Ammonium Removal by the Oxygen-Limited Autotrophic Nitrification-Denitrification System

The present lab-scale research reveals the potential of implementation of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with normal nitrifying sludge as the biocatalyst for the removal of nitrogen from nitrogen-rich wastewater in one step. In a sequential batch reactor, synthetic wastewater containing 1 g of NH₄⁺-N liter⁻¹ and minerals was treated. Oxygen supply to the reactor was double-controlled with a pH controller and a timer. At a volumetric loading rate (B_v) of 0.13 g of NH₄⁺-N liter⁻¹ day⁻¹, about 22% of the fed NH₄⁺-N was converted to NO₂⁻-N or NO₃⁻-N, 38% remained as NH₄⁺-N, and the other 40% was removed mainly as N₂. The specific removal rate of nitrogen was on the order of 50 mg of N liter⁻¹ day⁻¹, corresponding to 16 mg of N g of volatile suspended solids⁻¹ day⁻¹. The microorganisms which catalyzed the OLAND process are assumed to be normal nitrifiers dominated by ammonium oxidizers. The loss of nitrogen in the OLAND system is presumed to occur via the oxidation of NH₄⁺ to N₂ with NO₂⁻ as the electron acceptor. Hydroxylamine stimulated the removal of NH₄⁺ and NO₂⁻. Hydroxylamine oxidoreductase (HAO) or an HAO-related enzyme might be responsible for the loss of nitrogen.     

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H₂S) and methane (CH₄) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H₂S and CH₄ production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H₂S emission was notably high (290.7 ± 72.3 mg S-H₂S liter⁻¹ day⁻¹), whereas emissions of CH₄ remained low (17.9 ± 15.9 mg COD-CH₄ liter⁻¹ day⁻¹). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH₄ emissions increased after 1 year of biofilm growth (327.6 ± 16.6 mg COD-CH₄ liter⁻¹ day⁻¹), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.     

Effects of pH and Oxygen and Ammonium Concentrations on the Community Structure of Nitrifying Bacteria from Wastewater

Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter⁻¹), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant. The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined. Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis. Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level. The smaller changes in community structure brought about by the two pH extremes, however, were irreversible. Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative, Nitrosomonas eutropha C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the β subgroup of the class Proteobacteria. A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles.Autotrophic nitrifying bacteria that oxidize ammonium to nitrite and nitrate are found in soils, sediments, wastewaters, freshwater, and marine water and on building facades. They are essential components of the nitrogen (N) cycle, linking the most reduced and most oxidized forms of inorganic N. Nitrification occurs as a two-step process carried out by two distinct groups of bacteria; ammonia-oxidizing bacteria convert ammonia to nitrite, and then nitrite oxidizers convert nitrite to nitrate (22, 30). Environmental factors control the rate of nitrification. The most significant environmental factors are substrate concentration, pH, temperature, and oxygen availability (12, 23). Nitrifying bacteria exhibit different substrate concentration sensitivities (26). Media containing low substrate concentrations (10 mg of NH₄⁺ liter⁻¹) can give larger most-probable-number counts of ammonia oxidizers than media containing higher NH₄⁺ concentrations (6, 26). Also, ammonia oxidation is inhibited at high substrate concentrations. The growth rates of Nitrosomonas spp. cultures were reduced in the presence of 1,050 to 2,800 mg of NH₄⁺-N liter⁻¹ (16). Substrate inhibition of ammonia oxidation has also been observed in studies of wastewater systems (23). Natural environments, such as soil and water, usually contain 1 to 10 mg of NH₄⁺-N liter⁻¹ (22), yet liquid wastes from animal farms give rise to concentrations up to 1,600 or 5,600 mg of NH₄⁺-N liter⁻¹ (5, 17). Free ammonia (NH₃) rather than the total ammonium concentration inhibits ammonia oxidizers (1). As the ratio between the ionized form and the nonionized form depends on pH, the toxicity of ammonium also depends on the environmental pH.The pH range for growth of pure cultures of ammonia oxidizers is 5.8 to 8.5, and the pH range for growth of nitrite oxidizers is 6.5 to 8.5 (30). Nitrification was inhibited at pH values below 5.8 in our preliminary experiments performed with an enriched culture of nitrifiers obtained from wastewater. Yet in natural environments, such as soil, nitrification has been reported to occur at pH values below 4.0 (7, 29).Limiting amounts of dissolved oxygen (concentrations below 2 mg liter⁻¹) inhibit nitrification and cause nitrite accumulation or nitrous and nitric oxide production (9, 21). Ammonia-oxidizing bacteria are the key functional group in removing ammonium from wastewaters. Knowledge of the effect of oxygen on nitrification and nitrifying populations has economic importance since aeration of activated sludge is one of the most costly items in the operation of a wastewater treatment plant (21).In environments with high inputs of ammonium, such as wastewaters, biooxidation of this substrate increases the oxygen uptake and lowers the pH. Such modifications of the environment not only affect the production of nitrite and nitrate but can also select a different nitrifying community that is perhaps specialized for these new conditions. Nitrification does occur in extreme environments that pure cultures of nitrifiers cannot tolerate (4). In this study we examined extreme environments in which nitrifying bacteria may be viable but have not been cultured thus far.Because of the difficulty of obtaining nitrifier isolates, nucleic acid-based methods have greatly aided studies of the diversity of nitrifiers (11, 20, 27, 28). Recent molecular investigations have provided valuable information concerning the diversity of ammonia oxidizers in natural environments (5, 15, 20, 25). However, no previous study has focused on the structural or compositional responses of nitrifying communities to perturbations in the environment. In the present laboratory study we examined the effects of high ammonium concentrations, different pH values, and different oxygen concentrations on nitrification and on the community structure of nitrifying bacteria from wastewater. To test the abilities of the communities to regain their original structures, growth of nitrifying communities under the new conditions was followed by incubation under the original conditions.     

