Microbial manganese and sulfate reduction in Black Sea shelf sediments

The microbial ecology of anaerobic carbon oxidation processes was investigated in Black Sea shelf sediments from mid-shelf with well-oxygenated bottom water to the oxic-anoxic chemocline at the shelf-break. At all stations, organic carbon (C(org)) oxidation rates were rapidly attenuated with depth in anoxically incubated sediment. Dissimilatory Mn reduction was the most important terminal electron-accepting process in the active surface layer to a depth of approximately 1 cm, while SO(4)(2-) reduction accounted for the entire C(org) oxidation below. Manganese reduction was supported by moderately high Mn oxide concentrations. A contribution from microbial Fe reduction could not be discerned, and the process was not stimulated by addition of ferrihydrite. Manganese reduction resulted in carbonate precipitation, which complicated the quantification of C(org) oxidation rates. The relative contribution of Mn reduction to C(org) oxidation in the anaerobic incubations was 25 to 73% at the stations with oxic bottom water. In situ, where Mn reduction must compete with oxygen respiration, the contribution of the process will vary in response to fluctuations in bottom water oxygen concentrations. Total bacterial numbers as well as the detection frequency of bacteria with fluorescent in situ hybridization scaled to the mineralization rates. Most-probable-number enumerations yielded up to 10(5) cells of acetate-oxidizing Mn-reducing bacteria (MnRB) cm(-3), while counts of Fe reducers were <10(2) cm(-3). At two stations, organisms affiliated with Arcobacter were the only types identified from 16S rRNA clone libraries from the highest positive MPN dilutions for MnRB. At the third station, a clone type affiliated with Pelobacter was also observed. Our results delineate a niche for dissimilatory Mn-reducing bacteria in sediments with Mn oxide concentrations greater than approximately 10 micromol cm(-3) and indicate that bacteria that are specialized in Mn reduction, rather than known Mn and Fe reducers, are important in this niche.     

Microbial Iron Reduction by Enrichment Cultures Isolated from Estuarine Sediments

Microbial Fe reduction in acetate- and succinate-containing enrichment cultures initiated with an estuarine sediment inoculum was studied. Fe reduction was unaffected when SO₄²⁻ reduction was inhibited by MoO₄²⁻, indicating that both processes could occur independently. Bacterially produced sulfide precipitated as FeS but was not completely responsible for Fe reduction. The separation of oxidized Fe particles from bacteria by dialysis tubing demonstrated that direct bacterial contact was necessary for Fe reduction. Fe reduction in cultures amended with NO₃⁻ was delayed until NO₃⁻ and NO₂⁻ were removed. However, bacterial attachment to oxidized Fe particles in NO₃⁻-amended cultures occurred early during growth in a manner similar to NO₃⁻-free cultures. During late stages of growth, bacteria not attached to Fe particles became pale and swollen, while attached cells remained bright blue when examined by 4′,6-diamidine-2-phenylindole epifluo-rescence microscopy. The presence of added oxidized Mn had no effect on Fe reduction. The results suggested that enzymatic Fe reduction was responsible for reducing Fe in these cultures even in the presence of sulfide and that cells incapable of Fe reduction became unhealthy when Fe(III) was the only available electron acceptor.     

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO₃⁻ inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO₃⁻ biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO₃⁻ and synthetic, high-surface-area goethite. Results showed that the presence of NO₃⁻ inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO₃⁻ reduction, a 10-fold decrease in the rate of NO₂⁻ reduction, and a 20-fold increase in the amounts of N₂O produced. Nitrogen stable isotope experiments that utilized δ¹⁵N values of N₂O to distinguish between chemical and biological reduction of NO₂⁻ revealed that the N₂O produced during NO₂⁻ or NO₃⁻ reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO₃⁻ reduction produces NO₂⁻ and Fe(II), which then abiotically react to reduce NO₂⁻ to N₂O with the subsequent oxidation of Fe(II) to Fe(III).     

