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Experimental evolution of resistance in Paramecium caudatum against the bacterial parasite Holospora undulata.

Host-parasite coevolution is often described as a process of reciprocal adaptation and counter adaptation, driven by frequency-dependent selection. This requires that different parasite genotypes perform differently on different host genotypes. Such genotype-by-genotype interactions arise if adaptation to one host (or parasite) genotype reduces performance on others. These direct costs of adaptation can maintain genetic polymorphism and generate geographic patterns of local host or parasite adaptation. Fixation of all-resistant (or all-infective) genotypes is further prevented if adaptation trades off with other host (or parasite) life-history traits. For the host, such indirect costs of resistance refer to reduced fitness of resistant genotypes in the absence of parasites. We studied (co)evolution in experimental microcosms of several clones of the freshwater protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. After two and a half years of culture, inoculation of evolved and naive (never exposed to the parasite) hosts with evolved and founder parasites revealed an increase in host resistance, but not in parasite infectivity. A cross-infection experiment showed significant host clone-by-parasite isolate interactions, and evolved hosts tended to be more resistant to their own (local) parasites than to parasites from other hosts. Compared to naive clones, evolved host clones had lower division rates in the absence of the parasite. Thus, our study indicates de novo evolution of host resistance, associated with both direct and indirect costs. This illustrates how interactions with parasites can lead to the genetic divergence of initially identical populations.

Animals↗

Capacity for biodegradation of CFCs and HCFCs in a methane oxidative counter-gradient laboratory system simulating landfill soil covers.

The attenuation of methane and four chlorofluorocarbons was investigated in a dynamic methane and oxygen counter-gradient system simulating a landfill soil cover. Soil was sampled at Skellingsted Landfill, Denmark. The soil columns showed a high capacity of methane oxidation with oxidation rates of 210 g m(-2) d(-1) corresponding to a removal efficiency of 81%. CFC-11 and to a lesser extent also CFC-12 were degraded in the active soil columns. The average removal efficiency was 90% and 30% for CFC-11 and CFC-12, respectively. Soil gas concentration profiles indicated that the removal was due to anaerobic degradation, which was verified in anaerobic batch experiments where CFC-11 was rapidly degraded. HCFC-21 and HCFC-22 were also degraded in active soil columns (61% and 41%, respectively), but compared to the CFCs, the degradation was located in the upper oxic part of the column with overlapping gradients of methane and oxygen. High oxidation rates of methane and HCFCs were obtained in soil microcosms incubated with methane. When increasing the column inlet flow, the oxidation zone was moved upward in the column, and the removal efficiency of methane and HCFCs decreased. The removal of CFCs was, however, less affected since the anaerobic zone expanded with increasing inlet flow rates. This study demonstrates the complexity of landfill soil cover systems and shows that both anaerobic and aerobic bacteria may play a very important role in reducing the emission of not only methane but also trace components into the atmosphere.

Bacteria, Aerobic↗

Resuscitation rate in different enterococcal species in the viable but non-culturable state.

AIMS: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. METHODS AND RESULTS: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1:10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). CONCLUSIONS: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.

Bacterial Proteins↗

Insurability of the adolescent and young adult with heart disease. Report from the Fifth Conference on Insurability, October 3-4, 1991, Columbus, Ohio.

By the mid-1990s there will be more than 500,000 young adults in the United States over the age of 21 with a cardiac malformation. Presently more than half of this population is denied insurance coverage entirely or in part because of their preexisting condition. Because some did not have coverage and because of uncertainty about whom to see for their cardiology care, patients assessed in NHS-II who were evaluated by their physician on an annual basis before the age of 21 were seen by a cardiologist only every 10 years after the age of 21. However, they have been shown by NHS-II to be well-educated, productive in the workplace, and to share an equal place in society with the general population. Their health-care costs are decidedly lower after the age of 21 than before. This group represents a microcosm of a general society of more than 37 million Americans who, for various reasons, are not insured. Dr. Wiener described an American health-care system in crisis. Smaller companies are no longer able to afford health insurance for all their employees, especially for those with preexisting conditions, because of an industry pricing concept based on a claims-experience standard rather than a community standard. The insurance industry, the government, and patients are demanding medical cost-containment. Health-care costs, 12.2% of the gross national product in 1990, are climbing, and no end to this increase is presently in sight.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Interaction modification among decomposers impairs ecosystem processes in lead-polluted soil.