Identification and Activities In Situ of Nitrosospira and Nitrospira spp. as Dominant Populations in a Nitrifying Fluidized Bed Reactor

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O₂, NH₄⁺, NO₂⁻, and NO₃⁻ demonstrated the occurrence of complete nitrification in the outer 125 μm of the aggregates. The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter, respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis. 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of Nitrospira spp. and Nitrosospira spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of Nitrospira spp. was restricted to the outer nitrifying zones.     

Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO₃⁻-N liter⁻¹. Traces of ¹⁵NO₃⁻ were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day⁻¹. Both assimilatory and dissimilatory reduction rates were estimated from analyses of ¹⁵N₂, ¹⁵N₂O, ¹⁵NH₄⁺, and ¹⁵N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N₂ and N₂O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO₃⁻ and NH₄⁺ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-³H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g⁻¹ day⁻¹. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N₂, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO₃⁻ was reduced to NH₄⁺. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month.     

Initial Effects of the Mount St. Helens Eruption on Nitrogen Cycle and Related Chemical Processes in Ryan Lake

Ryan Lake, a 1.6-hectare basin lake near the periphery of the tree blowdown area in the blast zone 19 km north of Mount St. Helens, was studied from August to October 1980 to determine the microbial and chemical response of the lake to the eruption. Nutrient enrichment through the addition of fresh volcanic material and the organic debris from the surrounding conifer forest stimulated intense microbial activity. Concentrations of such nutrients as phosphorus, sulfur, manganese, iron, and dissolved organic carbon were markedly elevated. Nitrogen cycle activity was especially important to the lake ecosystem in regulating biogeochemical cycling owing to the limiting abundance of nitrogen compounds. Nitrogen fixation, both aerobic and anaerobic, was active from aerobic benthic and planktonic cyanobacteria with rates up to 210 nmol of N₂ cm⁻¹ h⁻¹ and 667 nmol of N₂ liter⁻¹ h⁻¹, respectively, and from anaerobic bacteria with rates reaching 220 nmol of N₂ liter⁻¹ h⁻¹. Nitrification was limited to the aerobic epilimnion and littoral zones where rates were 43 and 261 nmol of NO₂ liter⁻¹ day⁻¹, respectively. Potential denitrification rates were as high as 30 μmol of N₂O liter⁻¹ day⁻¹ in the anaerobic hypolimnion. Total bacterial numbers ranged from 1 × 10⁶ to 3 × 10⁸ ml⁻¹ with the number of viable sulfur-metal-oxidizing bacteria reaching 2 × 10⁶ ml⁻¹ in the hypolimnion. A general scenario for the microbial cycling of nitrogen, carbon, sulfur, and metals is presented for volcanically impacted lakes. The important role of nitrogen as these lakes recover from the cataclysmic eruption and proceed back towards their prior status as oligotrophic alpine lakes is emphasized.     

