Transformation of a tundra river from heterotrophy to autotrophy by addition of phosphorus.

Continuous enrichment of an arctic river with only 10 parts per billion phosphate-phosphorus caused an immediate growth of attached algae for more than 10 kilometers downstream, showing that phosphorus alone limited photosynthesis. As a result of the increased photosynthesis, there was an increase in bacterial activity in films on rocks on the bottom of the stream. The major source of energy became the photosynthetic carbon fixed in the stream rather than the organic material entering from the surrounding tundra, and the overall metabolism of the stream shifted from heterotrophy to autotrophy. An increase in the size and developmental stage of some of the dominant aquatic insects illustrates the food limitation in this nutrient-poor habitat.

The ubiquitin-ligase system in Trypanosoma brucei brucei.

The question of whether the African trypanosome Trypanosoma brucei brucei utilizes a ubiquitin-ligase system to conjugate the 8,500-dalton protein ubiquitin to other proteins has not been investigated. Using 125I-labeled ubiquitin and gel electrophoresis (sodium dodecyl sulfate and acetic acid, urea, Triton X-100), we looked for the incorporation of label into proteins larger than ubiquitin to determine ubiquitin-ligase system activity in cytosolic and nuclear lysates of long slender, intermediate, and short stumpy bloodstream-form trypanosomes. We present data suggesting that there is cytosolic activity of a ubiquitin-ligase system in all three bloodstream forms of T. brucei brucei. There are indications that the three bloodstream forms of T. brucei brucei differ in their cytosolic ubiquitin-ligase system activity. Our assay showed no activity of this system in the nucleus of T. brucei brucei. Further studies on the ubiquitin-ligase system in T. brucei brucei may define differences between the three bloodstream forms, the parasite, and its host, leading to development of novel chemotherapeutic strategies.

Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA.

Microbial growth rates in subsurface sediment from three sites were measured using incorporation of tritiated thymidine into DNA. Sampling sites included Lula, Oklahoma, Traverse City, Michigan, and Summit Lake, Wisconsin. Application of the thymidine method to subsurface sediments required (1) thymidine concentrations greater than 125 nM, (2) incubation periods of less than 4 hours, (3) addition of SDS and EDTA for optimum macromolecular extraction, and (4) DNA purification, in order to accurately measure the rate of thymidine incorporation into DNA. Macromolecule extraction recoveries, as well as the percentage of tritium label incorporated into the DNA fraction, were variable and largely dependent upon sediment composition. In general, sandy sediments yielded higher extraction recoveries and demonstrated a larger percentage of label incorporated into DNA than sediments that contained a high silt-clay component. Reported results also indicate that the acid-base hydrolysis procedure routinely used for macromolecular fractionation in water samples may not be routinely applicable to the modified sediment procedure where addition of SDS and EDTA are required for macromolecule extraction. Growth rates exhibited by subsurface communities are relatively slow, ranging from 5.1 to 10.2×10(5) cells g(-1) day(-1). These rates are 2-1,000-fold lower than growth rates measured in surface sediments. These data lend support to the supposition that subsurface microbial communities are nutritionally stressed.

Mineralization of surfactants by the microbiota of submerged plant detritus.

In wetlands and canopied bodies of water, plant detritus is an important source of carbon and energy. Detrital materials possess a large surface area for sorption of dissolved organics and are colonized by a large and diverse microbiota. To examine the biodegradation of surfactants by these microorganisms, submerged oak leaves were obtained from a laundromat wastewater pond, its overflow, and a pristine control pond. Leaves were cut into disks and incubated in sterile water amended with 50 mug of C-labeled linear alkylbenzene sulfonate (LAS), linear alcohol ethoxylate, stearyltrimethyl ammonium chloride, distearyldimethyl ammonium chloride, benzoic acid, or mixed amino acids per liter. Sorption of the test compounds to the detritus and evolution of CO(2) were followed with time. All of the compounds sorbed to the detritus to various degrees, with LAS and stearyltrimethyl ammonium chloride the most sorptive and benzoic acid the least. All compounds were mineralized without a lag. With leaves from the laundromat wastewater pond, half-lives were 12.6 days for LAS, 8.4 days for linear alcohol ethoxylate, 14.2 days for stearyltrimethyl ammonium chloride, 1.0 days for benzoic acid, and 2.7 days for mixed amino acids. Mineralization of LAS and linear alcohol ethoxylate by control pond leaves was slower and exhibited an S-shaped rather than a typical first-order pattern. This study shows that detritus represents a significant site of surfactant removal in detritus-rich systems.