Environmental pollution by toxicants is generally believed to lead both to direct toxic effects and indirect effects via altered species interactions in a stressed community. We examined how contamination of a coniferous forest soil floor by lead (from an abandoned shooting range area) may alter trophic interactions and decomposition processes. We constructed laboratory microcosms containing microbial-based food webs with various trophic structures: microbes alone; microbes with microbivorous-detritivorous enchytraeid worms (Cognettia sphagnetorum, a potential keystone species of boreal forest soil); and microbes, worms, and predatory mites (Lysigamasus spp.) together. The direct toxic effect of lead and the effectiveness of the predators in preying upon the worms were studied in separate experiments. Polluted humic soil (acid-extractable lead concentration was 2,720 +/- 349 mg/kg of dry soil [mean +/- standard error]) was toxic to enchytraeids, and predators effectively reduced worm populations. Positive density-dependent effects of worms on microbes and a trophic cascade from predatory mites to microbes, observed in the unpolluted humus, were not observed in the polluted humus, indicating pollution-induced interaction modification among organisms. Concomitantly, lead pollution altered the process of nutrient mineralization in the humic soil. However, causality and relative importance of direct toxicity and indirect effects of lead on the dynamics of the decomposer food web could not be pointed out. Examination of our results indicates that traditional food web models are incapable of predicting mechanisms that alter dynamics of pollution-stressed belowground food webs.

Animals↗

Pyrene mineralization capacity increases with compost maturity.

Experiments were conducted to determine the effects of composting or simple addition of compost to the mineralization of n-hexadecane, pyrene and benzo(a)pyrene in soil. Soil (contaminated or clean) was composted with maple leaves and alfalfa. Samples from different composting phases were spiked with radiolabeled and cold n-hexadecane, pyrene or benzo(a)pyrene, placed in aerated microcosms at different temperatures, and monitored for mineralization. It was determined that neither composting nor the addition of compost had any effect on n-alkane or benzo(a)pyrene mineralization. In contrast, the pyrene mineralization rate increased dramatically with the amount of time that soil had been composted. Highest pyrene mineralization rates and extents (more than 60% after 20 days) were obtained when pyrene was in contact with composted soil from the curing stage. Neither thermophiles (55 degrees C) nor fungi were responsible for pyrene mineralization.

Air↗

[Measuring the electricity frequency properties of blood].

In order to understand the electricity frequency specialties of blood, we have developed a wide frequency electricity characteristic testing system and used it to test the amplitude frequency property and phase frequency property of the blood in different states and constituents at 1 Hz to 20 MHz. Further analysis on the results of tests helped us know some important properties of blood at even more microcosmic levels from a new angle. Meanwhile, some problems and considerations on the improvement of the electricity model of biotic tissue and blood were pointed out. (1) From 1 Hz to 5 KHz, the impedance of blood descended 99%. (2) Simple equivalent circuit of resistance and capacitance must be used in series equivalent but not in usual parallel connection equivalent. (3) Experiment indicated, equivalent circuits of blood need more analysis, because simple equivalent circuit of resistance and capacitance is liable to gross error. (4) When the three element model is used for measuring the resistance of inside liquid, capacitance of cell membrane and the resistance of outside liquid of blood, the three testing frequencies must be very similar.

Blood↗

Anaerobic benzene biodegradation linked to nitrate reduction.

