Epsilonproteobacteria Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas

Recent studies have indicated that chemoautotrophic Epsilonproteobacteria might play an important role, especially as anaerobic or microaerophilic dark CO₂-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO₂-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of Epsilonproteobacteria to dark CO₂ fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [¹⁴C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10^{5 14}CO₂-fixing prokaryotic cells ml⁻¹ were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10^{4 14}CO₂-fixing cells ml⁻¹ were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance. ¹⁴CO₂-incorporating cells belonged exclusively to the domain Bacteria. Among these, members of the Epsilonproteobacteria were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the Sulfurimonas subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO₂-fixing group. In conclusion, Epsilonproteobacteria were found to be mainly responsible for chemoautotrophic activity in the dark CO₂ fixation maxima of the Black Sea and central Baltic Sea redoxclines. These Epsilonproteobacteria might be relevant in similar habitats of the world's oceans, where high dark CO₂ fixation rates have been measured.     

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.     

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.     

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.     

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.     

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.     

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).     

Hydrogen Metabolism by Decomposing Cyanobacterial Aggregates in Big Soda Lake, Nevada

Hydrogen production by incubated cyanobacterial epiphytes occurred only in the dark, was stimulated by C₂H₂, and was inhibited by O₂. Addition of NO₃⁻ inhibited dark, anaerobic H₂ production, whereas the addition of NH₄⁺ inhibited N₂ fixation (C₂H₂ reduction) but not dark H₂ production. Aerobically incubated cyanobacterial aggregates consumed H₂, but light-incubated rates (3.6 μmol of H₂ g⁻¹ h⁻¹) were statistically equivalent to dark uptake rates (4.8 μmol of H₂ g⁻¹ h⁻¹), which were statistically equivalent to dark, anaerobic production rates (2.5 to 10 μmol of H₂ g⁻¹ h⁻¹). Production rates of H₂ were fourfold higher for aggregates in a more advanced stage of decomposition. Enrichment cultures of H₂-producing fermentative bacteria were recovered from freshly harvested, H₂-producing cyanobacterial aggregates. Hydrogen production in these cyanobacterial communities appears to be caused by the resident bacterial flora and not by the cyanobacteria. In situ areal estimates of dark H₂ production by submerged epiphytes (6.8 μmol of H₂ m⁻² h⁻¹) were much lower than rates of light-driven N₂ fixation by the epiphytic cyanobacteria (310 μmol of C₂H₄ m⁻² h⁻¹).     

Effects of Polychlorinated Biphenyl Congener Concentration and Sediment Supplementation on Rates of Methanogenesis and 2,3,6-Trichlorobiphenyl Dechlorination in an Anaerobic Enrichment

We have employed a method of enrichment that allows us to significantly increase the rate of reductive polychlorinated biphenyl (PCB) dechlorination. This method shortens the time required to investigate the effects that culture conditions have on dechlorination and provides an estimate of the potential activity of the PCB-dechlorinating anaerobes. The periodic supplementation of sterile sediment and PCB produced an enhanced, measurable, and sustained rate of dechlorination. We observed volumetric rates of the dechlorination of 2,3,6-trichlorobiphenyl (2,3,6-CB) to 2,6-dichlorobiphenyl (2,6-CB) of more than 300 μmol liter^-1 day^-1 when the cultures were supplemented daily. A calculation of this activity that is based on an estimate of the number of dechlorinating anaerobes present indicates that 1.13 pmol of 2,3,6-CB was dechlorinated to 2,6-CB day^-1 bacterial cell^-1. This rate is similar to that of the reductive dechlorination of 3-chlorobenzoate by Desulfomonile tiedjei. Methanogenesis declined from 585.3 to 125.9 μmol of CH₄ liter^-1 day^-1, while dechlorination increased from 8.2 to 346.0 μmol of 2,3,6-CB dechlorinated to 2,6-CB liter^-1 day^-1.     

Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM⁻¹ under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO₃⁻ per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N₂ per mol of NO₃⁻). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O₂ per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS⁻¹ day⁻¹, the maximum specific growth rate (μ_max) was 0.0506 day⁻¹, and the Monod half-saturation coefficient (K_s) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ_max, and K_s were 10.7 (±0.11) μmol of CM mg of TSS⁻¹ day⁻¹, 0.145 day⁻¹, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Effect of Sulfide on Nitrogen Fixation in a Stream Sediment-Water System

Nitrogen fixation (C₂H₂ reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C₂H₄ production occurring in the presence of 8 μmol of S²⁻ per ml of stream water. Sodium sulfide at concentrations of 16 μmol of S²⁻ per ml or greater inhibited nitrogen fixation, with 64 μmol of S²⁻ per ml being completely inhibitory. Sulfide at levels of 16 μmol/ml or above inhibited CO₂ production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO₂ production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 μmol of S²⁻ per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H₂, H₂S, and other sulfur species at various pH values, it appeared that H₂S was the toxic entity and that HS⁻ was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS⁻ from H₂S.     

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).     

Denitrification Rates in the Low-Oxygen Waters of the Stratified Baltic Proper

Denitrification activity was shown in the deep, low-oxygen waters of the Baltic proper by both in vitro and in situ methods. The vertical distribution of NO₃⁻ in the water column showed nitrate consumption and NO₂⁻ and N₂O maxima in the deep waters when O₂ was below 0.2 ml liter⁻¹, which is suggestive evidence for denitrification. Direct in situ evidence for denitrification was obtained by finding an N₂ saturation of up to 108% in the deep waters. When these waters were incubated with ¹⁵NO₃⁻, ¹⁵N₂ was produced. Quantification of the denitrification rate done by the addition of C₂H₂ to water samples from the active depths showed a rate of about 0.10 μmol liter⁻¹ day⁻¹.     

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.     

Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea

The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophyll e (BChl e) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter⁻¹. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e. Only traces of BChl e (8 ng liter⁻¹) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m⁻² s⁻¹ at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of the Chlorobiaceae and was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m⁻² s⁻¹ for photosynthetic ¹⁴CO₂ fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.     

Annual Cycle of Bacterial Secondary Production in Five Aquatic Habitats of the Okefenokee Swamp Ecosystem

Rates of bacterial secondary production by free-living bacterioplankton in the Okefenokee Swamp are high and comparable to reported values for a wide variety of marine and freshwater ecosystems. Bacterial production in the water column of five aquatic habitats of the Okefenokee Swamp was substantial despite the acidic (pH 3.7), low-nutrient, peat-accumulating character of the environment. Incorporation of [³H]thymidine into cold-trichloroacetic acid-insoluble material ranged from 0.03 to 2.93 nmol liter⁻¹ day⁻¹) and corresponded to rates of bacterial secondary production of 3.4 to 342.2 μg of carbon liter⁻¹ day⁻¹ (mean, 87.8 μg of carbon liter⁻¹ day⁻¹). Bacterial production was strongly seasonal and appeared to be coupled to annual changes in temperature and primary production. Bacterial doubling times ranged from 5 h to 15 days and were fastest during the warm months of the year, when the biomass of aquatic macrophytes was high, and slowest during the winter, when the plant biomass was reduced. The high rates of bacterial turnover in Okefenokee waters suggest that bacterial growth is an important mechanism in the transformation of dissolved organic carbon into the nutrient-rich bacterial biomass which is utilized by microconsumers.     

Differential responses of the sunn4 and rdn1-1 super-nodulation mutants of Medicago truncatula to elevated atmospheric CO2

Background and AimsNitrogen fixation in legumes requires tight control of carbon and nitrogen balance. Thus, legumes control nodule numbers via an autoregulation mechanism. ‘Autoregulation of nodulation’ mutants super-nodulate are thought to be carbon-limited due to the high carbon-sink strength of excessive nodules. This study aimed to examine the effect of increasing carbon supply on the performance of super-nodulation mutants.MethodsWe compared the responses of Medicago truncatula super-nodulation mutants (sunn-4 and rdn1-1) and wild type to five CO₂ levels (300–850 μmol mol⁻¹). Nodule formation and nitrogen fixation were assessed in soil-grown plants at 18 and 42 d after sowing.Key ResultsShoot and root biomass, nodule number and biomass, nitrogenase activity and fixed nitrogen per plant of all genotypes increased with increasing CO₂ concentration and reached a maximum at 700 μmol mol⁻¹. While the sunn-4 mutant showed strong growth retardation compared with wild-type plants, elevated CO₂ increased shoot biomass and total nitrogen content of the rdn1-1 mutant up to 2-fold. This was accompanied by a 4-fold increase in nitrogen fixation capacity in the rdn1-1 mutant.ConclusionsThese results suggest that the super-nodulation phenotype per se did not limit growth. The additional nitrogen fixation capacity of the rdn1-1 mutant may enhance the benefit of elevated CO₂ for plant growth and N₂ fixation.     

