Effects of storage on measurements of potential microbial activities in stream fine benthic organic matter.

Sample storage can significantly influence measured microbial activities in stream fine benthic organic matter (FBOM), possibly confounding effects of sample variability and short-term changes in activity. Denitrification potential, acetylene reduction and respiration rates, mineralizable N and extractable ammonium concentrations, and beta-glucosidase and phosphatase enzyme activities of FBOM from first-order mountain streams in the western Oregon Cascade Mountains were assayed at various times after collection to determine potential storage effects. Denitrification potential, phosphatase activity, and extractable ammonium remained stable over a minimum of 11 h of storage at 5 degrees C. Mineralizable N concentrations, respiration rates, and beta-glucosidase activity all decreased within 12 h of collection. Results varied for acetylene reduction. Once assay conditions were established, denitrification potential and respiration rates were linear with incubation time. Based on paired t-tests, measures of acetylene reduction, denitrification potential, respiration rate, beta-glucosidase activity, and phosphatase activity were generally similar at a 1-wk interval within the same stream reaches.

Long-term starvation-induced loss of antibiotic resistance in bacteria.

Escherichia coli, Pseudomonas fluorescens, and aPseudomonas sp. strain 133B containing the pSa plasmid were starved in well water for up to 523 days. There were two patterns of apparent antibiotic resistance loss observed. InPseudomonas sp. strain 133B, there was no apparent loss of antibiotic resistance even after starvation for 340 days. InE. coli, by day 49 there was a ten-fold difference between the number of cells that would grow on antibiotic- and nonantibiotic-containing plates. However, over 76% of the cells that apparently lost their antibiotic resistance were able to express antibiotic resistance after first being resuscitated on non-selective media. By day 523, only 12% of these cells were able to express their antibiotic resistance after being resuscitated. After starvation for 49 days, cells that could not grow on antibiotic medium even after resuscitation, showed a permanent loss of chloramphenicol (Cm) resistance but retained resistance to kanamycin (Km) and streptomycin (Sm). Restriction enzyme digests show that a 2.5 to 3.0 Kb region from map location 12.5 to 15.5 Kb was deleted. This coincides with the 2.5 Kb reduction in plasmid size observed in 3 isolates that had lost antibiotic resistance after starvation for 49 days.

Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water.

Strains of enteric bacteria and pseudomonads containing plasmid R388::Tnl721 (Tpr, Tcr) or pRO101 (Hgr, Tcr) were starved for over 250 days in sterile well water to evaluate effects of starvation-survival on plasmid expression and maintenance. Viable populations dropped to between approximately 0.1 and 1% of the initial populations. Escherichia coli(pRO101) and Pseudomonas cepacia(pRO101) lost both viability and plasmid expression at a lower rate than strains containing R388::Tnl721. Three patterns of host-plasmid interaction were detected: (i) no apparent loss of plasmid expression, (ii) loss of plasmid expression on initial recovery with subsequent expression upon resuscitation, and (iii) loss of capability to produce functional plasmid resistance.

Observations on microbial percent respiration values in arctic and subarctic marine waters and sediments.

Percent respiration was measured in over 1,100 arctic and subarctic marine water and sediment samples using(14)C-labeled glucose and glutamate. These measurements were made at different times of the year in 4 regions. Percent respiration values were typically lower in regions where the waters of large rivers mixed with seawater. They were also lower in sediments and in waters collected near the bottom than in surface waters. They were higher in winter arctic waters than water samples collected in the summer; however, a similar seasonal trend was not observed in subarctic waters. There were a number of studies in which there were significant positive rank correlations between percent respiration and salinity and between percent respiration and temperature. From what is known about the range of temperature and salinity encountered in samples collected during these studies and the results of temperature and salinity effects experiments, it was concluded that changes in these 2 variables did not explain the variation observed in percent respiration. Correlations between percent respiration and the inorganic nutrients PO4 (-3), NH4 (+) and NO3 (-) showed that of the 3 variables, only NO3 (-) showed relatively high correlations with all the same sign. From this it was concluded that there may be situations in which NO3 (-) levels may influence percent respiration in nearshore marine waters. It is also likely that qualitative characteristics of the available organic nutrients may also influence percent respiration levels. Although no organic nutrient data is available for statistical analysis, the patterns of percent respiration near river plumes and the relatively strong negative correlation often observed between uptake rates (heterotrophic activity) and percent respiration suggests that organic nutrients may be a factor in controlling percent respiration. It is suggested that there are situations in which percent respiration measurements may be used to document stress in natural microbial populations due to nutrient deficiencies.

Field observations of methane concentrations and oxidation rates in the southeastern bering sea.

