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).     

A New Sensitive Bioassay for Determination of Microbially Available Phosphorus in Water

The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water. However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters. Even a very low concentration of phosphorus (below 1 μg of P liter⁻¹) can promote extensive microbial growth. We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 μg of P liter⁻¹. The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of Pseudomonas fluorescens P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth. Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 ± 9,400 CFU/μg of PO₄-P. A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 μg of PO₄-P liter⁻¹. The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 μg of P liter⁻¹ (median, 0.60 μg of P liter⁻¹).     

Assessing Phytoplankton and Bacterioplankton Production During Early Spring in Lake Erken, Sweden

The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5°C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The ¹⁴C and O₂ methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO₃ N assimilation ratio was 1.57. The total integrated incorporation of [¹⁴C]bicarbonate into particulate material (>1 μm) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 × 10⁹ to 1.52 × 10⁹ cells liter⁻¹ (~0.5 μg of C liter⁻¹ day⁻¹). Estimates of bacterioplankton production from rates of [³H]thymidine incorporation were ca. 1.2 to 1.7 μg of C liter⁻¹ day⁻¹. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 μg of C liter⁻¹ day⁻¹, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [³H]thymidine incorporation method.     

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “Protaminobacter rubrum”

“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a ³²⁵RLDRD³²⁹ motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg³²⁵ and Arg³²⁸ in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R³²⁵Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter⁻¹ and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter⁻¹) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g⁻¹ with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Ethylene Removal at Low Temperatures under Biofilter and Batch Conditions

Removal of the plant hormone ethylene (C₂H₄) is often required by horticultural storage facilities, which are operated at temperatures below 10°C. The aim of this study was to demonstrate an efficient, biological C₂H₄ removal under such low-temperature conditions. Peat-soil, acclimated to degradation of C₂H₄, was packed in a biofilter (687 cm³) and subjected to an airflow (~73 ml min⁻¹) with 2 ppm (μl liter⁻¹) C₂H₄. The C₂H₄ removal efficiencies achieved at 20, 10, and 5°C, respectively, were 99.0, 98.8, and 98.4%. This corresponded to C₂H₄ levels of 0.022 to 0.032 ppm in the biofilter outlet air. At 2°C, the average C₂H₄ removal efficiency dropped to 83%. The detailed temperature response of C₂H₄ removal was tested under batch conditions by incubation of 1-g soil samples in a temperature gradient ranging from 0 to 29°C with increments of 1°C. The C₂H₄ removal rate was highest at 26°C (0.85 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹), but remained at levels of 0.14 to 0.28 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹ at 0 to 10°C. At 35 to 40°C, the C₂H₄ removal rate was negligible (0.02 to 0.06 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹). The Q₁₀ (i.e., the ratio of rates 10°C apart) for C₂H₄ removal was 1.9 for the interval 0 to 10°C. In conclusion, the present results demonstrated microbial C₂H₄ removal, which proceeded at 0 to 2°C and produced a moderately psychrophilic temperature response.     

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.     

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).     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

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.     

Minimization of Glycerol Production during the High-Performance Fed-Batch Ethanolic Fermentation Process in Saccharomyces cerevisiae, Using a Metabolic Model as a Prediction Tool

On the basis of knowledge of the biological role of glycerol in the redox balance of Saccharomyces cerevisiae, a fermentation strategy was defined to reduce the surplus formation of NADH, responsible for glycerol synthesis. A metabolic model was used to predict the operating conditions that would reduce glycerol production during ethanol fermentation. Experimental validation of the simulation results was done by monitoring the inlet substrate feeding during fed-batch S. cerevisiae cultivation in order to maintain the respiratory quotient (RQ) (defined as the CO₂ production to O₂ consumption ratio) value between 4 and 5. Compared to previous fermentations without glucose monitoring, the final glycerol concentration was successfully decreased. Although RQ-controlled fermentation led to a lower maximum specific ethanol production rate, it was possible to reach a high level of ethanol production: 85 g · liter⁻¹ with 1.7 g · liter⁻¹ glycerol in 30 h. We showed here that by using a metabolic model as a tool in prediction, it was possible to reduce glycerol production in a very high-performance ethanolic fermentation process.     

Continuous and Batch Production of Chloroperoxidase by Mycelial Pellets of Caldariomyces fumago in an Airlift Fermentor

Batch and continuous production of the extracellular heme glycoprotein chloroperoxidase (CPO) was studied with an airlift fermentor. We induced Caldariomyces fumago CMI 89362 to form pellets by transferring a small inoculum volume in preculture prior to growth in a 1-liter fermentor. Continuous replacement of the fructose-salts medium (dilution rate, 0.008 h⁻¹) supported continuous CPO formation at an average concentration of 128 ± 10 mg of CPO liter⁻¹ for 8 days. Optimum CPO production rates averaged 1.2 ± 0.1 mg of CPO h⁻¹ at dilution rates below 0.033 h⁻¹. Varying the carbohydrate content of the feed solution or the time of starting the feed did not significantly alter the amount of CPO produced. Batch fermentation in the airlift fermentor resulted in maximum CPO concentrations of 280 ± 80 mg of CPO liter⁻¹, although on two separate occasions CPO concentrations reached 400 to 450 mg liter⁻¹, which was double the amount obtained by free hyphae in shake flask culture.     

