Gerald K. Sims

Microscale determination of inorganic nitrogen in water and soil extracts

Abstract Rapid, sensitive analysis of NH4 ‐ NO3 ‐, and NO2 ‐ in 1–150 μL of soil extract or water was achieved using a modified indophenol blue technique adapted to microtiter plate format. The microplate technique was similar to conventional steam distillation in accuracy and precision. By varying aliquot volume, a wide linear dynamic range (0.05 to 1000 mg of NH4 +‐ or NO3 ‐‐NL‐1) was achieved without the need for sample dilution or concentration. High sample throughput (250–500 NH4 + analyses d‐1) was accomplished manually, but could be significantly increased by automation. Of considerable importance was the very low waste stream produced by the method. All equipment and supplies required are commercially available and need no modifications for this use. The microtiter plate format could be used for other soil colorimetric analyses with little or no down time for equipment setup, a major consideration for commercial soil‐testing laboratories. The method and equipment used are well suited to quality co...

Degradation of pyridines in the environment

Pyridine and pyridine derivatives occur in the environment as a result of industrial and agricultural activities. The fate of pyridines in the environment is a function of both abiotic and biotic processes, including photochemical transformations, complexation, surface attenuation, transport, and biological degradation. Pyridine is readily degraded in soil, and numerous bacteria isolated from soils or sludges are capable of growing on pyridines as sole sources of carbon and/or nitrogen. Numerous substituted pyridines are also susceptible to biodegradation, although major changes in biodegrada‐bility of the pyridine ring result from slight modification of the nature orposition of ring substituents. Bacteria apparently degrade most simple pyridine derivatives, particularly hydroxypyridines and pyridinecarboxylic acids, via pathways involving hydroxylated intermediates. The initial hydroxylation step in biodegradation of many pyridines is unusual in the incorporation of oxygen derived from water. Data sugges...

Biodegradation of atrazine under denitrifying conditions.

Abstract Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by 14CO2 evolution. Denitrification was confirmed by detection of 15N2 in headspace samples of K15NO3-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [14C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media.

Biodegradation of 2-methyl, 2-ethyl, and 2-hydroxypyridine by an Arthrobacter sp. isolated from subsurface sediment

A bacterium capable of degrading 2-methylpyridine was isolated by enrichment techniques from subsurface sediments collected from an aquifer located at an industrial site that had been contaminated with pyridine and pyridine derivatives. The isolate, identified as an Arthrobacter sp., was capable of utilizing 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine as primary C, N, and energy sources. The isolate was also able to utilize 2-, 3-, and 4-hydroxybenzoate, gentisic acid, protocatechuic acid and catechol, suggesting that it possesses a number of enzymatic pathways for the degradation of aromatic compounds. Degradation of 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine was accompanied by growth of the isolate and release of ammonium into the medium. Degradation of 2-methylpyridine was accompanied by overproduction of riboflavin. A soluble blue pigment was produced by the isolate during the degradation of 2-hydroxypyridine, and may be related to the diazadiphenoquinones reportedly produced by other Arthrobacter spp. when grown on 2-hydroxypyridine. When provided with 2-methylpyridine, 2-ethylpyridine, and 2-hydroxypyridine simultaneously, 2-hydroxypyridine was rapidly and preferentially degraded; however there was no apparent biodegradation of either 2-methylpyridine or 2-ethylpyridine until after a seven day lag. The data suggest that there are differences between the pathway for 2-hydroxypyridine degradation and the pathway(s) for 2-methylpyridine and 2-ethylpyridine.

Isolation, characterization, and substrate utilization of a quinoline-degrading bacterium

Abstract A Gram (+) rod-shaped organism identified as a Rhodococcus sp. capable of growth utilizing quinoline as the dominant carbon, energy, and nitrogen source was isolated from soil. The isolate, designated as Rhodococcus sp. Q1 was also capable of growth on 2-hydroxyquinoline, pyridine, 2,3-dimethylpyridine, catechol, benzoate, and protocatechuic acid, suggesting a diverse capacity for aromatic ring degradation. Concentrations of quinoline in excess of 3.88 mM were toxic. Although ring nitrogen was released into the growth medium as ammonium, quinoline degradation was not limited by the availability of inorganic N. A degradation product was isolated and identified as 2-hydroxyquinoline on the basis of ultraviolet, fluorescence emission, and mass spectroscopy. When grown on quinoline or 2-hydroxyquinoline, this bacterium produced pigmented compounds.

