Membrane lipids of a psychrophilic and barophilic deep-sea bacterium

In a psychrophilic and barophilic marine bacterial isolate of the genusAlteromonas, the ratio of total unsaturated versus saturated fatty acids in the membrane lipids increased when the organism was grown at increasing hydrostatic pressures and decreasing temperatures. This regulatory capacity, as well as the presence of relatively large amounts of 20:5 polyunsaturated fatty acid, appear to be functional in maintaining membrane fluidity within a range of pressures distinctly below and above the specific optimum and at typical deep sea temperatures.     

Ammonia-induced injury in pure cultures and natural populations of coliform bacteria

Ammonia-induced injury was investigated in pure cultures of Escherichia coli and Enterobacter aerogenes, and in natural coliform populations obtained from the oligotrophic Luxapallila and the eutrophic Sunflower Rivers in northern Mississippi. Pure cultures were affected by ammonia exposure as indicated by changes in the injury ratio (IR) of CFU on m-T7 agar/CFU on m-Endo agar. Ammonia concentrations between 0 and 20 (mg NH3-N/1) had little or no effect and concentrations between 40 and 80 caused the greatest injury. Natural coliform populations from the oligotrophic river were more prone to ammonia-induced injury than those from the eutrophic river. The results stress the need for the routine use of m-T7 media and the enumeration of injured cells when using the membrane filter procedure to ascertain domestic water quality.     

Evolutionary relationships of cultivated psychrophilic and barophilic deep-sea bacteria

Evolutionary relationships of cultivated barophilic bacteria were determined. All psychrophilic and barophilic isolates were affiliated with one of five genera of the gamma subdivision of the class Proteobacteria ((gamma)-Proteobacteria): Shewanella, Photobacterium, Colwellia, Moritella, and a new group containing strain CNPT3. The data indicate that the barophilic phenotype has evolved independently in different (gamma)-Proteobacteria genera.     

A high-pressure thermal gradient block for investigating microbial activity in multiple deep-sea samples

Details about the construction and use of a high-pressure thermal gradient block for the simultaneous incubation of multiple samples are presented. Most parts used are moderately priced off-the-shelf components that easily obtainable. In order to keep the pressure independent of thermal expansion of the sample vessels, a back-pressure system with a constant leak rate was installed. Pressure is applied through high-pressure liquid chromatography (HPLC) pumps that run in constant pressure mode with variable flow rate, thereby regulating any pressure fluctuations. The device allows incubations along a wide range of temperatures and pressures and can easily be modified to accommodate different experiments, either biological or chemical. As an application, we present measurements of bacterial sulfate reduction rates in hydrothermal sediments from Guyamas Basin over a wide range of temperatures and pressures. Sulfate reduction rates increase with increasing pressure and show maximum values at pressures higher than in situ.     

Power generation from cellulose using mixed and pure cultures of cellulose-degrading bacteria in a microbial fuel cell

Microbial fuel cells (MFCs) have been used to generate electricity from various organic compounds such as acetate, glucose, and lactate. We demonstrate here that electricity can be produced in an MFC using cellulose as the electron donor source. Tests were conducted using two-chambered MFCs, the anode medium was inoculated with mixed or pure culture of cellulose-degrading bacteria Nocardiopsis sp. KNU (S strain) or Streptomyces enissocaesilis KNU (K strain), and the catholyte in the cathode compartment was 50mM ferricyanide as catholyte. The power density for the mixed culture was 0.188mW (188mW/m(2)) at a current of 0.5mA when 1g/L cellulose was used. However, the power density decreased as the cellulose concentration in the anode compartment decreased. The columbic efficiencies (CEs) ranged from 41.5 to 33.4%, corresponding to an initial cellulose concentration of 0.1-1.0g/L. For the pure culture, cellobioase enzyme was added to increase the conversion of cellulose to simple sugars, since electricity production is very low. The power densities for S and K strain pure cultures with cellobioase were 162mW/m(2) and 145mW/m(2), respectively. Cyclic voltammetry (CV) experiments showed the presence of peaks at 380, 500, and 720mV vs. Ag/AgCl for the mixed bacterial culture, indicating its electrochemical activity without an external mediator. Furthermore, this MFC system employs a unique microbial ecology in which both the electron donor (cellulose) and the electron acceptor (carbon paper) are insoluble.     

Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples

Several barophilic and barotolerant bacteria were isolated from deep-sea mud samples of Suruga Bay (2485 m depth), the Ryukyu Trench (5110 m depth), and the Japan Trench (land-side 6356 m, and sea-side 6269 m depth, respectivelys. The barophilic bacteria, strains DB5501, DB6101, DB6705 and DB6906, were albe to grow better under high hydrostatic pressures than under atmospheric pressure (0.1 megapascals; MPa). The optimal growth pressures for the barophilic bacteria were approximately 50 MPa at 10°C. The barotolerant strains DSK1 and DSS12 were determined to be psychrophilic, and had optimal growth temperatures of 10°C and 8°C, respectively. The degree of barophily and barotolerance was shown to be very dependent on temperature. For example, at 4°C the barophilic strains were indistinguishable from barotolerant bacteria, whereas at 15°C the barotolerant strains behaved more like the barophilic strains. Based on sequence analysis of 16S ribosomal DNA, all of the strains included in this study belong to the gamma subgroup of the Proteobacteria. Phylogenetic relations between the isolated strains and the known gamma subgroup bacteria suggested that the isolated strains belong to a new sub-branch of this group.     

Degradation of paracetamol by pure bacterial cultures and their microbial consortium

Three bacterial strains utilizing paracetamol as the sole carbon, nitrogen, and energy source were isolated from a paracetamol-degrading aerobic aggregate, and assigned to species of the genera Stenotrophomonas and Pseudomonas. The Stenotrophomonas species have not included any known paracetamol degraders until now. In batch cultures, the organisms f1, f2, and fg-2 could perform complete degradation of paracetamol at concentrations of 400, 2,500, and 2,000 mg/L or below, respectively. A combination of three microbial strains resulted in significantly improved degradation and mineralization of paracetamol. The co-culture was able to use paracetamol up to concentrations of 4,000 mg/L, and mineralized 87.1 % of the added paracetamol at the initial of 2,000 mg/L. Two key metabolites of the biodegradation pathway of paracetamol, 4-aminophenol, and hydroquinone were detected. Paracetamol was degraded predominantly via 4-aminophenol to hydroquinone with subsequent ring fission, suggesting new pathways for paracetamol-degrading bacteria. The degradation of paracetamol could thus be performed by the single isolates, but is stimulated by a synergistic interaction of the three-member consortium, suggesting a possible complementary interaction among the various isolates. The exact roles of each of the strains in the consortium need to be further elucidated.     

Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

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.     

Co-metabolism of fluorobenzoates by natural microbial populations.

Co-metabolic degradation of monofluorobenzoates was carried out by a mixed soil population in a basal salts medium. The monofluorobenzoates did not support growth of microorganisms but were shown to be subject to ring cleavage as a result of microbial activity. Rate of ring cleavage was increased by use of the co-substrate enrichment technique using glucose as the co-substrate. Results indicate that the monofluorobenzoates were subject to an initial co-metabolic attack with glucose, providing the energy necessary for co-metabolism to proceed to a point where complete metabolism became possible.     

Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes

A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.     

Competition of two microbial populations for a single resource in a chemostat when one of them exhibits wall attachment

It is known that two microbial populations competing for a single resource in a homogeneous environment with time-invariant inputs cannot coexist in a steady state. The case where two microbial populations compete for a single resource in a chemostat but one of them exhibits attachment to the chemostat walls is studied theoretically. Because of the cells' attachment to the walls, the environment is no longer homogeneous. The present article considers the case where the attached cells form no more than a monolayer. Other situations occur, often frequently, but we do not consider them here. Two models are used to represent the attachment to the walls: the Topiwala-Hamer model and a model which assumes that the attachment of microbial cells to the solid surfaces is a reversible process. The first model does not allow the population that exhibits wall attachment to wash out from the chemostat, in contrast to the second model (which nevertheless reduces to the first one in the limit). It has been found that in most of the possible cases for both models, the two competitors can coexist in a stable steady state for a wide range of the operating parameters space. The results of the stability analysis are discussed and analytical expressions for the conditions and the boundaries of the domains of stable coexistence are given for all the possible situations that may arise.     

