Todd M. Kana

METHODS FOR MEASURING DENITRIFICATION: DIVERSE APPROACHES TO A DIFFICULT PROBLEM

Denitrification, the reduction of the nitrogen (N) oxides, nitrate (NO3-) and nitrite (NO2-), to the gases nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2), is important to primary production, water quality, and the chemistry and physics of the atmosphere at ecosystem, landscape, regional, and global scales. Unfortunately, this process is very difficult to measure, and existing methods are problematic for different reasons in different places at different times. In this paper, we review the major approaches that have been taken to measure denitrification in terrestrial and aquatic environments and discuss the strengths, weaknesses, and future prospects for the different methods. Methodological approaches covered include (1) acetylene-based methods, (2) 15N tracers, (3) direct N2 quantification, (4) N2:Ar ratio quantification, (5) mass balance approaches, (6) stoichiometric approaches, (7) methods based on stable isotopes, (8) in situ gradients with atmospheric environmental tracers, and (9) molecular approaches. Our review makes it clear that the prospects for improved quantification of denitrification vary greatly in different environments and at different scales. While current methodology allows for the production of accurate estimates of denitrification at scales relevant to water and air quality and ecosystem fertility questions in some systems (e.g., aquatic sediments, well-defined aquifers), methodology for other systems, especially upland terrestrial areas, still needs development. Comparison of mass balance and stoichiometric approaches that constrain estimates of denitrification at large scales with point measurements (made using multiple methods), in multiple systems, is likely to propel more improvement in denitrification methods over the next few years.

PHOTOACCLIMATION OF PHOTOSYNTHESIS IRRADIANCE RESPONSE CURVES AND PHOTOSYNTHETIC PIGMENTS IN MICROALGAE AND CYANOBACTERIA1

The photosynthesis‐irradiance response (PE) curve, in which mass‐specific photosynthetic rates are plotted versus irradiance, is commonly used to characterize photoacclimation. The interpretation of PE curves depends critically on the currency in which mass is expressed. Normalizing the light‐limited rate to chl a yields the chl a‐specific initial slope (αchl). This is proportional to the light absorption coefficient (achl), the proportionality factor being the photon efficiency of photosynthesis (φm). Thus, αchl is the product of achl and φm. In microalgae αchl typically shows little (<20%) phenotypic variability because declines of φm under conditions of high‐light stress are accompanied by increases of achl. The variation of αchl among species is dominated by changes in achl due to differences in pigment complement and pigment packaging. In contrast to the microalgae, αchl declines as irradiance increases in the cyanobacteria where phycobiliproteins dominate light absorption because of plasticity in the phycobiliprotein:chl a ratio. By definition, light‐saturated photosynthesis (Pm) is limited by a factor other than the rate of light absorption. Normalizing Pm to organic carbon concentration to obtain PmC allows a direct comparison with growth rates. Within species, PmC is independent of growth irradiance. Among species, PmC covaries with the resource‐saturated growth rate. The chl a:C ratio is a key physiological variable because the appropriate currencies for normalizing light‐limited and light‐saturated photosynthetic rates are, respectively, chl a and carbon. Typically, chl a:C is reduced to about 40% of its maximum value at an irradiance that supports 50% of the species‐specific maximum growth rate and light‐harvesting accessory pigments show similar or greater declines. In the steady state, this down‐regulation of pigment content prevents microalgae and cyanobacteria from maximizing photosynthetic rates throughout the light‐limited region for growth. The reason for down‐regulation of light harvesting, and therefore loss of potential photosynthetic gain at moderately limiting irradiances, is unknown. However, it is clear that maximizing the rate of photosynthetic carbon assimilation is not the only criterion governing photoacclimation.

Effect of irradiances up to 2000 μE m−2 s−1 on marine Synechococcus WH7803—I. Growth, pigmentation, and cell composition

Abstract We grew Synechococcus WH7803 at rates exceeding 1.4 d −1 at irradiances from 200 to 2000 μE m −2 s −1 under continuous light in nutrient replete media with no evidence of photoinhibition. Concentrations of the photosynthetic pigments phycoerythrin, phycocyanin, and chlorophyll a , were inversely related to growth irradiance. Phycoerythrin exhibited the greatest plasticity with the concentration in cells adapted to 30 μE m −2 s −1 being ca . 20 times greater than that in cells adapted to 700 μE m −2 s −1 . Changes in the phycoerythrin: phycocyanin ratio as well as their respective concentrations indicate that phycobilisomes underwent changes in size at irradiances which saturated or nearly saturated growth and underwent changes in number at irradiances which limited growth. Phycoerythrin in high light adapted cells contained 20% in light limited cells. Results support the notion that nutrient replete Synechococcus have the capacity to grow at maximal growth rates in brightly lit oceanic surface mixed layers.