Dehalogenation of the Herbicides Bromoxynil (3,5-Dibromo-4-Hydroxybenzonitrile) and Ioxynil (3,5-Diiodino-4-Hydroxybenzonitrile) by Desulfitobacterium chlororespirans

ABSTRACT Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 μM) and DBHB (250 to 260 μM) were used as electron acceptors for growth. Doubling times for growth (means ± standard deviations for triplicate cultures) on bromoxynil (18.4 ± 5.2 h) and DBHB (11.9 ± 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.

Availability of urea to autotrophic ammonia-oxidizing bacteria as related to the fate of 14C- and 15N-labeled urea added to soil

Nitrate has been found to accumulate more rapidly in soils fertilized with urea than with inorganic sources of NH4+, despite the fact that nitrification must be preceded by hydrolytic decomposition. For acidic conditions, this finding has been attributed to limited uptake of NH4+ by ammonium-oxidizing bacteria (also reported herein), suggesting an advantage for direct utilization of a nonionizable N substrate such as urea. If the same advantage applies to urea-C, nitrification of urea-N would also be promoted in neutral or alkaline soils, as reported in numerous studies. To ascertain whether urea-C can be utilized directly by nitrifying organisms, NO2− production was measured for Nitrosomonas europaea and Nitrosospira sp. NPAV in minimal media with urea as the sole source of either C or C and N. Nitrite accumulated only with the latter organism, in which case nearly quantitative recovery was observed for N added as NH4+ and/or urea. In a subsequent study, recovery of 14C and 15N in gaseous, extractable, and hydrolyzable forms was determined after incubation with labeled urea for up to 29 days, by using two soils that differed markedly in physiochemical properties affecting nutrient availability. Results obtained in correlating 14C incorporation in the amino acid fraction with 15N accumulation as NO3− were consistent with the stoichiometry that would be expected if C fixation were driven by autotrophic nitrification. Our findings demonstrate unequivocally that urea is utilized as a source of C and N by nitrifying microorganisms, which may account for rapid nitrification of urea-N in soils.

Proteolytic activity under nitrogen or sulfur limitation

Sand cultures were used to evaluate the effect of C, N, and S ratio on protein degradation by soil microorganisms. Sand was inoculated with soil and amended with defined nutrient media to produce limitation for C, N, or S. Limitation for N or S resulted in reduced biomass (total protein) and increased proteolytic activity as indicated by measurements of dye released from a commercial protease substrate (azocoll). Carbon limitation had little effect on proteolytic activity. As expected, utilization of carbon (glucose) was dependent upon the availability of N or S. Protein synthesis inhibitors (chloramphenicol and cycloheximide) suppressed proteolytic activity, suggesting a need for new gene expression in the response of organisms to N or S stress. Correlations of proteolytic activity and biomass among treatments revealed distinctly different relationships depending upon the availability of C, N, or S. The results of this experiment support a role of proteolytic activity in response of microorganisms to N or S deprivation and suggest that protease activity in soil is more strongly influenced by regulatory signals than by standing biomass.

Factors controlling degradation of pesticides in soil

Rates of pesticide degradation in soil exhibit a high degree of variability, the sources of which are usually unclear. Combining data from incubations performed using a range of soil properties and environmental conditions has resulted in greater understanding of factors controlling such degradation. The herbicides clomazone, flumetsulam, atrazine, and cloransulam-methyl, as well as the former insecticide naphthalene offer examples of degradation kinetics controlled by coupling competing processes which may in turn be regulated separately by environmental conditions and soil properties. The processes of degradation and volatilization appear to compete for clomazone in solution; sorbed clomazone is degraded only after the solution phase is depleted. Similarly, volatilization of naphthalene is enhanced when degradation has been inhibited by high nutrient levels. Degradation of the herbicide flumetsulam has been shown to be regulated by sorption, even though the compound has a relatively low affinity for the soil. The fate pathway for cloransulam-methyl shifts from mineralization to formation of metabolities, bound residues and physically occluded material as temperature increases. Atrazine degradation in soil may be controlled in part by the presence of inorganic nitrogen, as the herbicide appears to be used as a nitrogen source by micro-organisms. New insight gained from measurement of multiple fate processes is demonstrated by these examples.

A microscale method for colorimetric determination of urea in soil extracts

Abstract The diacetyl monoxime colorimetric method of determining urea in soil extracts was modified for microplate format. A 100‐μL aliquot of extract was treated with color reagent in a disposable plastic microtiter plate (96 wells/plate), and color was developed by heating the plate in a low‐temperature oven at 87°C for 55 min. After cooling for 20 min at ambient temperature, absorbance measurements were simultaneously performed on all 96 wells using a microplate reader. The microscale method was faster and more convenient than the conventional method; moreover, the volume of waste was markedly reduced. Studies to compare the two methods showed very little difference in accuracy, precision, or sensitivity.