Characterization and distribution of esterase activity in activated sludge.

The location and activity of esterase enzymes in activated sludge from three municipal wastewater treatment plants were characterized using model substrates and denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) of particulate, freeze-thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze-thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C2-C18 monoesters of p-nitrophenol and C2-C8 diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C4) and decreased as the chain lengths increased beyond C8. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple bands were observed upon analysis of particulate and freeze-thaw fractions by non-denaturing PAGE in combination with activity staining using various alpha-naphthol ester substrates (C2-C8). Our results indicate that esterase enzymes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent for the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular material.

Biodegradation of [S,S], [R,R] and mixed stereoisomers of ethylene diamine disuccinic acid (EDDS), a transition metal chelator.

An in-depth biodegradation test program was executed on the hexadentate ligand Ethylene Diamine Di Succinate (EDDS). The EDDS structure contains two chiral carbon atoms, and has three stereoisomers ([R,R], [R,S]/[S,R], [S,S]). Our research has focused on the isomer mixture (i.e. 25%[S,S]; 25%[R,R]; 50%[S,R]/[R,S], as produced from the reaction of ethylene diamine with maleic anhydride) and on the single [S,S]- and [R,R]-isomers. Biodegradation screening of the 14C-labelled EDDS isomer mixture in a Batch Activated Sludge (BAS) test with various inocula revealed incomplete mineralization, up to ca. 65% after 28 days. N-(2-aminoethyl) aspartic acid (AEAA), probably the d-isomer, was identified as the major portion of the 14C-material remaining in solution. Further testing revealed that the [S,S]-isomer is rapidly and completely mineralized in all test systems. By contrast, [R,R]-EDDS remained undegraded in a Sturm (OECD 301B) test, but was very slowly biotransformed into the recalcitrant metabolite AEAA in a BAS test. The [S,R]/[R,S] form undergoes biotransformation to AEAA in both high and low biomass systems. In a sewage treatment simulation test (OECD 303) the steady state DOC removal of mixture-EDDS in a CAS test was limited to 25-35%, even after extensive pre-acclimation, while the [S,S]-isomer achieved nearly complete removal (96%). This study illustrates the importance stereospecificity may have on the biodegradation and metabolite formation of a chemical. A biodegradation scheme for the different EDDS stereoisomers is proposed.

A simplified modeling approach using microbial growth kinetics for predicting exposure concentrations of organic chemicals in treated wastewater effluents.

Various mathematical relationships have been used to assess exposure concentrations of organic chemicals when emissions occur via wastewater treatment. These relationships range from a simple removal factor calculation to more sophisticated approaches using kinetic based mathematical models. While these existing approaches have been used by decision makers to screen new chemicals for exposure assessments, they all have limitations in the predictive capabilities. Thus, a simplified modeling approach grounded in sound scientific fundamentals that utilizes relatively easy to obtain input parameters is needed. In this paper a simplified modeling approach that utilizes microbial growth kinetics was developed for predicting effluent concentrations in secondary biological wastewater treatment systems. Receiving water predicted exposure concentrations (PEC) are assessed by using a dilution factor. One advantage of this approach is that it allows for wastewater treatment plant effluent concentrations, and therefore receiving water exposure levels, to be predicted with a minimum amount of experimental data. It also provides quantitative data that can be used to assess the relative biodegradability of different chemicals for use in regulatory and risk assessment activities.

Impact of biodegradation test methods on the development and applicability of biodegradation QSARs.

The biodegradability of a substance depends on the structure and physical form of the substance, the time that has been available for acclimation, and the environmental conditions. Importantly, these later factors can be just as important as structure in determining the outcome of a biodegradation test. The development of appropriate QSARs for biodegradation and the ultimate value of the final QSAR depends on understanding these factors. This paper will describe what is known about the effect of test conditions on the results of biodegradation tests. The ability of these tests to reflect real environmental conditions will also be examined. Finally, we will discuss what we believe, in the light of this information, should be the goal of biodegradation QSARs and how these QSARs can be most appropriately used in fate assessments.

Microbial colonization and decomposition of carex litter in an arctic lake.

The decomposition and microbial colonization of Carex leaf litter were examined in an arctic lake in Alaska during the summer of 1978. Dried leaf segments in screen bags were placed at various locations and depths for 13 and 26 days. Weight loss varied from 24.15 to 33.56% and from 27.69 to 65.01% after 13 and 26 days, respectively. Abiotic controls lost approximately 19.5% with no subsequent change. Weight loss significantly correlated with microbial colonization as measured by alkaline phosphatase activity (r = 0.780), cellulase activity (r = 0.613), heterotrophic CO(2) fixation (r = 0.835), and acetate incorporation into microbial lipids (r = 0.618). Alkaline phosphatase activity correlated with cellulase activity (r = 0.889), and heterotrophic CO(2) fixation correlated with acetate incorporation into lipids (r = 0.712). Weight loss after 26 days inversely correlated with the logarithm of the depth of incubation regardless of whether incubation occurred on the sediment surface or in the water column. These findings suggest that a rapid initial period of microbial colonization is driven by nutrients derived from the litter and that the rate of these processes is controlled by a factor(s) inversely related to the logarithm of depth, such as light intensity, primary production, or turbulence.

