Roy M. Ventullo

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.

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 10.2×105 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.

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.

Phosphorous limitation in an arctic river biofilm—A whole ecosystem experiment

Abstract A fourth-order arctic river was experimentally enriched with phosphate (7.7 ± 7.0 μ g 1−1) to determine the effect of such a loading (equivalent to a community of 10,000 people) upon the trophically important biofilm. The effect upon a light-grown biofilm (an autotrophic/heterotrophic assemblage) and a dark-grown biofilm (predominantly heterotrophic assemblage) was determined after 28 days of colonization. Seven attributes of the biofilms were monitored, 2 autotrophic indices, chlorophyll α, [14C]HCO3 incorporation into lipids and 5 heterotrophic indices; [14C]acetate incorporation into lipids, metabolic heat output, turn-over times of microbially labile glucose and glutamate and mineralization of microbially recalcitrant ring-labelled [14C]hydroxybenzoic acid. The findings showed that the addition of phosphorus resulted in a substantial stimulation of both autotrophic and heterotrophic processes suggesting that arctic rivers of this type would be liable to cultural eutrophication.

Denitrification potential of epilithic communities in a lotic environment

Denitrification potentials of epilithic microbial populations were assessed using the acetylene inhibition method, in which acetylene is used to block the reduction of nitrous oxide (N2O) to nitrogen (N2). Samples of the epilithic community were incubated in filtered river water containing modified Bushnell-Haas salts, glycerol, and yeast extract—under aerobic (0.2 atm O2) and anaerobic (0.2 atm He) acetylene atmospheres. N2O was produced under both atmospheres only if exogenous nitrate of nitrite was added. Denitrification potentials were typically higher when nitrite was the added electron acceptor. The rates of denitrification were temperature-and carbon-dependent and the maximum rate, 8.53 μg N2O−N per cm2 per day occurred at 23°C when nitrite was the electron acceptor.

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.

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.

Response of Aquatic Microbial Communities to Surfactants

The response of microbial communities to three classes of surfactants was studied in a stream and lake ecosystem. Dodecyltrimethylammonium chloride (TMAC, cationic) had no effect on bacterial biomass or carbon turnover inthe sediment, water column, or epilithon in a first-order stream that was continuously dosed with TMAC for 23 days. Biodegradation of TMAC was evident at all sites and showed a time-dependent increase only at the highest (0.25 mg/L) dose site. The effects of long-chain nonionic (linear primary alcohol ethoxylate: Neodol 45-7) and anionic (linear alkylbenzene sulfonate) surfactants were studied in situ in Acton Lake, Ohio. The chemicals were tested over a concentration range of 0.001 to 10 mg/L in both short-term (3 h) and long-term (21 day) exposures. Neither of the chemicals had averse effects on the microbial biomass as measured by acridine orange direct counts. In the acute test of these chemicals, decreased turnover of glucose was noted in the concentration range of 0.1 to 10 mg/L surfactant. However, in chronic tests (21 day) the communities recovered, and carbon turnover returned to preexposure levels. Chronic exposure also resulted in an adaptive response as indicated by increased degradation of the chemicals after exposure. Our studies indicate that exposure of lake and stream microbial communities to surfactants had no long-term detrimental effects on bacterial heterotrophy and resulted in increased biodegradation of these materials.