Benzene oxidation to carbon dioxide linked to nitrate reduction was observed in enrichment cultures developed from soil and groundwater microcosms. Benzene biodegradation occurred concurrently with nitrate reduction at a constant ratio of 10 mol of nitrate consumed per mol of benzene degraded. Benzene biodegradation linked to nitrate reduction was associated with cell growth; however, the yield, 8.8 g (dry weight) of cells per mol of benzene, was less than 15% of the predicted yield for benzene biodegradation linked to nitrate reduction. In experiments performed with [14C]benzene, approximately 92 to 95% of the label was recovered in 14CO2, while the remaining 5 to 8% was incorporated into the nonvolatile fraction (presumably biomass), which is consistent with the low measured yield. In benzene-degrading cultures, nitrite accumulated stoichiometrically as nitrate was reduced and then was slowly reduced to nitrogen gas. When nitrate was depleted and only nitrite remained, the rate of benzene degradation decreased to almost zero. Based on electron balances, benzene biodegradation appears to be coupled more tightly to nitrate reduction to nitrite than to further reduction of nitrite to nitrogen gas.

Anaerobiosis↗

Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments.

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

Comamonas↗

[Effects of nitrogen source and aeration mode on algae growth in freshwater].

Aquarium microcosms were used to study the effects of nitrogen source and aeration mode on the growth and species changes of algae in freshwater. Nitrate nitrogen(NO3(-) -N) and ammonia nitrogen(NH4(+) -N) were used as nitrogen sources. For each nitrogen source, four modes of aeration were selected, including control, continuous aeration, aeration during the day, and aeration at night. In the early stage of the experiment, algae in the NH4(+) -N treatment experiment grew well. In the later stage, algae in the NO3(-) -N treatment experiment grew better. For different aeration modes, continuous aeration show varied effects on algae growth in the two nitrogen source treatments. Day-only aeration had little effect on algae growth. Night-only aeration inhibited algae growth considerably. In NH(+) -N treatments, cyanophyta became dominant species easily. In contrast, chlorophyta dominated in NO3(-) -N treatments.

Ammonia↗

Carbon isotopes as a tool to evaluate the origin and fate of vinyl chloride: laboratory experiments and modeling of isotope evolution.

Accumulation of vinyl chloride (VC) is often a main concern at sites contaminated with chlorinated ethenes and ethanes due to its high toxicity. Since there can be several possible sources of VC and ethene at such sites, assessing the origin and fate of VC can be complicated. Aim of this study was to evaluate carbon isotope fractionation associated with various anaerobic processes that lead to the production of VC and ethene in view of using isotopes to evaluate the origin and fate of these compounds in groundwater. Microcosms were constructed using sediments and groundwater from a contaminated site and amended with potential precursors for VC and ethene production. In the microcosms with dichloroethene isomers, sequential reductive dechlorination was observed, and isotopic enrichmentfactors of -19.9 +/- 1.5 per thousand for cis-1,2-dichloroethene, -30.3 +/- 1.9 per thousand for trans-1,2-dichloroethene, and -7.3 +/- 0.4 per thousand for 1,1-dichloroethene were obtained. In microcosms with chlorinated ethanes, 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) were predominantly transformed by dichloroelimination to ethene and VC, respectively, and enrichmentfactors of -32.1 +/- 1.1 per thousand for 1,2OCA and -2.0 +/- 0.2 per thousand for 1,1,2-TCA were observed. Except for 1,1,2-TCA, a strong 13C enrichment in each of the potential precursor of VC was observed, which opens the possibility to trace the origin of VC based on the isotope ratio of potential precursors. Furthermore, it was possible to model the isotope evolution of VC present as substrate or intermediate product as a function of time. The study demonstrates that carbon isotope ratios can potentially be used for qualitative and possibly quantitative evaluation of the origin and fate of VC at sites with complex contaminant mixtures.

Carbon Isotopes↗

Biodegradation of the nitramine explosive CL-20.

The cyclic nitramine explosive CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) was examined in soil microcosms to determine whether it is biodegradable. CL-20 was incubated with a variety of soils. The explosive disappeared in all microcosms except the controls in which microbial activity had been inhibited. CL-20 was degraded most rapidly in garden soil. After 2 days of incubation, about 80% of the initial CL-20 had disappeared. A CL-20-degrading bacterial strain, Agrobacterium sp. strain JS71, was isolated from enrichment cultures containing garden soil as an inoculum, succinate as a carbon source, and CL-20 as a nitrogen source. Growth experiments revealed that strain JS71 used 3 mol of nitrogen per mol of CL-20.

Aniline Compounds↗

Evolution of specialists in an experimental microcosm.