Lignocellulose mineralization by arctic lake sediments in response to nutrient manipulation.

Mineralization of specifically labeled C-cellulose- and C-lignin-labeled lignocelluloses by Toolik Lake, Alaska, sediments was examined in response to manipulation of various environmental factors. Mineralization was measured by quantifying the amount of labeled CO(2) released from the specifically labeled substrates. Nitrogen (NH(4)NO(3)) and, to a greater degree, phosphorus (PO(4)) additions enhanced the mineralization of white pine (Pinus strobus) cellulose during the summer of 1978. Nitrogen and phosphorus together had no cumulative effect. During the summer of 1979, nitrogen or phosphorus alone had only a slight stimulatory effect on the mineralization of a sedge (Carex aquatilis) cellulose; however, together, they had a dramatic effect. This variable response of mineralization to nutrient addition between 1978 and 1979 was probably attributable to year-to-year variation in nutrient availability within the lake. Cellobiose addition and oxygen depletion inhibited the amount of pine cellulose mineralized. Whereas addition of nitrogen to oxygen-depleted treatments had limited effect, addition of phosphorus resulted in mineralizations equal to or greater than that of the controls. Nitrogen had no effect on mineralization of pine or Carex lignins. Phosphorus, however, inhibited mineralization of both lignins. With Carex lignin, the phosphorus inhibition occurred at a concentration as low as 0.1 muM. The antagonistic role of phosphorus in cellulose and lignin mineralizations may be of significance in understanding the increased proportion of lignin relative to cellulose in decomposing litter.

Preservation of estuarine sediments for lipid analysis of biomass and community structure of microbiota.

Various methods were tested for preserving estuarine sediments in the field before biochemical analysis of the microbiota. Total microbial biomass was determined as lipid phosphate (LP), and the fatty acids of the microbial lipids were used as indicators of community structure. Control samples were sieved to remove macroinvertebrates and plant materials and were extracted immediately in the field. Other samples were preserved both before and after sieving and stored for 5 days before analysis. Freezing resulted in a 50% decline in LP and significant decreases in many fatty acids. Refrigeration resulted in a 19% decrease in LP but no change in the fatty acids. Samples preserved with Formalin before sieving exhibited no significant change in LP but substantial increases in many fatty acids, which were probably derived from the macroinvertebrates. Sieved samples preserved with Formalin showed a 17 to 18% decline in LP but no change in the fatty acids. Ideally, samples should be sieved and extracted immediately in the field. However, short-term refrigeration and longer-term preservation of sieved samples with Formalin may be acceptable compromises.

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.

Improvements in and environmental applications of double-vial radiorespirometry for the study of microbial mineralization.

Several variations in the scintillation mixture and the filter paper arrangements for double-vial radiorespirometry were compared. Improved efficiencies (44%) and shorter response times were found by adding wetting agents and methanolic NaOH to the scintillation mixture in the filter paper. The scintillation chemicals used did not contain dioxane and were found to be nontoxic to the test microbiota in this system. Covering the inner reaction vial with aluminum foil minimized the reduction in counting efficiency when testing colored or dense environmental samples. Mineralization rates were determined with C-labeled glucose, acetate, and glutamate and [C]cellulose- and [C]lignin-labeled lignocellulose for composting cow manure, forest soil, and arctic lake sediment microbiota. This improved method can be used in a variety of procedures involving the measurement of microbial mineralizations of organic compounds. Since no liquid scintillation cocktail is used for counting, the radioactive wastes are aqueous or can be incinerated, making disposal easy.

Metabolism of the benzidine-based azo dye Direct Black 38 by human intestinal microbiota.

Benzidine-based azo dyes are proven mutagens and have been linked to bladder cancer. Previous studies have indicated that their initial reduction is the result of the azo reductase activity of the intestinal microbiota. Metabolism of the benzidine-based dye Direct Black 38 was examined by using a semicontinuous culture system that simulates the lumen of the human large intestine. The system was inoculated with freshly voided feces, and an active flora was maintained as evidenced by volatile fatty acid and gas production. Within 7 days after exposure to the dye, the following metabolites were isolated and identified by gas chromatography-mass spectrometry:benzidine, 4-aminobiphenyl, monoacetylbenzidine, and acetylaminobiphenyl. Benzidine reached its peak level after 24 h, accounting for 39.1% of the added dye. Its level began to decline, and by day 7 the predominant metabolite was acetylaminobiphenyl, which accounted for 51.1% of the parent compound. Formation of the deaminated and N-acetylated analogs of benzidine, which have enhanced mutagenicity and lipophilicity, previously has not been attributed to the intestinal microbiota.