Kinetics of biodegradation of nitrilotriacetic acid (NTA) in an estuarine environment.

The effects of salinity and dissolved organic carbon (DOC) on the kinetics of biodegradation of nitrilotriacetic acid (NTA) were studied in a Canadian estuary with a prior history of NTA exposure. Kinetic parameters for degradation of 14C-labeled NTA, maximum velocity (Vmax) and first-order rate constant (k1), were estimated by nonlinear regression models from velocity and time-course plots, respectively. The distribution of bacteria with NTA-degrading capability was also determined at various salinities and DOC levels by the 14C-most-probable-number (14C-MPN) technique. In general, NTA degradation was rapid in estuarine water over the range of salinities and DOC levels tested. Mean Vmax and k1 values (+/- standard deviation) across several sampling periods averaged 4753 +/- 2849 ng liter-1 hr-1 and 0.32 +/- 0.19 day-1, respectively. The estimated half-life for NTA degradation in estuarine water, based on the mean k1 value, was approximately 2 days. Degradation rates for NTA were relatively insensitive to changes in salinity or DOC values, and neither of these two parameters had significant effects on NTA degradation at the microbial community or individual cell levels. Based on 14C-MPN results, the distribution of estuarine bacteria capable of degrading NTA was broad and not related to salinity or DOC levels. The NTA degraders appeared to be indigenous members of the estuarine microbial community and not wastewater-associated microorganisms.

Adaptation of aquatic microbial communities to quaternary ammonium compounds.

The effects of long-chain (C(12) to C(18)) quaternary ammonium compounds (QACs) on the density, heterotrophic activity, and biodegradation capabilities of heterotrophic bacteria were examined in situ in a lake ecosystem. Monoalkyl and dialkyl substituted QACs were tested over a range of concentrations (0.001 to 10 mg/liter) in both acute (3 h) and chronic (21 day) exposures. In general, none of the QACs tested had significant adverse effects on bacterial densities in either acute or chronic studies. However, significant decreases in bacterial heterotrophic activity were noted in acute studies at QAC concentrations from 0.1 to 10 mg/liter. Chronic exposure of lake microbial communities to a specific monoalkyl QAC resulted in an adaptive response and recovery of heterotrophic activity. No-observable-effect level in the adapted populations was >10 mg/liter. Chronic exposure also resulted in significant increases in the number and activity of bacteria capable of biodegrading the material. The increase in biodegradation capability was observed at low (microgram per liter) concentrations which are approximately the same as realistic environmental levels. In general, our studies indicated that exposure of lake microbial communities to QACs results in the development of adapted communities which are less sensitive to potential toxic effects and more active in the biodegradation of these materials.

Degradation of olestra, a non caloric fat replacer, by microorganisms isolated from activated sludge and other environments.

Olestra is a non-caloric fat substitute consisting of fatty acids esterified to sucrose. Previous work has shown that olestra is not metabolized in the gut and is excreted unmodified in human feces. To better understand the fate of olestra in engineered and natural environments, aerobic bacteria and fungi that degrade olestra were enriched from sewage sludges, soils and municipal solid waste compost not previously exposed to olestra. Various mixed and pure cultures were obtained from these sources which were able to utilize olestra as a sole carbon and energy source. The fastest growing enrichment was obtained from activated sludge and later yielded an olestra-degrading pure culture of Pseudomonas aeruginosa. This mixed culture extensively degraded both 14C-fatty acid labeled olestra and 14C-sucrose labeled olestra during 8 days of incubation. Longer-term incubation with pure cultures of P.aeruginosa demonstrated that > 98% of 14C-sucrose labeled olestra and > 72% of 14C-fatty acid labeled olestra was mineralized to CO2 after 69 days. These results indicate that olestra degraders are present in environments not previously exposed to olestra and that olestra can serve as a sole carbon and energy source. Furthermore, a common bacterial species was isolated from activated sludge and shown to have the ability to degrade olestra.