Kinetic study and mathematical modeling of methanogenesis of acetate using pure cultures of methanogens

Kinetics of methanogenesis from acetate was studied using pure cultures of Methanosarcina barkeri and Methanosarcina mazei. Methane formation was found to be associated with cell growth. Nearly equimolar methane was produced from acetate during the methanogenic growth, and about 1.94 g of cells were formed from each mole of acetate consumed. Cell growth can be estimated from methane production. Significant substrate inhibition was found when acetate concentration was higher than 0.12 M. Among the three methanogenic strains studied, M. mazei strain S6 had the highest specific growth rate at all acetate concentrations studied and was least sensitive to environmental factors investigated (e.g., acetate concentration). The maximum specific growth rate found for strain S6 was 0.022 hr(-1) at acetic acid concentration around 7 g/L. The other two strains studied were M. barkeri strain 227 and strain MS. Growth of M. barkeri was completely inhibited at sodium acetate concentrations higher than 0.24 M. The maximum specific growth rate found for strains 227 and MS was 0.019 and 0.021 h(-1) at acetic acid concentrations of 3.6 and 6.8 g/L, respectively. A kinetic model with substrate inhibition was developed and can be used to simulate the methane formation from M. mazei strain S6 grown on acetate at 35 degrees C, pH 7.     

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.     

Separation and quantification of pigments from natural phototrophic microbial populations

Abstract A simple high-performance liquid chromatography (HPLC) method is described for the rapid (approx. 20 min) simultaneous separation and identification of the major chlorophylls and carotenoids from phytoplankton cells and phototrophic sulfur bacteria. Lyophilized samples were extracted with acetone in the dark at room temperature. Pigments were eluted from a silica column with a hexane-acetone mixture (80: 20, v/v). About 20 algal and bacterial chlorophyll and carotenoid pigments could be separated in one run. The method allowed for the detection of trace amounts of major carotenoids (> approx. 6 ng · 1⁻¹) and of chlorophylls and pheophytins (> approx. 200 ng · 1⁻¹) in natural samples. The method has been applied to samples from the metalimnion of Lake Vechten (The Netherlands) and has proved very useful in estimating algal and bacterial pigments simultaneously with respect to depth distribution and biomass changes of the microbial populations.     

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     

A robust plate assay for detection of extracellular microbial protease activity in metagenomic screens and pure cultures

A robust, efficient and cost-effective agar that utilises lactose free milk powder for identification of bacterial protease activity in pure cultures and metagenomic screens has been developed and tested on protease positive bacteria, selected strains and false protease positives isolated from a previously constructed metagenomic library.     

The effects of wall populations on coexistence of bacteria in the liquid phase of chemostat cultures

We have examined the effects of wall populations on coexistence between strains of Escherichia coli in the liquid phase of mixed (two-strain) chemostats. The wall populations of the two competing strains became established soon after the start of the cultures and, although the relative abundance of the strains in the liquid phase could change over time by several orders of magnitude, the composition of an established wall population did not change markedly. The bacterial strains examined could not displace an established wall population of a competing strain. The presence of a permanent wall population allowed a strain that was less fit in the liquid phase to coexist with a superior strain. The resulting coexistence did not require that the inferior strain attached to the vessel wall better than the superior strain. We believe that the coexistence developed because the inferior strain survived and reproduced on the vessel wall. The progeny from that wall population then provided replacements for the bacteria that the inferior strain lost through a selective disadvantage in the liquid phase of the culture. By replacing the chemostat vessel, hence eliminating the wall populations, we could distinguish between cases where the coexistence depended on the presence of a wall population and where it resulted from some alternative mechanism.