Nitrogen dynamics in stream wood samples incubated with [C]lignocellulose and potassium [N]nitrate

Surface wood samples obtained from a Douglas fir log (Pseudotsuga menziesii) in a Pacific Northwest stream were incubated in vitro with [C]lignocellulose in a defined mineral salts medium supplemented with 10 mg of N liter of N-labeled NO(3) (50 atom% N). Evolution of CO(2), distribution and isotopic dilution of N, filtrate N concentrations, and the rates of denitrification, N(2) fixation, and respiration were measured at 6, 12, and 18 days of incubation. The organic N content of the lignocellulose-wood sample mixture had increased from 132 mug of N to a maximum of 231 mug of N per treatment after 6 days of incubation. Rates of [C]lignocellulose decomposition were greatest during the first 6 days and then began to decline over the remaining 12 days. Total CO(2) evolution was also highest at day 6 and declined steadily over the remaining duration of the incubation. Filtrate NH(4)-N increased from background levels to a final value of 57 mug of N per treatment. Filtrate NO(3) N completely disappeared by day 6, and organic N showed a slight decline between days 12 and 18. The majority of the N that could be recovered appeared in the particulate organic fraction by day 6 (41 mug of N), and the filtrate NH(4) N fraction contained 11 mug of N by day 18. The N enrichment values of the filtrate NH(4) and the inorganic N associated with the particulate fraction had increased to approximately 20 atom% N by 18 days of incubation, whereas the particulate organic fraction reached its highest enrichment by day 6. Measurements of N(2) fixation and denitrification indicated an insignificant gain or loss of N from the experimental system by these processes. The data show that woody debris in stream ecosystems might function as a rapid and efficient sink for exogenous N, resulting in stimulation of wood decomposition and subsequent activation of other N cycling processes.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N2O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N₂O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg⁻¹. Without added C, peak NO emissions of 4 μg of N kg⁻¹ h⁻¹ were increased to 15 μg of N kg⁻¹ h⁻¹ by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 μg of N kg⁻¹ h⁻¹ in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 μg of N kg⁻¹ h⁻¹ after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N₂O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N₂O produced during nitrification in soil.     

Use of Aliphatic n-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria

Ammonia (NH₃)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH₃ oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C₈ and C₉. In contrast, NH₃ oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C₈ (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH₄⁺-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH₄⁺-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH₄⁺-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.     

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.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.     

Microcystin Production by Microcystis aeruginosa in a Phosphorus-Limited Chemostat

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C₁₈ cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C₁₈ reverse-phase column. The specific growth rate (μ) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing μ. The MC-LR and MC-RR contents on a dry weight basis were highest at μ of 0.1/day at 339 and 774 μg g⁻¹, respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower μ, whereas the MC-producing rate was linearly proportional to μ. The C fixation rate at an ambient irradiance (160 microeinsteins m⁻² s⁻¹) increased with μ. The ratios of the MC-producing rate to the C fixation rate were higher at a lower μ. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.     

Influence of Starvation on Potential Ammonia-Oxidizing Activity and amoA mRNA Levels of Nitrosospira briensis

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NO_x biosensor. The apparent half-saturation constant [K_m(app)] value of the NH₃ oxidation of N. briensis was 3 μM NH₃ for cultures grown both in continuous and batch cultures as determined by a NO_x biosensor. Cells grown on the wall of the vessel had a lower K_m(app) value of 1.8 μM NH₃. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 μM N h⁻¹, and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH₄⁺, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH₄⁺ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH₄⁺ availability.     

Steady-State Growth and Chemical Composition of the Marine Chlorophyte Dunaliella tertiolecta in Nitrogen-Limited Continuous Cultures

The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, and urea-N limitations. The effect of the nitrogen cell quota (Q_n) on the steady-state growth rate (μ) was the same regardless of the N source. The relationship between μ and Q_n was well described by the Droop equation, but only up to the true maximum growth rate ^μ (= cell washout rate). The ratio between the minimum cell quota (k_Q) and the maximum cell quota (Q_m) was 0.19. Hence, there is no substitute for determining ^μ experimentally. That there was no difference in growth response to different N sources suggests that no internal pooling of inorganic nitrogen occurred. Both the carbon (Q_c) and phosphorus (Q_p) cell quotas under N limitation increased with increasing μ in a threshold fashion: virtually no change in either cell quota up to ~0.8 ^μ, followed by a rapid and large increase up to ^μ. In addition, in the region of low μ, there was an increase in Q_p with a decreasing medium N/P ratio of between 15 and 5 (by atoms). The results generally indicate the physiological limits in cellular constituency under N limitation. The usefulness of this information, however, in describing the response of natural populations of marine phytoplankton to transient nutrient exposures on the temporal and spatial microscales that most likely exist is of limited value.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Denitrification and Ammonia Formation in Anaerobic Coastal Sediments

Simultaneous determinations of nitrogen gas production, ammonia, and particulate organic nitrogen formation in the coastal sediments of Mangoku-Ura, Simoda Bay, and Tokyo Bay were made by using the ¹⁵N-label tracer method. The rate of nitrogen gas production in the sediment surface layer was about 10⁻² μg atom of N per g per h, irrespective of the location of the sediments examined. [¹⁵N]ammonia and -particulate organic nitrogen accounted for 20 to 70% of the three products, and after several hours of incubation, the major fraction of nondenitrified ¹⁵N in Mangoku-Ura and Simoda Bay sediments was recovered as ammonia. In Tokyo Bay sediments, particulate organic nitrogen was produced at a greater rate than was ammonia. The reduction rate data suggest that the pathway of nitrate reduction to ammonia is important in coastal sediments.     

