes were measured in deep-sea sediment cores at three stations under in situ temperature and pressure in the NE-Atlantic in March/April and July/August 1992. Generally, activity profiles declined vertically in the upper 10 cm of the cores. Experiments under in situ pressure were not significantly different from measurements under surface conditions. The ranking of potential activity rates in the top sediment horizon was: aminopeptidase > esterase > chitobiase > β-glucosidase > α-glucosidase with ratios of 687/174/11/3/1. This is similar to ratios obtained in marine aggregates from the upper mixed layer, thus supporting the idea of pelagic-benthic coupling in the open ocean. The vertical activity profiles show that the biochemical composition, and thereby the nutritive quality of the degradable material, changed with depth in the sediment cores. About 518 mg carbon was potentially mobilized in the 0–1 cm sediment horizon per square meter per day. This contrasts with the input of particulate organic carbon to the sea floor in this area of only 2.74 mg C m² d⁻¹, determined by sediment traps, which indicates that the deep-sea benthic community can rapidly utilize sedimenting particulate organic material and highlights the importance of extracellular enzyme activity in the sediment biogeochemical loop.     

Early steps in microbial colonization processes at deep-sea hydrothermal vents

A pluri-disciplinary in situ colonization experiment was performed to study early stages of colonization in deep-sea vent Alvinella spp. worm habitats. Four colonization devices were deployed onto Alvinella spp. colonies of different chimneys of the East-Pacific Rise (EPR 13 degrees N), for two different periods: a short (less than a week) and a longer one (3 weeks). Video imagery and monitoring of the thermal and physico-chemical conditions were performed during the colonization experiments. Numerous microorganisms bearing specialized adhesion-appendages and/or high amounts of polymeric extracellular matrix were observed on devices, which may efficiently contribute to the colonization of new surfaces. The microbial cohorts preceding and accompanying Alvinella spp. settlement were identified. In all cases, Archaea could not be detected and the microbial mats were essentially composed of e-Proteobacteria. Within this group, one phylotype (AlviH2) was found to dominate the libraries of three colonization devices. Dominance of e-Proteobacteria in the libraries may reflect the wide physiological variety encountered within this group or an adaptability of these microorganisms towards their changing environment. Bacteria affiliated to the Cytophaga-Flavobacterium-Bacteroides group or to the e-Proteobacteria, that grow either chemo-organoheterotrophically by fermentation or chemolithoautotrophically with H2 as an electron donor and S degrees /S2O32- or NO3- as a terminal electron acceptor, were isolated from one of the microbial mat formed in 20 days.     

A steep thermal gradient thermotaxis assay for the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans with its well-described nervous system is one of the multicellular organisms of choice to study thermotaxis. The neuronal circuitry for thermosensation has been analyzed at the level of individual cells. Two methods have previously been described to study the behavior of C. elegans with respect to temperature: 1) isothermal tracking assays and 2) linear thermal gradients (Hedgecock and Russell, 1975). Here we present a short linear thermal gradient assay which is faster and which allows statistical evaluation of different populations using a thermotaxis index. Thin agar plates are used on which a temperature gradient from about 10 degrees to 30 degrees is induced over the distance of about 5 cm. The short linear thermal gradient uses inexpensive materials so that multiple tests can be performed in parallel in a short period of time.     

Comparison of deep-sea sediment microbial communities in the Eastern Mediterranean

Bacterial and archaeal communities in sediments obtained from three geographically-distant mud volcanoes, a control site and a microbial mat in the Eastern Mediterranean deep-sea were characterized using direct 16S rRNA gene analyses. The data were thus in relation to the chemical characteristics of the (stratified) habitats to infer community structure-habitat relationships. The bacterial sequences in the different habitats were related to those of Actinobacteria, Bacilli, Chloroflexi, Alpha-, Beta-, Gamma-, Delta- and Epsilonproteobacteria and unclassified bacteria, including the JS1 group. The archaeal sequences found were affiliated with those of the Methanosarcinales, Thermoplasmales, Halobacteriales and Crenarchaea belonging to marine benthic group I and B, as well as MCG group archaea. In each sample, the communities were diverse and unique at the phylotype level. However, at higher taxonomic levels, similar groups were found in different sediments, and similar depth layers tended to contain similar communities. The sequences that dominated in all top layers (as well as in the mat) probably represented organisms involved in aerobic heterotrophy, sulfide-based chemoautotrophy and methanotrophy and/or methylotrophy. Sequences of organisms most likely involved in anaerobic methane oxidation, sulfate reduction and anaerobic heterotrophy were predominantly found in deeper layers. The data supported the notion of (1) uniqueness of each habitat at fine taxonomic levels, (2) stratification in depth and (3) conservation of function in the sediments.     

Phylogenetic relationships ofThiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genusThiomicrospira. To identify these ‘phylotypes’ in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three describedThiomicrospira species, viz.,Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate,Thiomicrospira sp. strain MA2-6. AllThiomicrospira species exceptTms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria.Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated withThiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivisionThiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence ofTms. crunogena. Identifying a dominant molecular isolate asTms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another ‘phylotype’ represented a newThiomicrospira species, phylogenetically in an intermediate position betweenTms. crunogena andTms. pelophila. The third ‘phylotype’ was identified as aDesulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.     

A salt-concentration gradient method for the determination of the maximum salt concentration for microbial growth

A method is described for establishing a discontinuous salt concentration gradient which is useful for determining the maximum salt concentration which can be tolerated by growing microorganisms. The technique can be used aerobically or anaerobically, and has a standard error varying from ± 0.1% to ± 0.4%, depending on the organism. Data are given for several yeasts and forSerratia marcescens. The results indicate that the method has a potential usefulness for separating and identifying closely related microorganisms.