Regulation and energization of nitrate transport in a halophilic Pseudomonas stutzeri.

Nitrate transport and its regulation by oxygen was studied in denitrifying halophilic Pseudomonas stutzeri, strain Zobell, and a Tn-5 transposon nitrite reductase mutant of this organism. The rate of nitrate transport was found to be 130 nanomoles nitrate min-1 mg protein-1 and 150 nanomoles nitrate min-1 mg protein-1 in the wildtype and the nitrite reductase mutant respectively as compared to 26.4 nanomoles nitrate min-1 mg protein-1 in a non-halophilic Pseudomonas stutzeri. Asparagine was found to be the best energy source for nitrate uptake. The ratio of nitrate import to nitrite export was established by measuring extracellular nitrate and nitrite concentrations using HPLC/UV analysis. There was a 1.3:1 (NO3-/NO2-) exchange. High concentrations of nitrate during growth was found to have a negative effect on nitrite metabolism. Oxygen exerted an inhibitory effect on nitrate uptake which was reversible and more pronounced in cells grown on low concentrations of nitrate compared to cells grown at high concentrations of nitrate.

Spatial distribution of microbial biomass, activity, community structure, and the biodegradation of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) in the subsurface.

The vertical distribution of microbial biomass, activity, community structure and the mineralization of xenobiotic chemicals was examined in two soil profiles in northern Wisconsin. One profile was impacted by infiltrating wastewater from a laundromat, while the other served as a control. An unconfined aquifer was present 14 meters below the surface at both sites. Biomass and community structure were determined by acridine orange direct counts and measuring concentrations of phospholipid-derived fatty acids (PLFA). Microbial activity was estimated by measuring fluorescein diacetate (FDA) hydrolysis, thymidine incorporation into DNA, and mixed amino acid (MAA) mineralization. Mineralization kinetics of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) were determined at each depth. Except for MAA mineralization rates, measures of microbial biomass and activity exhibited similar patterns with depth. PLFA concentration and rates of FDA hydrolysis and thymidine incorporation decreased 10-100 fold below 3 m and then exhibited little variation with depth. Fungal fatty acid markers were found at all depths and represented from 1 to 15% of the total PLFAs. The relative proportion of tuberculostearic acid (TBS), an actinomycete marker, declined with depth and was not detected in the saturated zone. The profile impacted by wastewater exhibited higher levels of PLFA but a lower proportion of TBS than the control profile. This profile also exhibited faster rates of FDA hydrolysis and amino acid mineralization at most depths. LAS was mineralized in the upper 2 m of the vadose zone and in the saturated zone of both profiles. Little or no LAS biodegradation occurred at depths between 2 and 14 m. LAE was mineralized at all depths in both profiles, and the mineralization rate exhibited a similar pattern with depth as biomass and activity measurements. In general, biomass and biodegradative activities were much lower in groundwater than in soil samples obtained from the same depth.

Characterization of the sediment bacterial community in groundwater discharge zones of an alkaline fen: a seasonal study.