The impact of adaptation on the persistence of a balanced polymorphism was explored using the lactose operon of Escherichia coli as a model system. Competition in chemostats for two substitutable resources, methylgalactoside and lactulose, generates stabilizing frequency-dependent selection when two different naturally isolated lac operons (TD2 and TD10) are used. The fate of this balanced polymorphism was tracked over evolutionary time by monitoring the frequency of fhuA-, a linked neutral genetic marker that confers resistance to the bacteriophage T5. In four out of nine chemostats the lac polymorphism persisted for 400-600 generations when the experiments were terminated. In the other five chemostats the fhuA polymorphism, and consequently the lac operon polymorphism, was lost between 86 and 219 generations. Four of 13 chemostat cultures monomorphic for the lac operon retained the neutral fhuA polymorphism for 450-550 generations until they were terminated; the remainder became monomorphic at fhuA between 63 and 303 generations. Specialists on each galactoside were isolated from chemostats that maintained the fhuA polymorphism, whether polymorphic or monomorphic at the lac operon. Strains isolated from three of four chemostats in which the lac polymorphism was preserved had switched their galactoside preference. Most of the chemostats where the fhuA polymorphism was lost also contained specialists. These results demonstrate that the initial polymorphism at lac was of little consequence to the outcome of long-term adaptive evolution. Instead, the fitnesses of evolved strains were dominated by mutations arising elsewhere in the genome, a fact confirmed by showing that operons isolated from their evolved backgrounds were alone unable to explain the presence of both specialists. Our results suggest that, once stabilized, ecological specialization prevented selective sweeps through the entire population, thereby promoting the maintenance of linked neutral polymorphisms.

Bacterial Outer Membrane Proteins↗

A review of in situ measurement of organic compound transformation in groundwater.

Laboratory assessments of the rate of degradation of organic compounds in groundwater have been criticized for producing unrepresentative results. The potential for organic compounds to be transformed in groundwater has been measured using in situ methods, which avoid problems of attempting to duplicate aquifer conditions in the laboratory. In situ assessments of transformation rates have been accomplished using transport studies and in situ microcosms (ISMs); a review of these methods is given here. In transport studies, organic solutes are injected into an aquifer and the concentrations are monitored as they are transported downgradient. The change in mass of a solute is determined by the area contained under the breakthrough curve (plot of concentration versus time). ISMs isolate a portion of the aquifer from advective flow and act as in situ batch reactors. Experiments using ISMs involve removing water from the ISM, amending it with the solutes of interest, re-injecting the amended water, and monitoring the solute concentrations with time. In both transport and ISM studies, the loss of organic solutes from solution does not allow a distinction to be made between sorptive, abiotic and biotic transformation losses. Biological activity can be chemically suppressed in ISMs and the results from those experiments used to indicate sorption and abiotic loss. Transformation products may be monitored to provide additional information on transformation mechanisms and rates.

Adsorption↗

Enhanced PCE dechlorination by biobarrier systems under different redox conditions.

The industrial solvent tetrachloroethylene (PCE) is among the most ubiquitous chlorinated compounds found in groundwater contamination. The objective of this study was to evaluate the (1) feasibility of enhancing PCE biodegradation using cane molasses and sludge cakes as the primary substrates under methanogenic and iron reducing conditions, and (2) potential of installation a sludge cake/cane molasses biobarrier to clean up PCE-contaminated aquifers. The biodegradability of sludge cake (from secondary wastewater treatment system) and cane molasses was tested using bioavailability experiments. Results show that biodegradable materials were released from sludge cake/cane molasses and utilized by microbial consortia. Based on the chemical oxygen demand (COD) tests, approximately 28 and 248 mg of biodegradable COD can be released from 1g of sludge cake and 1g of cane molasses under anaerobic conditions, which have the potential to convert 70 and 620 mg of PCE to ethylene (ETH), respectively. Reductive dechlorination was evaluated using microcosms containing primary substrates (sludge cake/cane molasses) and inocula (aquifer sediments). Results indicate that sludge cake and cane molasses can serve as the diffusion sources of primary substrates, and enhance the reductive dechlorination of PCE under methanogenic processes. However, results from this study were not sufficient enough to show that reductive dechlorination of PCE would occur under iron-reducing conditions. This indicates that more studies need to be performed to further evaluate the role of iron reduction on the PCE dechlorination. Results reveal that it is feasible and applicable to install a sludge cake or cane molasses biobarrier to clean up PCE contaminated aquifers. From an engineering point of view, the sludge cake/cane molasses biobarrier has the potential to become an environmentally and economically acceptable technology for PCE bioremediation.