Carbon gain and photosynthetic response of chrysanthemum to photosynthetic photon flux density cycles

Most models of carbon gain as a function of photosynthetic irradiance assume an instantaneous response to increases and decreases in irradiance. High- and low-light-grown plants differ, however, in the time required to adjust to increases and decreases in irradiance. In this study the response to a series of increases and decreases in irradiance was observed in Chrysanthemum × morifolium Ramat. “Fiesta” and compared with calculated values assuming an instantaneous response. There were significant differences between high- and low-light-grown plants in their photosynthetic response to four sequential photosynthetic photon flux density (PPFD) cycles consisting of 5-minute exposures to 200 and 400 micromoles per square meter per second (μmol m⁻²s⁻¹). The CO₂ assimilation rate of high-light-grown plants at the cycle peak increased throughout the PPFD sequence, but the rate of increase was similar to the increase in CO₂ assimilation rate observed under continuous high-light conditions. Low-light leaves showed more variability in their response to light cycles with no significant increase in CO₂ assimilation rate at the cycle peak during sequential cycles. Carbon gain and deviations from actual values (percentage carbon gain over- or underestimation) based on assumptions of instantaneous response were compared under continuous and cyclic light conditions. The percentage carbon gain overestimation depended on the PPFD step size and growth light level of the leaf. When leaves were exposed to a large PPFD increase, the carbon gain was overestimated by 16 to 26%. The photosynthetic response to 100 μmol m⁻² s⁻¹ PPFD increases and decreases was rapid, and the small overestimation of the predicted carbon gain, observed during photosynthetic induction, was almost entirely negated by the carbon gain underestimation observed after a decrease. If the PPFD cycle was 200 or 400 μmol m⁻² s⁻¹, high- and low-light leaves showed a carbon gain overestimation of 25% that was not negated by the underestimation observed after a light decrease. When leaves were exposed to sequential PPFD cycles (200-400 μmol m⁻² s⁻¹), carbon gain did not differ from leaves exposed to a single PPFD cycle of identical irradiance integral that had the same step size (200-400-200 μmol m⁻² s⁻¹) or mean irradiance (200-300-200 μmol m⁻² s⁻¹).     

CO2 Uptake and Fixation by Endosymbiotic Chemoautotrophs from the Bivalve Solemya velum

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve Solemya velum. Symbiont-enriched suspensions were generated by homogenization of S. velum gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with ¹⁴C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO₂ and not with HCO₃⁻ concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO₂ was 28 ± 3 μM, and the average maximal velocity was 50.8 ± 7.1 μmol min⁻¹ g of protein⁻¹. The reliance of S. velum symbionts on extracellular CO₂ is consistent with their intracellular lifestyle, since HCO₃⁻ utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO₂ may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.     

Biodegradation of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Ring Cleavage Product 4-Nitro-2,4-Diazabutanal by Phanerochaete chrysosporium

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO₂ and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH₂NHNO₂, in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium. A soil sample taken from an ammunition plant contained RDX (342 μmol kg⁻¹), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 μmol kg⁻¹), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 μmol kg⁻¹), and traces of NDAB (3.8 μmol kg⁻¹). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 ± 22 μmol kg⁻¹) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N₂O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N₂O. To determine C stoichiometry, we first generated [¹⁴C]NDAB in situ by incubating [¹⁴C]RDX with strain DN22, followed by incubation with the fungus. The production of ¹⁴CO₂ increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus P. chrysosporium may constitute the basis for the development of bioremediation technologies.