The cell density, activity, and community structure of the bacterial community in wetland sediments were monitored over a 13-month period. The study was performed at Cedar Bog, an alkaline fen. The objective was to characterize the relationship between the sediment bacterial community in groundwater upwelling zones and the physical and chemical factors which might influence the community structure and activity. DNA, protein, and lipid synthesis were measured at three different upwelling zones by using [3H]thymidine, [14C]leucine, and [14C]glucose incorporation, respectively. The physiological status (apparent stress) of the consortium was assessed by comparing [14C]glucose incorporation into membrane and that into storage lipids. Bacterial cell density was determined by acridine orange direct counts, and gross bacterial community structure was determined by bisbenzimidazole-cesium chloride gradient analysis of total bacterial community DNA. Both seasonal and site-related covariation were observed in all estimates of bacterial biomass and activity. Growth rate estimates and cell density peaked in late July at 2.5 x 10(8) cells/g/day and 2.7 x 10(9) cells/g, respectively, and decreased in December to 2.0 x 10(7) cells/g/day and 1.5 x 10(9) cells/g, respectively. Across sites, membrane-to-storage-lipid ratios were generally highest in late spring and peaked in September for one site. Overall, the data indicate dynamic seasonal differences in sediment bacterial community activity and physiology, possibly in response to changing physical and chemical environmental factors which included the C/N/P ratios of the perfusing groundwater. By contrast, total cell numbers were rather constant, and community structure analysis indicated that the overall community structure was similar throughout the study.

Heterotrophic activity and biodegradation of labile and refractory compounds by groundwater and stream microbial populations.

The bacteriology and heterotrophic activity of a stream and of nearby groundwater in Marmot Basin, Alberta, Canada, were studied. Acridine orange direct counts indicated that bacterial populations in the groundwater were greater than in the stream. Bacteria that were isolated from the groundwater were similar to species associated with soils. Utilization of labile dissolved organic material as measured by the heterotrophic potential technique with glutamic acid, phenylalanine, and glycolic acid as substrates was generally greater in the groundwater. In addition, specific activity indices for the populations suggested greater metabolic activity per bacterium in the groundwater. 14C-labeled lignocellulose, preferentially labeled in the lignin fraction by feeding Picea engelmannii [14C]phenylalanine, was mineralized by microorganisms in both the groundwater and the stream, but no more than 4% of the added radioactivity was lost as 14CO2 within 960 h. Up to 20% of [3'-14C]cinnamic acid was mineralized by microorganisms in both environments within 500 h. Both microbial populations appear to influence the levels of labile and recalcitrant dissolved organic material in mountain streams.

Stream periphytic biodegradation of the anionic surfactant C12-alkyl sulfate at environmentally relevant concentrations.

The effects of continuous exposure to C12-alkyl sulfate on a periphytic microbial community were determined in an 8-week stream mesocosm study. C12-alkyl sulfate concentrations ranged from environmentally relevant (< 10-20 micrograms/liter) to unrealistically high concentrations (> 1500 micrograms/liter). Endpoints evaluated included turnover rates, bacterial cell density, heterotrophic mixed amino acid uptake, and fatty acid profile evaluations. Predosed periphyton demonstrated a mean turnover rate for C12-alkyl sulfate of 0.08/hr. During the 8-week dosing period, a significant increase in mean turnover rates was observed in streams dosed with > or = 61 micrograms C12-alkyl sulfate/liter, despite a 10 degrees C drop in stream temperature. A significant correlation between turnover rate and C12-alkyl sulfate concentration was also observed. While bacterial cell density increased during the study, it was determined that the biodegradation acclimation to C12-alkyl sulfate was not biomass-specific. Likewise, bacterial activity generally increased over the study, but it did not correlate with either biodegradation or bacterial cell density. Lastly, phospholipid fatty acid profiles indicate that a shift in the microbial community occurred in the high-dose stream as opposed to the control stream. This study demonstrates that C12-alkyl sulfate is rapidly degraded and induces a biodegradative acclimation response at environmentally relevant concentrations.

The effects of chlorinated municipal sewage and temperature on the abundance of bacteria in the Sheep River, Alberta.

The intersite and temporal differences in the abundance of sessile and planktonic bacteria subjected to a chlorinated municipal sewage discharge and a thermal discharge were investigated in the Sheep River, Alberta, from January through December 1978. Significantly higher densities of both sessile and planktonic cells generally occurred within the chlorinated effluent plume. Higher numbers of sessile bacteria were also found below the thermal discharge, with maximum densities occurring in the late winter-early spring and minimum densities following mountain runoff. The higher numbers of sessile bacteria in the chlorinated plume was attributed to nutrient enrichment. These results indicate that chlorinated sewage effluent has no adverse effects on the abundance of sessile bacteria.