Denitrification in coastal ecosystems: methods, environmental controls, and ecosystem level controls, a review

In this review of sediment denitrification in estuaries and coastal ecosystems, we examine current denitrification measurement methodologies and the dominant biogeochemical controls on denitrification rates in coastal sediments. Integrated estimates of denitrification in coastal ecosystems are confounded by methodological difficulties, a lack of systematic understanding of the effects of changing environmental conditions, and inadequate attention to spatial and temporal variability to provide both seasonal and annual rates. Recent improvements in measurement techniques involving 15 N techniques and direct N2 concentration changes appear to provide realistic rates of sediment denitrification. Controlling factors in coastal systems include concentrations of water column NO3−, overall rates of sediment carbon metabolism, overlying water oxygen concentrations, the depth of oxygen penetration, and the presence/absence of aquatic vegetation and macrofauna. In systems experiencing environmental change, either degradation or improvement, the importance of denitrification can change. With the eutrophication of the Chesapeake Bay, the overall rates of denitrification relative to N loading terms have decreased, with factors such as loss of benthic habitat via anoxia and loss of submerged aquatic vegetation driving such effects.

Harmful algal blooms in the Chesapeake and coastal bays of Maryland, USA: Comparison of 1997, 1998, and 1999 events

Harmful algal blooms in the Chesapeake Bay and coastal bays of Maryland, USA, are not a new phenomenon, but may be increasing in frequency and diversity. Outbreaks ofPfiesteria piscicida (Dinophyceae) were observed during 1997 in several Chesapeake Bay tributaries, while in 1998,Pfiesteria-related events were not found but massive blooms ofProrocentrum minimum (Dinophyceae) occurred. In 1999,Aureococcus anophagefferens (Pelagophyceae) developed in the coastal bays in early summer in sufficient densities to cause a brown tide. In 1997, toxicPfiesteria was responsible for fish kills at relatively low cell densities. In 1998 and 1999, the blooms ofP. minimum andA. anophagefferens were not toxic, but reached sufficiently high densities to have ecological consequences. These years differed in the amount and timing of rainfall events and resulting nutrient loading from the largely agricultural watershed. Nutrient loading to the eastern tributaries of Chesapeake Bay has been increasing over the past decade. Much of this nutrient delivery is in organic form. The sites of thePfiesteria outbreaks ranked among those with the highest organic loading of all sites monitored bay-wide. The availability of dissolved organic carbon and phosphorus were also higher at sites experiencingA. anophagefferens blooms than at those without blooms. The ability to supplement photosynthesis with grazing or organic substrates and to use a diversity of organic nutrients may play a role in the development and maintenance of these species. ForP. minimum andA. anophagefferens, urea is used preferentially over nitrate.Pfiesteria is a grazer, but also has the ability to take up nutrients directly. The timing of nutrient delivery may also be of critical importance in determining the success of certain species.

Simultaneous Measurement of Denitrification and Nitrogen Fixation Using Isotope Pairing with Membrane Inlet Mass Spectrometry Analysis

ABSTRACT A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N2 gas species (28N2,29N2, and 30N2) were monitored during incubation experiments after the addition of15NO3−. Formulas were developed to estimate the production (denitrification) and consumption (N2 fixation) of N2 gas from the fluxes of the different isotopic forms of N2. Proportions of the three isotopic forms produced from15NO3− and14NO3− agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 μg-atoms of N m−2 h−1. They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N2 production rates due to denitrification could be separated from high N2 consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 μg-atoms of N m−2 h−1) were lower than nitrogen fixation rates (average, 60 μg-atoms of N m−2 h−1). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N2 isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.

An in situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)

Direct comparisons between photosynthetic O2 evolution rate and electron transport rate (ETR) were made in situ over 24 h using the benthic macroalga Ulva lactuca (Chlorophyta), growing and measured at a depth of 1.8 m, where the midday irradiance rose to 400–600 μmol photons m−2 s−1. O2 exchange was measured with a 5-chamber data-logging apparatus and ETR with a submersible pulse amplitude modulated (PAM) fluorometer (Diving-PAM). Steady-state quantum yield ((Fm′−Ft)/Fm′) decreased from 0.7 during the morning to 0.45 at midday, followed by some recovery in the late afternoon. At low to medium irradiances (0–300 μmol photons m−2 s−1), there was a significant correlation between O2 evolution and ETR, but at higher irradiances, ETR continued to increase steadily, while O2 evolution tended towards an asymptote. However at high irradiance levels (600–1200 μmol photons m−2 s−1) ETR was significantly lowered. Two methods of measuring ETR, based on either diel ambient light levels and fluorescence yields or rapid light curves, gave similar results at low to moderate irradiance levels. Nutrient enrichment (increases in [NO3−], [NH4+] and [HPO42-] of 5- to 15-fold over ambient concentrations) resulted in an increase, within hours, in photosynthetic rates measured by both ETR and O2 evolution techniques. At low irradiances, approximately 6.5 to 8.2 electrons passed through PS II during the evolution of one molecule of O2, i.e., up to twice the theoretical minimum number of four. However, in nutrient-enriched treatments this ratio dropped to 5.1. The results indicate that PAM fluorescence can be used as a good indication of the photosynthetic rate only at low to medium irradiances.