Fe(II) Oxidation Is an Innate Capability of Nitrate-Reducing Bacteria That Involves Abiotic and Biotic Reactions

Phylogenetically diverse species of bacteria can catalyze the oxidation of ferrous iron [Fe(II)] coupled to nitrate (NO₃⁻) reduction, often referred to as nitrate-dependent iron oxidation (NDFO). Very little is known about the biochemistry of NDFO, and though growth benefits have been observed, mineral encrustations and nitrite accumulation likely limit growth. Acidovorax ebreus, like other species in the Acidovorax genus, is proficient at catalyzing NDFO. Our results suggest that the induction of specific Fe(II) oxidoreductase proteins is not required for NDFO. No upregulated periplasmic or outer membrane redox-active proteins, like those involved in Fe(II) oxidation by acidophilic iron oxidizers or anaerobic photoferrotrophs, were observed in proteomic experiments. We demonstrate that while “abiotic” extracellular reactions between Fe(II) and biogenic NO₂⁻/NO can be involved in NDFO, intracellular reactions between Fe(II) and periplasmic components are essential to initiate extensive NDFO. We present evidence that an organic cosubstrate inhibits NDFO, likely by keeping periplasmic enzymes in their reduced state, stimulating metal efflux pumping, or both, and that growth during NDFO relies on the capacity of a nitrate-reducing bacterium to overcome the toxicity of Fe(II) and reactive nitrogen species. On the basis of our data and evidence in the literature, we postulate that all respiratory nitrate-reducing bacteria are innately capable of catalyzing NDFO. Our findings have implications for a mechanistic understanding of NDFO, the biogeochemical controls on anaerobic Fe(II) oxidation, and the production of NO₂⁻, NO, and N₂O in the environment.     

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10⁶ acetate-utilizing manganese-reducing cells cm⁻³ in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.     

Nitrite oxidation in the upper water column and oxygen minimum zone of the eastern tropical North Pacific Ocean

Nitrogen (N) is an essential nutrient in the sea and its distribution is controlled by microorganisms. Within the N cycle, nitrite (NO₂⁻) has a central role because its intermediate redox state allows both oxidation and reduction, and so it may be used by several coupled and/or competing microbial processes. In the upper water column and oxygen minimum zone (OMZ) of the eastern tropical North Pacific Ocean (ETNP), we investigated aerobic NO₂⁻ oxidation, and its relationship to ammonia (NH₃) oxidation, using rate measurements, quantification of NO₂⁻-oxidizing bacteria via quantitative PCR (QPCR), and pyrosequencing. ¹⁵NO₂⁻ oxidation rates typically exhibited two subsurface maxima at six stations sampled: one located below the euphotic zone and beneath NH₃ oxidation rate maxima, and another within the OMZ. ¹⁵NO₂⁻ oxidation rates were highest where dissolved oxygen concentrations were <5 μM, where NO₂⁻ accumulated, and when nitrate (NO₃⁻) reductase genes were expressed; they are likely sustained by NO₃⁻ reduction at these depths. QPCR and pyrosequencing data were strongly correlated (r²=0.79), and indicated that Nitrospina bacteria numbered up to 9.25% of bacterial communities. Different Nitrospina groups were distributed across different depth ranges, suggesting significant ecological diversity within Nitrospina as a whole. Across the data set, ¹⁵NO₂⁻ oxidation rates were decoupled from ¹⁵NH₄⁺ oxidation rates, but correlated with Nitrospina (r²=0.246, P<0.05) and NO₂⁻ concentrations (r²=0.276, P<0.05). Our findings suggest that Nitrospina have a quantitatively important role in NO₂⁻ oxidation and N cycling in the ETNP, and provide new insight into their ecology and interactions with other N-cycling processes in this biogeochemically important region of the ocean.     

The Impact of Gamma Radiation on Sediment Microbial Processes

Microbial communities have the potential to control the biogeochemical fate of some radionuclides in contaminated land scenarios or in the vicinity of a geological repository for radioactive waste. However, there have been few studies of ionizing radiation effects on microbial communities in sediment systems. Here, acetate and lactate amended sediment microcosms irradiated with gamma radiation at 0.5 or 30 Gy h⁻¹ for 8 weeks all displayed NO₃⁻ and Fe(III) reduction, although the rate of Fe(III) reduction was decreased in 30-Gy h⁻¹ treatments. These systems were dominated by fermentation processes. Pyrosequencing indicated that the 30-Gy h⁻¹ treatment resulted in a community dominated by two Clostridial species. In systems containing no added electron donor, irradiation at either dose rate did not restrict NO₃⁻, Fe(III), or SO₄²⁻ reduction. Rather, Fe(III) reduction was stimulated in the 0.5-Gy h⁻¹-treated systems. In irradiated systems, there was a relative increase in the proportion of bacteria capable of Fe(III) reduction, with Geothrix fermentans and Geobacter sp. identified in the 0.5-Gy h⁻¹ and 30-Gy h⁻¹ treatments, respectively. These results indicate that biogeochemical processes will likely not be restricted by dose rates in such environments, and electron accepting processes may even be stimulated by radiation.     