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.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

New and Fast Method To Quantify Respiration Rates of Bacterial and Plankton Communities in Freshwater Ecosystems by Using Optical Oxygen Sensor Spots

A new method of respiration rate measurement based on oxygen luminescence quenching in sensor spots was evaluated for the first time for aquatic bacterial communities. The commonly used Winkler and Clark electrode methods to quantify oxygen concentration both require long incubation times, and the latter additionally causes signal drift due to oxygen consumption at the cathode. The sensor spots proved to be advantageous over those methods in terms of precise and quick oxygen measurements in natural bacterial communities, guaranteeing a respiration rate estimate during a time interval short enough to neglect variations in organism composition, abundance, and activity. Furthermore, no signal drift occurs during measurements, and respiration rate measurements are reliable even at low temperatures and low oxygen consumption rates. Both a natural bacterioplankton sample and a bacterial isolate from a eutrophic river were evaluated in order to optimize the new method for aquatic microorganisms. A minimum abundance of 2.2 × 10⁶ respiring cells ml⁻¹ of a bacterial isolate was sufficient to obtain a distinct oxygen depletion signal within 20 min at 20°C with the new oxygen sensor spot method. Thus, a culture of a bacterial isolate from a eutrophic river (OW 144; 20 × 10⁶ respiring bacteria ml⁻¹) decreased the oxygen saturation about 8% within 20 min. The natural bacterioplankton sample respired 2.8% from initially 94% oxygen-saturated water in 30 min. During the growth season in 2005, the planktonic community of a eutrophic river consumed between 0.7 and 15.6 μmol O₂ liter⁻¹ h⁻¹. The contribution of bacterial respiration to the total plankton community oxygen consumption varied seasonally between 11 and 100%.     

(Per)chlorate Reduction by the Thermophilic Bacterium Moorella perchloratireducens sp. nov., Isolated from Underground Gas Storage

A thermophilic bacterium, strain An10, was isolated from underground gas storage with methanol as a substrate and perchlorate as an electron acceptor. Cells were gram-positive straight rods, 0.4 to 0.6 μm in diameter and 2 to 8 μm in length, growing as single cells or in pairs. Spores were terminal with a bulged sporangium. The temperature range for growth was 40 to 70°C, with an optimum at 55 to 60°C. The pH optimum was around 7. The salinity range for growth was between 0 and 40 g NaCl liter⁻¹ with an optimum at 10 g liter⁻¹. Strain An10 was able to grow on CO, methanol, pyruvate, glucose, fructose, cellobiose, mannose, xylose, and pectin. The isolate was able to respire with (per)chlorate, nitrate, thiosulfate, neutralized Fe(III) complexes, and anthraquinone-2,6-disulfonate. The G+C content of the DNA was 57.6 mol%. On the basis of 16S rRNA analysis, strain An10 was most closely related to Moorella thermoacetica and Moorella thermoautotrophica. The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell extracts. Strain An10 is the first thermophilic and gram-positive bacterium with the ability to use (per)chlorate as a terminal electron acceptor.     

Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM⁻¹, and there was 95% inhibition at ~30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM⁻¹; 95% inhibition at ~70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM⁻¹, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ~50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H2-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H₂-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 ± 0.19 mol%), a phospholipid pattern of type II, MK-9(H₄) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t_d = 8 h), H₂ plus CO₂ (t_d = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H₂, nickel was not necessary. The optimum growth temperature was 65°C; no growth was observed below 40°C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70°C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K_m was 88 μl of CO liter⁻¹ and V_max was 20.2 μl of CO h⁻¹ mg of protein⁻¹. The threshold value of S. thermoautotrophicus of 0.2 μl of CO liter⁻¹ was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 μmol min⁻¹ mg of protein⁻¹. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Fungal Growth, Production, and Sporulation during Leaf Decomposition in Two Streams

I examined the activity of fungi associated with yellow poplar (Liriodendron tulipifera) and white oak (Quercus alba) leaves in two streams that differed in pH and alkalinity (a hardwater stream [pH 8.0] and a softwater stream [pH 6.7]) and contained low concentrations of dissolved nitrogen (<35 μg liter⁻¹) and phosphorus (<3 μg liter⁻¹). The leaves of each species decomposed faster in the hardwater stream (decomposition rates, 0.010 and 0.007 day⁻¹ for yellow poplar and oak, respectively) than in the softwater stream (decomposition rates, 0.005 and 0.004 day⁻¹ for yellow poplar and oak, respectively). However, within each stream, the rates of decomposition of the leaves of the two species were not significantly different. During the decomposition of leaves, the fungal biomasses determined from ergosterol concentrations, the production rates determined from rates of incorporation of [¹⁴C]acetate into ergosterol, and the sporulation rates associated with leaves were dynamic, typically increasing to maxima and then declining. The maximum rates of fungal production and sporulation associated with yellow poplar leaves were greater than the corresponding rates associated with white oak leaves in the hardwater stream but not in the softwater stream. The maximum rates of fungal production associated with the leaves of the two species were higher in the hardwater stream (5.8 mg g⁻¹ day⁻¹ on yellow poplar leaves and 3.1 mg g⁻¹ day⁻¹ on oak leaves) than in the softwater stream (1.6 mg g⁻¹ day⁻¹ on yellow poplar leaves and 0.9 mg g⁻¹ day⁻¹ on oak leaves), suggesting that effects of water chemistry other than the N and P concentrations, such as pH or alkalinity, may be important in regulating fungal activity in streams. In contrast, the amount of fungal biomass (as determined from ergosterol concentrations) on yellow poplar leaves was greater in the softwater stream (12.8% of detrital mass) than in the hardwater stream (9.6% of detrital mass). This appeared to be due to the decreased amount of fungal biomass that was converted to conidia and released from the leaf detritus in the softwater stream.     

Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments

Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at μg liter⁻¹ levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 μg C liter⁻¹ in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10⁻² liter·μg⁻¹ of C·h⁻¹) ≫ maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10⁻² liter·μg⁻¹ of C·h⁻¹). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell⁻¹ level was achieved at ≤10 μg C liter⁻¹ than at >10 μg C liter⁻¹ with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at μg liter⁻¹ levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.