Measurements of methane oxidation rates were made in southeastern Bering Sea water samples with [C]methane. The rate at which CO(2) evolved from samples exposed to one methane concentration was defined as the relative methane oxidation rate. Rate determinations at three methane concentrations were used to estimate methane oxidation kinetics. The rate constant calculated from the kinetics and the observed methane concentration in the same water sample were used to calculate an in situ methane oxidation rate and the turnover time. First-order kinetics were observed in essentially all experiments in which methane oxidation kinetics were measured. Relative methane oxidation rates were greater in waters collected at inshore stations than at the offshore stations and were greater in bottom samples than in surface samples. In most water samples analyzed, there was essentially no radioactivity associated with the cells. The resulting respiration percentages were therefore very high with a mean of >98%. These data suggest that most of the methane was used by the microflora as an energy source and that very little of it was used in biosynthesis. The relative methane oxidation rates were not closely correlated with methane concentrations and did not appear to be linked to either oxygen or dissolved inorganic nitrogen concentrations. However, there was a significant correlation with relative microbial activity. Our data suggest that the methane oxidizers were associated with the general microbial heterotrophic community. Since these organisms did not appear to be using methane as a carbon source, it is unlikely that they have been isolated and identified as methane oxidizers in the past.

Long-term effects of crude oil on uptake and respiration of glucose and glutamate in arctic and subarctic marine sediments.

The effects of crude oil on uptake and respiration (mineralization) of glucose and glutamate in marine sediments were investigated. After the sediments were treated with crude oil, they were replaced at or near the collection site by scuba divers. These sediments remained in situ until they were retrieved for analysis. Glucose and glutamate uptake rates were found to decrease, and the percent respired was found to increase in Arctic and subarctic marine sediments that had been exposed to fresh crude oil. These same changes were also observed when "weathered" crude oil was used and when untreated sediments were overlaid with oiled sediments. When the kinetics of glutamate uptake were determined, both the maximum potential uptake rate and the turnover time were significantly affected. A comparison between the proportion of glucose taken into the cells and that respired as CO(2) indicated that crude oil affected biosynthetic mechanisms. A study of sediments that had been exposed to crude oil for at least 5 months showed that glutamate transport into the cells was affected more extensively than biosynthetic mechanisms. In the initial months of exposure, bacterial concentrations and total adenylate concentrations were found to decrease in the presence of crude oil. Our data suggest that secondary productivity in the marine environment could be adversely affected by the presence of crude oil in marine sediments.

Field observations on the acute effect of crude oil on glucose and glutamate uptake in samples collected from arctic and subarctic waters.

The acute effects of crude oil on glucose uptake rates by marine microorganisms were studied in 215 water and 162 sediment samples collected from both arctic and subarctic marine waters. The mean percentage reduction of glucose uptake rates ranged from 37 to 58 in the water samples exposed to crude oil and from 14 to 36 in the sediment samples. Substrate uptake kinetic studies indicated that the observed reductions by microbial populations exposed to crude oil were caused by metabolic inhibition. The effect of crude oil was less in sediments than in the water samples, with the difference being significant at the P < 0.0002 level. With the exception of one sediment study, all of the differences observed in the uptake rates between treated and nontreated samples were statistically significant. A high degree of variability was observed in the degree which glucose and glutamate uptake rates were altered in water samples exposed to crude oil. In some cases, uptake rates were greater in the samples exposed to crude oil. Data on samples collected in Cook Inlet suggested that areas where pelagic microorganisms are most probably chronically exposed to crude oil are also the areas where the effects of crude oil on glucose uptake are the lowest. Two studies indicated that after pelagic populations are exposed to crude oil for several days, the heterotrophic population adjusts to the presence of crude oil.

Bacterial ecology of an old-growth douglas fir canopy.

Microbial populations associated with the major substrates of the canopy of a single 70 m old-growth Douglas fir were studied to determine potential activities. Seasonal samples from bark, foliage, epiphytic moss, lichens, and litter accumulations were collected to: (a) obtain population data, (b) isolate the major groups of microorganisms present, (c) measure enzymatic activities associated with cellulose and xylan degradation, and (d) examine the potential for nitrogen fixation. We tested 562 bacterial isolates for utilization of 25 compounds associated with the canopy substrates, and for activities in nitrogen and sulfur cycle transformations. Total bacterial populations, reflecting seasonal temperature and moisture conditions, were lowest on bark and foliage [21-266×10(3) colony-forming units (CFU/g)] and highest on moss and lodged litter (19-610×10(5) CFU/g). Lichens contained intermediate numbers of bacteria (3.3-270×10(5) CFU/g). The majority of the bacteria were classified as species ofArthrobacter, Bacillus, Flavobacterium, andXanthomonas. Isolates ofAlcaligenes (Achromobacter), Aeromonas, Chromobacterium, Micrococcus, andPseudomonas were less common. No measurable rates of nitrogen fixation attributable to free-living bacteria were detected by acetylene reduction. Eleven species in six genera of lichens containing a blue-green algal phycobiont showed positive acetylene reduction. One species,Lobaria oregana, accounted for 51% of the total lichen biomass of the canopy. Cellulase and xylanase activity was routinely detected in moss and litter samples, and less frequently in lichens. There was a strong correlation between the two activities for moss (r=0.94) and litter (r=0.81).

Isolation of a unique marine bacterium capable of growth on a wide variety of polysaccharides from macroalgae.

A unique marine bacterium has been isolated which can be cultured on a variety of polysaccharides and cell wall preparations from red and brown algae.