Evidence for Microbial Fe(III) Reduction in Anoxic, Mining-Impacted Lake Sediments (Lake Coeur d'Alene, Idaho)

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg⁻¹, suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter⁻¹) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe₃O₄), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg⁻¹. Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 × 10⁵ cells g (dry weight) of sediment⁻¹. Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Mycorrhiza and soil bacteria influence extractable iron and manganese in soil and uptake by soybean

Excess manganese (Mn) in soil is toxic to crops, but in some situations arbuscular mycorrhizal fungi (AMF) alleviate the toxic effects of Mn. Besides the increased phosphorus (P) uptake, mycorrhiza may affect the balance between Mn-reducing and Mn-oxidizing microorganisms in the mycorrhizosphere and affect the level of extractable Mn in soil. The aim of this work was to compare mycorrhizal and non-mycorrhizal plants that received extra P in relation to alleviation of Mn toxicity and the balance between Mn-oxidizing and Mn-reducing bacteria in the mycorrhizosphere. A clayey soil containing 508 mg kg⁻¹ of extractable Mn was fertilized with 30 mg kg⁻¹ (P1) or 45 mg kg⁻¹ (P2) of soluble P. Soybean (Glycine max L. Merrill, cv. IAC 8-2) plants at P1 level were non-inoculated (CP1) or inoculated with either Glomus etunicatum (GeP1) or G. macrocarpum (GmP1), while plants at P2 level were left non-inoculated (CP2). Plants were grown in a greenhouse and harvested after 80 days. In the mycorrhizosphere of the GmP1 and GeP1 plants a shift from Mn-oxidizing to Mn-reducing bacteria coincided with higher soil extractability of Mn and Fe. However, the occurrence of Mn-oxidizing/reducing bacteria in the (mycor)rhizosphere was unrelated to Mn toxicity in plants. Using 16S rDNA sequence homologies, the Mn-reducing isolates were consistent with the genus Streptomyces. The Mn-oxidizers were homologous with the genera Arthrobacter, Variovorax and Ralstonia. While CP1 plants showed Mn toxicity throughout the whole growth period, CP2 plants never did, in spite of having Fe and Mn shoot concentrations as high as in CP1 plants. Mycorrhizal plants showed Mn toxicity symptoms early in the growth period that were no longer visible in later growth stages. The shoot P concentration was almost twice as high in mycorrhizal plants compared with CP1 and CP2 plants. The shoot Mn and Fe concentrations and contents were lower in GmP1 and GeP1 plants compared with the CP2 treatment, even though levels of extractable metals increased in the soil when plants were mycorrhizal. This suggests that mycorrhiza protected its host plant from excessive uptake of Mn and Fe. In addition, higher tissue P concentrations may have facilitated internal detoxification of Mn in mycorrhizal plants. The exact mechanisms acting on alleviation of Mn toxicity in mycorrhizal plants should be further investigated.     

Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green Microalga Botryococcus braunii

Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C₃₀) is metabolized by methylation to give intermediates of C₃₁, C₃₂, C₃₃, and C₃₄, with C₃₄ being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm⁻¹ showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm⁻¹ were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm⁻¹ was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm⁻¹ are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm⁻¹ botryococcene Raman shift.     