Source and Magnitude of Ammonium Generation in Maize Roots

Studies with ¹⁵N indicate that appreciable generation of NH₄⁺ from endogenous sources accompanies the uptake and assimilation of exogenous NH₄⁺ by roots. To identify the source of NH₄⁺ generation, maize (Zea mays L.) seedlings were grown on ¹⁴NH₄⁺ and then exposed for 3 d to highly labeled ¹⁵NH₄⁺. More of the entering ¹⁵NH₄⁺ was incorporated into the protein-N fraction of roots in darkness (approximately 25%) than in the light (approximately 14%). Although the ¹⁴NH₄⁺ content of roots declined rapidly to less than 1 μmol per plant, efflux of ¹⁴NH₄⁺ continued throughout the 3-d period at an average daily rate of 14 μmol per plant. As a consequence, cumulative ¹⁴NH₄⁺ efflux during the 3-d period accounted for 25% of the total ¹⁴N initially present in the root. Although soluble organic ¹⁴N in roots declined during the 3-d period, insoluble ¹⁴N remained relatively constant. In shoots both soluble organic ¹⁴N and ¹⁴NH₄⁺ declined, but a comparable increase in insoluble ¹⁴N was noted. Thus, total ¹⁴N in shoots remained constant, reflecting little or no net redistribution of ¹⁴N between shoots and roots. Collectively, these observations reveal that catabolism of soluble organic N, not protein N, is the primary source of endogenous NH₄⁺ generation in maize roots.     

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).     

Differences in SOM Decomposition and Temperature Sensitivity among Soil Aggregate Size Classes in a Temperate Grasslands

The principle of enzyme kinetics suggests that the temperature sensitivity (Q₁₀) of soil organic matter (SOM) decomposition is inversely related to organic carbon (C) quality, i.e., the C quality-temperature (CQT) hypothesis. We tested this hypothesis by performing laboratory incubation experiments with bulk soil, macroaggregates (MA, 250–2000 μm), microaggregates (MI, 53–250 μm), and mineral fractions (MF, <53 μm) collected from an Inner Mongolian temperate grassland. The results showed that temperature and aggregate size significantly affected on SOM decomposition, with notable interactive effects (P<0.0001). For 2 weeks, the decomposition rates of bulk soil and soil aggregates increased with increasing incubation temperature in the following order: MA>MF>bulk soil >MI(P <0.05). The Q₁₀ values were highest for MA, followed (in decreasing order) by bulk soil, MF, and MI. Similarly, the activation energies (E_a) for MA, bulk soil, MF, and MI were 48.47, 33.26, 27.01, and 23.18 KJ mol⁻¹, respectively. The observed significant negative correlations between Q₁₀ and C quality index in bulk soil and soil aggregates (P<0.05) suggested that the CQT hypothesis is applicable to soil aggregates. Cumulative C emission differed significantly among aggregate size classes (P <0.0001), with the largest values occurring in MA (1101 μg g⁻¹), followed by MF (976 μg g⁻¹) and MI (879 μg g⁻¹). These findings suggest that feedback from SOM decomposition in response to changing temperature is closely associated withsoil aggregation and highlights the complex responses of ecosystem C budgets to future warming scenarios.     

Growth and Nitrogen Uptake Characteristics Reveal Outbreak Mechanism of the Opportunistic Macroalga Gracilaria tenuistipitata

Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources'' (NH₄ ⁺, NO₃ ⁻) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH₄ ⁺ than that with NO₃ ⁻ addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH₄ ⁺ and NO₃ ⁻ uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH₄ ⁺ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO₃ ⁻ uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters V_max = 37.2 µM g⁻¹ DM h⁻¹ and K_s = 61.5 µM. Further, based on the isotope ¹⁵N tracer method, we found that ¹⁵N from NH₄ ⁺ accumulated faster and reached an atom% twice than that of ¹⁵N from NO₃ ⁻, suggesting when both NH₄ ⁺ and NO₃ ⁻ were available, NH₄ ⁺ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

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.     

Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen

Glutamine is the first major organic product of assimilation of ¹³NH₄⁺ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of ¹³NO₃⁻ by tobacco cells cultured on nitrate. The percentage of organic ¹³N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. ¹³NO₃⁻, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of ¹³N in glutamine, and a concomitant increase in the percentage of ¹³N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NH₄⁺ into glutamine more extensively than it inhibits the incorporation of ¹³N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when ¹³NH₄⁺ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of ¹³NH₄⁺ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NO₃⁻ into glutamate more strongly than it inhibits the incorporation of ¹³N into glutamine, suggesting that the assimilation of ¹³NH₄⁺ derived from ¹³NO₃⁻ may be mediated solely by the glutamine synthetase-glutamate synthase pathway.     

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.     

New Insights into Methyl Bromide Cooxidation by Nitrosomonas europaea Obtained by Experimenting with Moderately Low Density Cell Suspensions

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (~6 × 10⁷ cells ml⁻¹) of the NH₃-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH₄⁺ and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH₄⁺, 18% of the NH₄⁺, and 35% of the NH₄⁺, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH₄⁺-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells⁻¹). Approximately 90% of the NH₃-dependent O₂ uptake activity and the NO₂⁻-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO₂⁻ production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO₂⁻ accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO₂⁻-producing and MeBr-oxidizing activities could not be attributed directly to NH₄⁺ or NH₃ limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO₂⁻ and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH₃-oxidizing activity.