Effects of petroleum hydrocarbons on plant litter microbiota in an arctic lake.

The effects of petroleum hydrocarbons on the microbial community associated with decomposing Carex leaf litter colonized in Toolik Lake, Alaska, were examined. Microbial metabolic activity, measured as the rate of acetate incorporation into lipid, did not vary significantly from controls over a 12-h period after exposure of colonized Carex litter to 3.0 ml of Prudhoe Bay crude oil, diesel fuel, or toluene per liter. ATP levels of the microbiota became elevated within 2 h after the exposure of the litter to diesel fuel or toluene, but returned to control levels within 4 to 8 h. ATP levels of samples exposed to Prudhoe Bay crude oil did not vary from control levels. Mineralization of specifically labeled C-[lignin]-lignocellulose and C-[cellulose]-lignocellulose by Toolik Lake sediments, after the addition of 2% (vol/vol) Prudhoe Bay crude oil, motor oil, diesel fuel, gasoline, n-hexane, or toluene, was examined after 21 days of incubation at 10 degrees C. Diesel fuel, motor oil, gasoline, and toluene inhibited C-[lignin]-lignocellulose mineralization by 58, 67, 67, and 86%, respectively. Hexane-treated samples displayed an increase in the rate of C-[lignin]-lignocellulose mineralization of 33%. C-[cellulose]-lignocellulose mineralization was inhibited by the addition of motor oil or toluene by 27 and 64%, respectively, whereas diesel fuel-treated samples showed a 17% increase in mineralization rate. Mineralization of the labeled lignin component of lignocellulose appeared to be more sensitive to hydrocarbon perturbations than was the labeled cellulose component.

Physical determinants of microbial colonization and decomposition of plant litter in an Arctic Lake.

Physical effects on the microbial colonization and decomposition of plant litter in an oligotrophic lake were studied in Toolik Lake, Alaska. Colonization, measured by adenosine triphosphate (ATP) analysis, and decomposition, measured by weight loss, were correlated with depth of incubation, light, and temperature. The effects of turbulence, light, and temperature were studied in microcosm experiments. A 10°C increase (above ambient) in temperature caused a doubling in the amount of microbial colonization and a 50% increase in decomposition. Light intensity had no quantitative effect on either colonization or decomposition. Turbulence experiments conducted in Toolik Lake showed significant differences in decomposition between natural turbulence and no turbulence. Elevated temperature (20°C, which was 10°C above ambient), high turbulence, and total darkness changed the composition of the litter microbiota, causing a shift from a bacterially dominated microbiota to one dominated by large filamentous forms, as revealed by scanning electron microscopy. This study shows the importance of these physical factors in determining the rate at which plant litter is colonized and degraded in aquatic environments.

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.

Biotransformation of 1-nitropyrene to 1-aminopyrene and N-formyl-1-aminopyrene by the human intestinal microbiota.

The nitropolycyclic aromatic hydrocarbon 1-nitropyrene (1-NP) is an environmental pollutant, a potent bacterial and mammalian mutagen, and a carcinogen. The metabolism of 1-NP by the human intestinal microbiota was studied using a semicontinuous culture system that simulates the colonic lumen. [3H]-1-Nitropyrene was metabolized by the intestinal microbiota to 1-aminopyrene (1-AP) and N-formyl-1-aminopyrene (FAP) as determined by high-performance liquid chromatography (HPLC) and mass spectrometry. Twenty-four hours after the addition of [3H]-1-NP, the formylated compound and 1-AP accounted for 20 and 80% of the total metabolism, respectively. This percentage increased to 66% for FAP after 24 h following 10 d of chronic exposure to unlabeled 1-NP, suggesting metabolic adaptation to 1-NP by the microbiota. Both 1-AP and FAP have been shown to be nonmutagenic towards Salmonella typhimurium TA98, which indicates that the intestinal microflora may potentially detoxify 1-NP.

Effect of mineral and organic soil constituents on microbial mineralization of organic compounds in a natural soil.