Effect of Fluoride on Nitrification of a Concentrated Industrial Waste

The potential for biological nitrification of an industrial waste containing 4,000 mg of ammonia N (NH₄⁺-N) and 10,000 mg of fluoride per liter was investigated. Ammonium sulfate and sodium fluoride were tested in various combinations of 100 to 2,000 mg of NH₄⁺-N per liter and 0 to 5,000 mg of F⁻ per liter in suspended-growth stirred-tank reactors containing enriched cultures of nitrifying bacteria from a municipal sewage treatment plant. The stirred-tank reactors were fed once per day at a constant hydraulic retention period and cell retention time of 10 days. Temperature was 23°C, and pH was 7.0 to 7.5. Clarified secondary effluent was used to make up feeds and to provide minor nutrients. Steady-state data, confirmed by mass balances, were obtained after five to six retention periods. In the absence of fluoride, nitrification efficiency was near 100% for up to 500 mg of NH₄⁺-N per liter. The influence of fluoride was studied at a low ammonia concentration (100 mg/liter) and exerted no significant effect on nitrification at concentrations of up to 200 mg/liter. Maximum effect of fluoride was reached at 800 mg of F⁻ per liter, and no greater inhibition was observed for up to 5,000 mg of F⁻ per liter. At the highest concentrations studied, ion pairing of ammonium and fluoride may exert a significant effect on kinetic coefficients. Kinetic analyses showed maximum specific substrate removal rates (q_max) of NH₄⁺-N to be about 2.3 mg of N per mg of volatile suspended solids per day in the absence of fluoride and 0.85 mg of N per mg of volatile suspended solids per day in the presence of fluoride. The form of inhibition due to the presence of fluoride was shown to be not competitive, conforming to a mixed inhibition model.     

Effects of Carbon Substrates on Nitrite Accumulation in Freshwater Sediments

The contribution of the biochemical pathways nitrification, denitrification, and dissimilatory NO₃⁻ reduction to NH₄⁺ (DNRA) to the accumulation of NO₂⁻ in freshwaters is governed by the species compositions of the bacterial populations resident in the sediments, available carbon (C) and nitrogen (N) substrates, and environmental conditions. Recent studies of major rivers in Northern Ireland have shown that high NO₂⁻ concentrations found in summer, under warm, slow-flowing conditions, arise from anaerobic NO₃⁻ reduction. Locally, agricultural pollutants entering rivers are important C and N sources, providing ideal substrates for the aquatic bacteria involved in cycling of N. In this study a range of organic C compounds commonly found in agricultural pollutants were provided as energy sources in 48-h incubation experiments to investigate if the chemical compositions of the pollutants affected which NO₃⁻ reduction pathway was followed and influenced subsequent NO₂⁻ accumulation. Carbon stored within the sediments was sufficient to support DNRA and denitrifier populations, and the resulting NO₂⁻ peak (80 μg of N liter⁻¹ [approximate]) observed at 24 h was indicative of the simultaneous activities of both bacterial groups. The value of glycine as an energy source for denitrification or DNRA appeared to be limited, but glycine was an important source of additional N. Glucose was an efficient substrate for both the denitrification and DNRA pathways, with a NO₂⁻ peak of 160 μg of N liter⁻¹ noted at 24 h. Addition of formate and acetate stimulated continuous NO₂⁻ production throughout the 48-h period, caused by partial inhibition of the denitrification pathway. The formate treatment resulted in a high NO₂⁻ accumulation (1,300 μg of N liter⁻¹ [approximate]), and acetate treatment resulted in a low NO₂⁻ concentration (<100 μg of N liter⁻¹).     