Biodegradation, Environmental↗

Attenuation mechanisms of N-nitrosodimethylamine at an operating intercept and treat groundwater remediation system.

The North Boundary Containment System (NBCS), an intercept-and-treat system, was established at Rocky Mountain Arsenal (RMA), Commerce City, CO, to remove low-level organic contaminants from a groundwater plume exiting RMA to the north and northwest. N-nitrosodimethylamine (NDMA) was detected in groundwater collected from the dewatering and recharge zones of the NBCS system. Concern over the fate of NDMA, in terms of potentially exiting the boundaries of the arsenal, prompted an investigation to evaluate potential attenuation mechanisms for NDMA within the alluvial aquifer system and within the NBCS itself. Groundwater, soil, and granular activated carbon (GAC) samples were taken from key locations in the NBCS system. Soil and GAC samples were assayed for sorption kinetics and for adsorption and desorption properties using 14C-labeled NDMA. NDMA biodegradation experiments were conducted by following 14CO(2) evolution from 14C-labeled NDMA in soils and GAC samples under aerobic and anaerobic conditions. The sorptive capacity of the site soils for NDMA was insignificant. Furthermore, the adsorption of the NDMA by the soil was almost completely reversible. Evaluation of the degradation potential of the native microbial consortia indicated a high level of NDMA mineralization when measured using bench-scale microcosms. The native consortia had capability to mineralize the NDMA under both aerobic and anaerobic incubations, indicating facultative characteristics. Testing of the local groundwater chemistry revealed that the area of the aquifer of interest was microaerobic and neutral in pH. These conditions were optimal for NDMA removal. While sorption was insignificant, degradation was a significant attenuation mechanism, which may be the reason that no NDMA has migrated off-site. This gives rise to the potential of a long-term sink for attenuating NDMA within the recharge zone of the treatment system.

Adsorption↗

Impact of lime, nitrogen and plant species on bacterial community structure in grassland microcosms.

A microcosm-based approach was used to study impacts of plant and chemical factors on the bacterial community structure of an upland acidic grassland soil. Seven perennial plant species typical of both natural, unimproved (Nardus stricta, Agrostis capillaris, Festuca ovina and F. rubra) and fertilized, improved (Holcus lanatus, Lolium perenne and Trifolium repens) grasslands were either left unamended or treated with lime, nitrogen, or lime plus nitrogen in a 75-day glasshouse experiment. Lime and nitrogen amendment were shown to have a greater effect on microbial activity, biomass and bacterial ribotype number than plant species. Liming increased soil pH, microbial activity and biomass, while decreasing ribotype number. Nitrogen addition decreased soil pH, microbial activity and ribotype number. Addition of lime plus nitrogen had intermediate effects, which appeared to be driven more by lime than nitrogen. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed that lime and nitrogen addition altered soil bacterial community structure, while plant species had little effect. These results were further confirmed by multivariate redundancy analysis, and suggest that soil lime and nitrogen status are more important controllers of bacterial community structure than plant rhizosphere effects.

Analysis of Variance↗

Ecological cultivation ark (ECA) project--mutation and evolution of micro-organisms in space.

Ecological cultivation capsules (ECC), that is a materially sealed microcosm. composed of primary producers, consumers and bacteria as a decomposer were developed in order to cultivate bacteria without any artificial operation for long duration more than 10 years in space. It is planned to be left on the space station to study the process that bacteria in MIR space station had acquired their resistance to cosmic ray radiation as well as ultra-violet light. As contrasted with the space experiment, bacteria are cultivating in the ECC on the ground to trace the changes of bacteria under the simulated radiation dose in Earth orbit.

Bacillus subtilis↗