Denitrification potential in stream sediments impacted by acid mine drainage: effects of pH, various electron donors, and iron.

Acid mine drainage (AMD) contaminates thousands of kilometers of stream in the western United States. At the same time, nitrogen loading to many mountain watersheds is increasing because of atmospheric deposition of nitrate and increased human use. Relatively little is known about nitrogen cycling in acidic, heavy-metal-laden streams; however, it has been reported that one key process, denitrification, is inhibited under low pH conditions. The objective of this research was to investigate the capacity for denitrification in acidified streams. Denitrification potential was assessed in sediments from several Colorado AMD-impacted streams, ranging from pH 2.60 to 4.54, using microcosm incubations with fresh sediment. Added nitrate was immediately reduced to nitrogen gas without a lag period, indicating that denitrification enzymes were expressed and functional in these systems. First-order denitrification potential rate constants varied from 0.046 to 2.964 day(-1). The pH of the microcosm water increased between 0.23 and 1.49 pH units during denitrification. Additional microcosm studies were conducted to examine the effects of initial pH, various electron donors, and iron (added as ferrous and ferric iron). Decreasing initial pH decreased denitrification; however, increasing pH had little effect on denitrification rates. The addition of ferric and ferrous iron decreased observed denitrification potential rate constants. The addition of glucose and natural organic matter stimulated denitrification potential. The addition of hydrogen had little effect, however, and denitrification activity in the microcosms decreased after acetate addition. These results suggest that denitrification can occur in AMD streams, and if stimulated within the environment, denitrification might reduce acidity.

Effects of various environmental conditions on the transformation of chlorinated solvents by Methanosarcina thermophila cell exudates.

Several microbiologically produced biomolecules have been shown to degrade chlorinated contaminants found in groundwater systems. It was discovered that the cell-free exudates of the methanogen Methanosarcina thermophila were capable of carbon tetrachloride (CT) and chloroform (CF) degradation. Characterization of the exudates suggested that the active agents were porphorinogen-type molecules, possibly containing zinc. This research was performed to determine if the exudates from M. thermophila could be used for remediation purposes. The cell exudates were found to be capable of degrading CT, CF, tetrachloroethene, trichloroethene, and 1,1,1-trichloroethane. CT degradation was used to gauge exudate activity under a variety of conditions that would be encountered in the environment. The cell exudates were active when incubated in two types of soil matrices and at temperatures ranging from 4 to 23 degrees C. Over a 35-day period approximately 10.2 micromoles of CT were degraded by M. thermophila exudates. To test the hypothesis that the exudates contained either a zinc porphorinogen or a quinone, experiments were performed with zinc 5,10,15,20-tetra (4-pyridyl)-21 H, 23 H-porphine tetrakis, protoporphyrin IX zinc, and juglone. The two zinc porphyrins were capable of mediating CT degradation at rates comparable to those observed with the M. thermophila exudates; however, juglone was only capable of very slow CT transformation. The electron-transfer activity of the M. thermophila cell exudates was therefore more consistent with the activity of porphorinogens rather than quinones. Finally, in two enrichment cultures established from aquifer material and marine sediment, the possibility of excreted agents capable of degrading CT was evident.

Investigation of cell exudates active in carbon tetrachloride and chloroform degradation.

Contamination of groundwater by chlorinated solvents such as carbon tetrachloride (CCl4) and chloroform (CHCl3) is a widespread problem. The cell exudates from the methanogen Methanosarcina thermophila are active in the degradation of CCl4 and CHCl3. This research was performed to characterize these exudates. Examination of the influence of pH indicated that activity was greater under alkaline conditions. Rapid CCl4 degradation occurred from 35-65 degrees C, with first-order degradation rate coefficients increasing as temperature increased. It was found that proteins were not responsible for CCl4 degradation. The active agents in the cell exudates were <10 kDa in size, with degradation activity present in both 1-10 kDa and <1 kDa size ranges. Upon purification of the <10 kDa size range of the cell exudates on a C(18) chromatography column, 17 fractions (out of 100) degraded >50% of the added CCl4 in 8 h. These 17 fractions were pooled into three samples based on their elution time from the C(18) column. One of these pooled samples contained elevated levels of cobalt, zinc, and iron, at 2, 3, and 13 times the levels measured in similarly fractionated and pooled samples of medium, respectively. The UV-visible spectrum of this pooled sample had an absorption maximum at 560-580 nm, which is similar to the absorption maxima of heme (approximately 550 and 575 nm). The two other pooled samples contained elevated levels of zinc at 11 and 22 times the concentration measured in similarly fractionated and pooled samples of medium, respectively, and also contained very low levels of nickel, cobalt, and iron. This research suggests that the cell exudates from M. thermophila contain porphorinogen-type molecules capable of dechlorination, possibly excreted corrinoids, hemes, and zinc-containing molecules.