Primary and Bacterial Secondary Production in a Southwestern Reservoir

Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH¹⁴CO₃ and [methyl-³H]thymidine incorporation, respectively. Peak instantaneous production ranged between 14.8 and 220.5 μg of C liter⁻¹ h⁻¹. There were two distinct periods of high rates of production. From May through July, production near the metalimnion exceeded 100 μg of C liter⁻¹ h⁻¹. During holomixis, production throughout the water column was in excess of 100 μg of C liter⁻¹ h⁻¹ and above 150 μg of C liter⁻¹ h⁻¹ near the surface. Annual areal primary production was 588 g of C m⁻². Bacterial production was markedly seasonal. Growth rates during late fall through spring were typically around 0.002 h⁻¹, and production rates were typically 5 μg of C liter⁻¹ h⁻¹. Growth rates were higher during warmer parts of the year and reached 0.03 h⁻¹ by August. The maximum instantaneous rate of bacterial production was approximately 45 μg of C liter⁻¹ h⁻¹. Annual areal bacterial production was 125 g of C m⁻². Temporal and spatial distributions of bacterial numbers and activities coincided with temporal and spatial distributions of primary production. Areal primary and bacterial secondary production were highly correlated (r = 0.77, n = 15, P < 0.002).     

Aerobic Fermentation of D-Xylose to Ethanol by Clavispora sp

Eleven strains of an undescribed species of Clavispora fermented D-xylose directly to ethanol under aerobic conditions. Strain UWO(PS)83-877-1 was grown in a medium containing 2% D-xylose and 0.5% yeast extract, and the following results were obtained: ethanol yield coefficient (ethanol/D-xylose), 0.29 g g⁻¹ (57.4% of theoretical); cell yield coefficient (dry biomass/D-xylose), 0.25 g g⁻¹; maximum ethanol concentration, 5.9 g liter⁻¹; maximum volumetric ethanol productivity, 0.11 g liter⁻¹ h⁻¹. With initial D-xylose concentrations of 40, 60, and 80 g liter⁻¹, maximum ethanol concentrations of 8.8, 10.9, and 9.8 g liter⁻¹ were obtained, respectively (57.2, 57.1, and 48.3% of theoretical). Ethanol was found to inhibit the fermentation of D-xylose (K_p = 0.58 g liter⁻¹) more than the fermentation of glucose (K_p = 6.5 g liter⁻¹). The performance of this yeast compared favorably with that reported for some other D-xylose-fermenting yeasts.     

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter⁻¹ and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K_m of 23 mg liter⁻¹ (437 μM) and a V_max of 0.98 mg of Cr h⁻¹ mg of protein⁻¹ (317 nmol min⁻¹ mg of protein⁻¹). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.     

Synergistic Interaction Between Anabaena and Zoogloea spp. in Carbon Dioxide-Limited Continuous Cultures

Flocs consisting of Anabaena and Zoogloea spp. were used as a model system for the study of planktonic phototroph-heterotroph interactions. In CO₂-limited continuous culture (3.2 μmol of NaHCO₃ liter⁻¹ h⁻¹, 1.5 μmol of glucose liter⁻¹ h⁻¹, pH 8.5, D = 0.026 h⁻¹), the biomass of the phototroph increased 8.6-fold due to association. However, direct CO₂ exchange accounted for only a 3.8-fold increase. When the glucose supply rate was increased to 7.5 μmol liter⁻¹ h⁻¹, there was a 26-fold increase in biomass. When CO₂ was supplied in excess, there was no difference due to association. In batch culture, using the same medium, the specific growth rate was 0.029 h⁻¹ for the phototroph alone and 0.047 h⁻¹ for the phototroph in association with the heterotroph. The stimulatory effect of the heterotroph was found only under CO₂-limiting conditions and was directly related to the concentration of organic matter supplied in the medium. Both the biomass and the growth rate of the Anabaena sp. were increased by association with the Zoogloea sp. Thus, dissolved organic matter may substitute for CO₂ to maximize both growth rate and biomass production by phototrophs when heterotrophic bacteria are present.     