This research addressed the effect of mineral and organic soil constituents on the fate of organic compounds in soils. Specifically, it sought to determine how the associations between organic chemicals and different soil constituents affect their subsequent biodegradation in soil. Four C-labeled surfactants were aseptically adsorbed to montmorillonite, kaolinite, illite, sand, and humic acids. These complexes were mixed with a woodlot soil, and CO(2) production was measured over time. The mineralization data were fitted to various production models by nonlinear regression, and a mixed (3/2)-order model was found to most accurately describe the mineralization patterns. Different mineralization patterns were observed as a function of the chemical and soil constituents. Surfactants that had been preadsorbed to sand or kaolinite usually showed similar mineralization kinetics to the control treatments, in which the surfactants were added to the soil as an aqueous solution. Surfactants that had been bound to illite or montmorillonite were typically degraded to lesser extents than the other forms, while surfactant-humic acid complexes were degraded more slowly than the other forms. The desorption coefficients (K(d)) of the soil constituent-bound surfactants were negatively correlated with the initial rates of degradation (k(1)) and estimates of CO(2) yield (P(o)) as well as actual total yields of CO(2). However, there was no relationship between K(d) and second-stage zero-order rates of mineralization (k(o)). Microbial community characteristics (biomass and activity) were not correlated with any of the mineralization kinetic parameters. Overall, this study showed that environmental form had a profound effect on the ultimate fate of biodegradable chemicals in soil. This form is defined by the physicochemical characteristics of the chemical, the composition and mineralogy of the soil, and the mode of entry of the chemical into the soil environment.

Composition and thermal properties of membrane lipids in cryptoendolithic lichen microbiota from antarctica.

In Antarctica there exists a cryptoendolithic microbiota which survives extremely low temperatures. Fatty acid analysis of the membrane phospholipids of this microbial community showed a predominance of polyunsaturated fatty acids. Artificial membranes made from the purified community phospholipids remained fluid to below -20 degrees C and had unusual hydration properties.

Mineralization of linear alkylbenzene sulfonate by a four-member aerobic bacterial consortium.

A bacterial consortium capable of linear alkylbenzene sulfonate (LAS) mineralization under aerobic conditions was isolated from a chemostat inoculated with activated sludge. The consortium, designated KJB, consisted of four members, all of which were gram-negative, rod-shaped bacteria that grew in pairs and short chains. Three isolates had biochemical properties characteristic of Pseudomonas spp.; the fourth showed characteristics of the Aeromonas spp. Cell suspensions were grown together in minimal medium with [C]LAS as the only carbon source. After 13 days of incubation, more than 25% of the [C]LAS was mineralized to CO(2) by the consortium. Pure bacterial cultures and combinations lacking any one member of the KJB bacterial consortium did not mineralize LAS. Three isolates carried out primary biodegradation of the surfactant, and one did not. This study shows that the four bacteria complemented each other and synergistically mineralized LAS, indicating catabolic cooperation among the four consortium members.

Conversion of 5-fluorocytosine to 5-fluorouracil by human intestinal microflora.

The mechanism of toxicity from 5-fluorocytosine chemotherapy is unclear. However, recent evidence suggests that the generation of 5-fluorouracil by a host may play an important role in the development of this toxicity. Using an in vitro semicontinuous culture system to mimic the intestinal microflora, we examined the capacity of this complex microbial community to convert 5-fluorocytosine to 5-fluorouracil. The system was dosed initially and after 2 weeks of chronic exposure to 5-fluorocytosine with radiolabeled 5-fluorocytosine. No detectable production of 5-fluorouracil was observed up to 8 h after the acute dose; however, at 24 h and at all time points thereafter, increasing levels of 5-fluorouracil were detected for 4 days. The chronic dose resulted in an increased rate of 5-fluorouracil production without the 8-h lag time. These findings suggest that the enzyme or enzymes responsible for the deamination of 5-fluorocytosine to 5-fluorouracil by the intestinal microflora can be induced by chronic exposure to 5-fluorocytosine and that this conversion may provide a mechanism through which 5-fluorocytosine toxicity is manifested.

Spatial distribution of biochemical parameters indicating biomass and community composition of microbial assemblies in estuarine mud flat sediments.

The spatial distribution of communities was examined in estuarine mud flat sediments by the biochemical analysis of the lipids and lipid components extracted from the sediments. Total phospholipid was used as a measure of total biomass, and fatty acids were used as indicators of community composition. Comparisons were made among 2- by 2-m (location) and 0.2- by 0.2-m (cluster) sampling plots by using a nested analysis of variance to design an optimal sampling strategy to define the microbial content of a large, relatively homogenous area. At two of the three stations, a 2- by 2-m plot was representative of the station, but 0.2- by 0.2-m areas were in no case representative of the station. The biomass measured by the extractable phospholipid and the total lipid palmitic acid showed excellent correlation with the fatty acid "signatures" characteristic of bacteria, but showed a lower correlation with the long-chain polyenoic fatty acids characteristic of the microfauna.