Sulfate Reduction in Freshwater Sediments Receiving Acid Mine Drainage

One arm of Lake Anna, Va., receives acid mine drainage (AMD) from Contrary Creek (SO₄²⁻ concentration = 2 to 20 mM, pH = 2.5 to 3.5). Acid-volatile sulfide concentrations, SO₄²⁻ reduction rates, and interstitial SO₄²⁻ concentrations were measured at various depths in the sediment at four stations in four seasons to assess the effects of the AMD-added SO₄²⁻ on bacterial SO₄²⁻ reduction. Acid-volatile sulfide concentrations were always an order of magnitude higher at the stations receiving AMD than at a control station in another arm of the lake that received no AMD. Summer SO₄²⁻ reduction rates were also an order of magnitude higher at stations that received AMD than at the control station (226 versus 13.5 mmol m⁻² day⁻¹), but winter values were inconclusive, probably due to low sediment temperature (6°C). Profiles of interstitial SO₄²⁻ concentrations at the AMD stations showed a rapid decrease with depth (from 1,270 to 6 μM in the top 6 cm) due to rapid SO₄²⁻ reduction. Bottom-water SO₄²⁻ concentrations in the AMD-receiving arm were highest in winter and lowest in summer. These data support the conclusion that there is a significant enhancement of SO₄²⁻ reduction in sediments receiving high SO₄²⁻ inputs from AMD.     

Impact of Different In Vitro Electron Donor/Acceptor Conditions on Potential Chemolithoautotrophic Communities from Marine Pelagic Redoxclines

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO₂ dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO₂ dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO₂ dark fixation rates, ranging from 0.7 to 1.4 μmol liter⁻¹ day⁻¹, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO₂ dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter⁻¹ CO₂ day⁻¹. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter⁻¹ NO₃⁻ day⁻¹. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.     

Seasonal Photochemical Transformations of Nitrogen Species in a Forest Stream and Lake

The photochemical release of inorganic nitrogen from dissolved organic matter is an important source of bio-available nitrogen (N) in N-limited aquatic ecosystems. We conducted photochemical experiments and used mathematical models based on pseudo-first-order reaction kinetics to quantify the photochemical transformations of individual N species and their seasonal effects on N cycling in a mountain forest stream and lake (Plešné Lake, Czech Republic). Results from laboratory experiments on photochemical changes in N speciation were compared to measured lake N budgets. Concentrations of organic nitrogen (N_org; 40–58 µmol L⁻¹) decreased from 3 to 26% during 48-hour laboratory irradiation (an equivalent of 4–5 days of natural solar insolation) due to photochemical mineralization to ammonium (NH₄ ⁺) and other N forms (N_x; possibly N oxides and N₂). In addition to N_org mineralization, N_x also originated from photochemical nitrate (NO₃ ⁻) reduction. Laboratory exposure of a first-order forest stream water samples showed a high amount of seasonality, with the maximum rates of N_org mineralization and NH₄ ⁺ production in winter and spring, and the maximum NO₃ ⁻ reduction occurring in summer. These photochemical changes could have an ecologically significant effect on NH₄ ⁺ concentrations in streams (doubling their terrestrial fluxes from soils) and on concentrations of dissolved N_org in the lake. In contrast, photochemical reactions reduced NO₃ ⁻ fluxes by a negligible (<1%) amount and had a negligible effect on the aquatic cycle of this N form.     

Investigation on Crude and High-Temperature Heated Coffee Oil by ATR-FTIR Spectroscopy along with Antioxidant and Antimicrobial Properties

The coffee oil has a promising potential to be used in food industry, but an efficient use, especially in products that required high-temperature heating, depends on its chemical composition and the changes induced by processing. Since there is little information on this topic, the aim of our study was to investigate the crude green and roasted coffee oil (GCO, RCO) and heated (HGCO, HRCO) for 1 h at 200°C, by Fourier Transform Infrared (FTIR) spectroscopy and in terms of antioxidant and antimicrobial properties. The results of FTIR spectroscopy revealed that no statistically significant differences (one-way ANOVA, p>0.05) in the oxidative status of GCO and RCO were found. The coffee oils heating induced significant spectral changes in the regions 3100–3600 cm^–1, 2800–3050 cm^–1 and 1680–1780 cm^–1 proved by the differences in the absorbance ratios A 3009 cm⁻¹/A 2922 cm⁻¹, A 3009 cm⁻¹/A 2853 cm⁻¹, A 3009 cm⁻¹/A 1744 cm⁻¹, A 1744 cm⁻¹/A 2922 cm⁻¹. These alterations were related to the reduction of the unsaturation degree due to primary and secondary oxidation processes of the lipid fraction. The radical scavenging ability of oils investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay revealed that the IC50 value of GCO was significantly lower than of RCO (p<0.05). The IC50 values of crude coffee oils were lower than those of heated samples. The antioxidant activity of oils was attributed to both antioxidant compounds with free-radical scavenging capacity and to lipids oxidation products generated by heating. In the first 6 h of incubation, the inhibitory activity of crude oils against E. coli and E. faecalis was not significantly different to the control (p>0.05). Also, HGCO and HRCO showed significantly different inhibitory potential related to the control (p<0.05). The heating induced statistically significant decreases in the effectiveness of coffee oils against the tested bacteria. GCO proved to be the most effective among investigated coffee oils against the tested bacteria.     

Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

Ethylene Removal by a Biofilter with Immobilized Bacteria

A biofilter which eliminated ethylene (C₂H₄) from the high parts-per-million range to levels near the limit for plant hormonal activity (0.01 to 0.1 ppm) was developed. Isolated ethylene-oxidizing bacteria were immobilized on peat-soil in a biofilter (687 cm³) and subjected to an atmospheric gas flow (73.3 ml min⁻¹) with 2 or 117 ppm of C₂H₄. Ethylene was eliminated to a minimum level of 0.017 ppm after operation with 2.05 ppm of C₂H₄ for 16 days. Also, the inlet C₂H₄ concentration of 117 ppm was reduced to <0.04 ppm. During operation with 2 and 117 ppm of C₂H₄, an increase in the C₂H₄ removal rate was observed, which was attributed to proliferation of the immobilized bacteria, notably in the first 0- to 5-cm segment of the biofilter. The maximal C₂H₄ elimination capacity of the biofilter was 21 g of C₂H₄ m⁻³ day⁻¹ during operation with 117 ppm of C₂H₄ in the inlet gas. However, for the first 0- to 5-cm segment of the biofilter, an elimination capacity of 146 g of C₂H₄ m⁻³ day⁻¹ was calculated. Transition of the biofilter temperature from 21 to 10°C caused a 1.6-fold reduction in the C₂H₄ removal rate, which was reversed during operation for 18 days. Batch experiments with inoculated peat-soil demonstrated that C₂H₄ removal still occurred after storage at 2, 8, and 20°C for 2, 3, and 4 weeks. However, the C₂H₄ removal rate decreased with increasing storage time and was reduced by ca. 50% after storage for 2 weeks at all three temperatures. The biofilter could be a suitable tool for C₂H₄ removal in, e.g., horticultural storage facilities, since it (i) removed C₂H₄ to 0.017 ppm, (ii) had a good operational stability, and (iii) operated efficiently at 10°C.     

Impact of Bacterial NO3? Transport on Sediment Biogeochemistry

Experiments demonstrated that Beggiatoa could induce a H₂S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO₃⁻ reduction from denitrification to dissimilatory NO₃⁻ reduction to ammonium. pH, O₂, and H₂S profiles indicated that the bacteria oxidized H₂S with NO₃⁻ and transported S⁰ to the sediment surface for aerobic oxidation.     

Root-Associated N2 Fixation (Acetylene Reduction) by Enterobacteriaceae and Azospirillum Strains in Cold-Climate Spodosols

N₂ fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C₂H₂ reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C₂H₄·g⁻¹ (dry weight)· h⁻¹ when incubated at pO₂ 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C₂H₄· g⁻¹ (dry weight)·h⁻¹ without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N₂-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Inhibition of NO3? and NO2? Reduction by Microbial Fe(III) Reduction: Evidence of a Reaction between NO2? and Cell Surface-Bound Fe2+

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO₃⁻ and NO₂⁻ (NO_x⁻) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe²⁺ and NO₂⁻. This coating could then inhibit reduction of NO_x⁻ by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of ~3 mM NO₃⁻ reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe²⁺ and NO₂⁻, demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe²⁺ onto Paracoccus denitrificans inhibited NO_x⁻ reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO₂⁻ was chemically reduced to N₂O by goethite or cell-sorbed Fe²⁺, but not at appreciable rates by aqueous Fe²⁺. Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe²⁺ and NO₂⁻. The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.