Impact of Nitrogen and Phosphorus on [14C]Lignocellulose Decomposition by Stream Wood Microflora

Nutritional and physical factors affecting the decomposition of [¹⁴C]lignocellulose prepared from Douglas fir (Pseudotsuga menziesii) were examined by incubating the labeled substrate with homogenized surface wood scrapings obtained from a Douglas fir log in a Pacific Northwest stream. Incubations were conducted in distilled water, in stream water collected from four different sources, or in a defined mineral salts solution with or without supplemental N (KNO₃). Decomposition rates of [¹⁴C]lignocellulose, as measured by ¹⁴CO₂ evolution, were greater in each of the four filter-sterilized sources of stream water than in distilled water alone. Decomposition experiments conducted in stream water media with the addition of defined mineral salts demonstrated that [¹⁴C]cellulose decomposition was stimulated 50% by the addition of either KNO₃ or KH₂PO₄/K₂HPO₄ and further enhanced (167%) by a combination of both. In contrast, [¹⁴C]lignin decomposition was stimulated (65%) only by the addition of both N and P. Decomposition of [¹⁴C]lignocellulose was greatest when supplemental KNO₃ was supplied in concentrations of at least 10.0 mg of N liter⁻¹ but not increased further by higher concentrations. The decomposition of [¹⁴C]lignocellulose increased as the incubation temperature was raised and NO₃⁻¹-N supplementation further increased these rates between three-and sevenfold over the range of temperatures examined (5 to 22°C). Accumulation of NH₄⁺ (2 to 4 mg of N liter⁻¹) was always observed in culture filtrates of incubations which had been supplemented with KNO₃, the quantity being independent of NO₃⁻ concentrations ≥ 10 mg of N liter⁻¹. The role of supplemental NO₃⁻ in the decomposition of [¹⁴C]lignocellulose is discussed in relation to wood decomposition and the low concentrations of N found in stream ecosystems of the Pacific Northwest.     

Nitrification Rates in the Baltic Sea: Comparison of Three Isotope Techniques

Simultaneous measurements of nitrification in the Baltic Sea were made at 10- to 30-m intervals in the months of June and November by three isotope techniques: [¹⁵N]nitrate dilution, N-serve sensitive [¹⁴C]bicarbonate incorporation, and [¹⁵N]ammonium oxidation to nitrite and nitrate. Nitrification rates of 1 to 280 nmol liter⁻¹ day⁻¹ were recorded, and each method showed that the highest rates of nitrification occurred below the halocline. Even in the presence of ammonium, dark incubations of mixed layer (above ca. 50 m) waters never yielded nitrification rates exceeding 45 nmol liter⁻¹ day⁻¹. The rates measured by the ammonium oxidation method were two- to sevenfold greater than those obtained by ¹⁴C incorporation or ¹⁵N dilution. The merits of each technique are discussed, and it is suggested that the [¹⁵N]ammonium oxidation method should be used in conjunction with the [¹⁴C]bicarbonate incorporation method.     

Ecology of Thioploca spp.: Nitrate and Sulfur Storage in Relation to Chemical Microgradients and Influence of Thioploca spp. on the Sedimentary Nitrogen Cycle