Fast repetition rate and pulse amplitude modulation chlorophyll a fluorescence measurements for assessment of photosynthetic electron transport in marine phytoplankton

Pulse amplitude modulation (PAM) and fast repetition rate (FRR) fluorescence are currently used to estimate photosynthetic quantum yields and photosynthetic rates in aquatic systems. Here we compare simultaneous measurements of the photochemical efficiency of photosystem II obtained from the two techniques and independent estimates of the rate of light absorption by photosystem II. We measured the light-dependencies of the photochemical efficiency of photosystem II (Fq′/Fm′) in five phytoplankters using FRR and Xe-PAM approaches. The FRR and PAM estimates were related in a non-linear fashion. At low irradiances, Fq′/Fm′ measured using PAM fluorescence exceeded Fq′/Fm′ measured using FRR fluorescence by about 20%. At high irradiances, measurements of Fq′/Fm′ from the two approaches converged. The differences in Fq′/Fm′ reflect the distinct techniques by which FRR and PAM protocols excite PSII and are amplified when estimating electron transfer rates as a result of the irradiance term. We also found that measurements of the effective light absorption cross-section for photosystem II obtained by FRR fluorescence compared well with estimates obtained from measured light absorption and photosynthetic unit size. Finally, we compared the photon efficiency of gross oxygen evolution from measurements of gross oxygen evolution and light absorption (ΦPO2 18) with FRR measurements of Fq′/Fm′. We found that measurements of Fq′/Fm′ were highly linearly correlated, but were lower by a factor of ∼1.5, than ΦPO2 18.

Effect of irradiances up to 2000 μE m−2 s−1 on marine Synechococcus WH7803—II. Photosynthetic responses and mechanisms

Abstract We investigated the photosynthetic behavior of Synechococcus WH7803 when grown over the irradiance range of 30–2000 μE m −2 s −1 in nutrient-replete, continuous light, pre-adapted batch cultures. For each of 8 growth irradiances investigated, we found a unique photosynthesis vs irradiance ( P vs I ) relationship. Cell- and carbon-specific photosynthetic light harvesting efficiencies (α (cell) and α (C) ) decreased 10-fold from the lowest to highest growth irradiances. Chlorophyll and phycocyanin-specific efficiencies also decreased but to a lesser extent. Phycoerythrin-specific efficiencies increased ca . 2-fold with increasing irradiances. Photosynthetic capacity ( P max ) approximated the in situ rate of photosynthesis ( P i ) only at growth irradiances which saturated growth rate; at light limiting irradiances, P max increased to 2.9 times P i . Photoinhibition of photosynthesis was only observed in light-limited cells. The mechanism of photoadaptation which resulted in the observed growth rate vs irradiance (μ vs I ) response involved regulation of α (cell) by changes in phycobilisome size over saturating irradiances and phycobilisome numbers over limiting irradiances. Our results are consistent with previously reported field observations of α (Chl) , α (cell) and P max increasing with depth, and of no photoinhibition of near-surface collected assemblages, suggesting that high irradiances in near-surface waters can support maximal growth rates of Synechococcus . These results raise important questions regarding the interpretation of parameters of P vs I measurement from field studies. Our results do not conform to the previously reported hypothesis that a significant percentage of phycoerythrin serves as a non-photosynthetic nitrogen storage compound in these cells.

Ecological Stoichiometry, Biogeochemical Cycling, Invasive Species, and Aquatic Food Webs: San Francisco Estuary and Comparative Systems

Eutrophication has altered food webs across aquatic systems, but effects of nutrient stoichiometry (varying nutrient ratios) on ecosystem structure and function have received less attention. A prevailing assumption has been that nutrients are not ecologically relevant unless concentrations are limiting to phytoplankton. However, changes in nutrient stoichiometry fundamentally affect food quality at all levels of the food web. Here, 30-year records of nitrogen and phosphorus concentrations and ratios, phytoplankton, zooplankton, macroinvertebrates, and fish in the San Francisco Estuary (Bay Delta) were examined to collectively interpret ecosystem changes within the framework of ecological stoichiometry. Changes in nutrient concentrations and nutrient ratios over time fundamentally affect biogeochemical nutrient dynamics that can lead to conditions conducive to invasions of rooted macrophytes and bivalve molluscs, and the harmful cyanobacterium Microcystis. Several other aquatic ecosystems considered here have exhibited similar changes in food webs linked to stoichiometric changes. Nutrient stoichiometry is thus suggested to be a significant driver of food webs in the Bay Delta by altering food quality and biogeochemical dynamics. Since nitrogen-to-phosphorus ratios have increased over time, an overall implication is that remediation of fish populations in the San Francisco Estuary will require significant nitrogen reductions to restore the historic ecological stoichiometric balance and the food web.