Microsensors, including a recently developed NO₃⁻ biosensor, were applied to measure O₂ and NO₃⁻ profiles in marine sediments from the upwelling area off central Chile and to investigate the influence of Thioploca spp. on the sedimentary nitrogen metabolism. The studies were performed in undisturbed sediment cores incubated in a small laboratory flume to simulate the environmental conditions of low O₂, high NO₃⁻, and bottom water current. On addition of NO₃⁻ and NO₂⁻, Thioploca spp. exhibited positive chemotaxis and stretched out of the sediment into the flume water. In a core densely populated with Thioploca, the penetration depth of NO₃⁻ was only 0.5 mm and a sharp maximum of NO₃⁻ uptake was observed 0.5 mm above the sediment surface. In sediments with only few Thioploca spp., NO₃⁻ was detectable down to a depth of 2 mm and the maximum consumption rates were observed within the sediment. No chemotaxis toward nitrous oxide (N₂O) was observed, which is consistent with the observation that Thioploca does not denitrify but reduces intracellular NO₃⁻ to NH₄⁺. Measurements of the intracellular NO₃⁻ and S⁰ pools in Thioploca filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO₃⁻ were found to a depth of at least 13 cm, providing final proof for the vertical shuttling of Thioploca spp. and nitrate transport into the sediment.     

Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination

The oxidation of NH₄⁺ by Nitrosomonas europaea was insensitive to 10 mM NaClO₃ (sodium chlorate) but was strongly inhibited by NaClO₂ (sodium chlorite; K_i, 2 μM). The oxidation of NO₂⁻ by Nitrobacter winogradskyi was inhibited by both ClO₃⁻ and ClO₂⁻ (K_i for ClO₂⁻, 100 μM). N. winogradskyi reduced ClO₃⁻ to ClO₂⁻ under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO₂⁻ was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH₄⁺ and NO₂⁻ was inhibited in the presence of 10 mM ClO₃⁻ after a 2-h lag period, despite the fact that, under these conditions, ClO₂⁻ was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH₄⁺ oxidation is inhibited by ClO₂⁻ produced by reduction of ClO₃⁻ by the NO₂⁻ oxidizer. The use of ClO₃⁻ inhibition of NO₂⁻ oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH₄⁺ is not affected.     

Biosensor Determination of the Microscale Distribution of Nitrate, Nitrate Assimilation, Nitrification, and Denitrification in a Diatom-Inhabited Freshwater Sediment

High-resolution NO₃⁻ profiles in freshwater sediment covered with benthic diatoms were obtained with a new microscale NO₃⁻ biosensor characterized by absence of interference from chemical species other than NO₂⁻ and N₂O. Analysis of the microprofiles obtained indicated no nitrification during darkness, high rates of nitrification and a tight coupling between nitrification and denitrification during illumination, and substantial rates of NO₃⁻ assimilation during illumination. Nitrification during darkness could be induced by purging the bulk water with O₂ gas, indicating that the stimulatory effect on nitrification by illumination was caused by algal production of O₂. NH₄⁺ addition did not stimulate nitrification during darkness when O₂ was restricted to the upper 1-mm layer, and there was thus a low nitrification potential in the permanently oxic top 1 mm of the sediment.     

Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H₂S, CO₂, and CH₄. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter⁻¹ day⁻¹. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.     

Transition from Anoxygenic to Oxygenic Photosynthesis in a Microcoleus chthonoplastes Cyanobacterial Mat

Benthic cyanobacterial mats with the filamentous Microcoleus chthonoplastes as the dominant phototroph grow in oxic hypersaline environments such as Solar Lake, Sinai. The cyanobacteria are in situ exposed to chemical variations between 200 μmol of sulfide liter⁻¹ at night and 1 atm pO₂ during the day. During experimental H₂S to O₂ transitions the microbial community was shown to shift from anoxygenic photosynthesis, with H₂S as the electron donor, to oxygenic photosynthesis. Microcoleus filaments could carry out both types of photosynthesis concurrently. Anoxygenic photosynthesis dominated at high sulfide levels, 500 μmol liter⁻¹, while the oxygenic reaction became dominant when the sulfide level was reduced below 100 to 300 μmol liter⁻¹ (25 to 75 μmol of H₂S liter⁻¹). An increasing inhibition of the oxygenic photosynthesis was observed upon transition to oxic conditions from increasing sulfide concentrations. Oxygen built up within the Microcoleus layer of the mat even under 5 mmol of sulfide liter⁻¹ (500 μmol of H₂S liter⁻¹) in the overlying water. The implications of such a localized O₂ production in a highly reducing environment are discussed in relation to the evolution of oxygenic photosynthesis during the Proterozoic era.     

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.