Determination of dissolved carbon dioxide by coulometric titration in modified atmosphere systems

The solubility of carbon dioxide (CO₂) in microbiological media at different pH values, water activities ( a_w ), temperatures, buffering capacities and ratios of headspace to media volumes was determined by using a coulometer. Buffering capacity and ratio of headspace to media volume were shown to be the major factors influencing the solubility of CO₂ in modified atmosphere model systems. The growth inhibitory effects of different dissolved CO₂ concentrations (0–50 μmol ml^-1) were determined for Pseudomonas fragi at 8°C and 22 C. Pseudomonas fragi was shown to be strongly affected by the CO₂ concentration in the media. A carbon dioxide concentration of 40 μmol ml^-1 was needed to inhibit Ps. fragi at 8°C. The importance of measuring dissolved CO₂ concentrations in modified atmosphere packaging applications was shown and the coulometer proved to be an excellent tool for this purpose.     

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat

The temperature dependence of C₃ photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO₂ on photosynthesis temperature response have been investigated in wheat ( Triticum aestivum L.) grown in controlled chambers with 370 or 700 μmol mol⁻¹ CO₂ from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO₂ slightly decreased the CO₂ compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V_cmax, although the latter effect was reversed at 15°C. With elevated CO₂, J_max decreased in the 15–25°C temperature range and increased at 30 and 35°C. The temperature response (activation energy) of V_cmax and J_max increased with growth in elevated CO₂. CO₂ enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO₂. We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO₂ on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.     

What physiological acclimation supports increased growth at high CO2 conditions?

Chlamydomonas acidophila Negoro is a green algal species abundant in acidic waters (pH 2–3.5), in which inorganic carbon is present only as CO₂. Previous studies have shown that aeration with CO₂ increased its maximum growth rate, suggesting CO₂ limitation under natural conditions. To unravel the underlying physiological mechanisms at high CO₂ conditions that enables increased growth, several physiological characteristics from high- and low-CO₂-acclimated cells were studied: maximum quantum yield, photosynthetic O₂ evolution (P_max), affinity constant for CO₂ by photosynthesis (K_0.5,p), a CO₂-concentrating mechanism (CCM), cellular Rubisco content and the affinity constant of Rubisco for CO₂ (K_0.5,r). The results show that at high CO₂ concentrations, C. acidophila had a higher K_0.5,p, P_max, maximum quantum yield, switched off its CCM and had a lower Rubisco content than at low CO₂ conditions. In contrast, the K_0.5,r was comparable under high and low CO₂ conditions. It is calculated that the higher P_max can already explain the increased growth rate in a high CO₂ environment. From an ecophysiological point of view, the increased maximum growth rate at high CO₂ will likely not be realised in the field because of other population regulating factors and should be seen as an acclimation to CO₂ and not as proof for a CO₂ limitation.     

Biomass Production and Carbohydrate Content of Arabidopsis thaliana at Atmospheric CO2 Concentrations from 390 to 1680 μl l-1

Abstract: The concentration dependency of the impact of elevated atmospheric CO₂ concentrations on Arabidopsis thaliana L. was studied. Plants were exposed to nearly ambient (390), 560, 810, 1240 and 1680 μl I^-1 CO₂ during the vegetative growth phase for 8 days. Shoot biomass production and dry matter content were increased upon exposure to elevated CO₂. Maximal increase in shoot fresh and dry weight was obtained at 560 μl I^-1 CU₂, which was due to a transient stimulation of the relative growth rate for up to 3 days. The shoot starch content increased with increasing CO₂ concentrations up to two-fold at 1680 μl I^-1 CO₂, whereas the contents of soluble sugars and phenolic compounds were hardly affected by elevated CO₂. The chlorophyll and carotenoid contents were not substantially affected at elevated CO₂ and the chlorophyll a/b ratio remained unaltered. There was no acclimation of photosynthesis at elevated CO₂; the photosynthetic capacity of leaves, which had completely developed at elevated CO₂ was similar to that of leaves developed in ambient air. The possible consequences of an elevated atmospheric CO₂ concentration to Arabidopsis thaliana in its natural habitat is discussed.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

Stomatal responses to CO2 during a diel Crassulacean acid metabolism cycle in Kalanchoe daigremontiana and Kalanchoe pinnata

To investigate the diurnal variation of stomatal sensitivity to CO₂, stomatal response to a 30 min pulse of low CO₂ was measured four times during a 24 h time-course in two Crassulacean acid metabolism (CAM) species Kalanchoe daigremontiana and Kalanchoe pinnata , which vary in the degree of succulence, and hence, expression and commitment to CAM. In both species, stomata opened in response to a reduction in p CO₂ in the dark and in the latter half of the light period, and thus in CAM species, chloroplast photosynthesis is not required for the stomatal response to low p CO₂. Stomata did not respond to a decreased p CO₂ in K. daigremontiana in the light when stomata were closed, even when the supply of internal CO₂ was experimentally reduced. We conclude that stomatal closure during phase III is not solely mediated by high internal p CO₂, and suggest that in CAM species the diurnal variability in the responsiveness of stomata to p CO₂ could be explained by hypothesizing the existence of a single CO₂ sensor which interacts with other signalling pathways. When not perturbed by low p CO₂, CO₂ assimilation rate and stomatal conductance were correlated both in the light and in the dark in both species.     

Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol⁻¹ CO₂) and double ambient (720 μmol mol⁻¹ CO₂) carbon dioxide (CO₂) mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density) and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO₂ concentration on the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf variation. In general, a doubling of the atmospheric CO₂ concentration enhanced plant growth and significantly increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated CO₂ concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate. However, no significant differences were found for the maximum rate of carboxylation ( V _cmax) and the light saturated rate of electron transport ( J _max) between the control and elevated CO₂ treatment.     

Light period of Crassulacean Acid Metabolism: Control of carbon transfer from malic acid to carbohydrates by CO2 concentration

In the CAM plants, Kalanchoë tubiflora (Harvey) Hasset, Sedum morganianum E. Walth and Sedum rubrotinctum R. T. Clausen, the effects of CO₂ concentrations on the light-dependent ¹⁴C transfer from the nocturnally synthetized [14C]-malic acid to starch have been studied. CO₂ concentrations up to 5 × 103 μ1 1–1 did not inhibit this carbon transfer. Higher CO₂ concentrations, however, were increasingly inhibitory. At 104 μl 1–1 CO₂, the carbon transfer was practically prevented.
The malic acid consumption in the light showed the same response to CO₂ concentrations as the [^l4C]-transfer. Photosynthesis itself was not inhibited by the CO₂ concentrations applied. It is assumed that, during phase III of CAM, light controls the internal CO₂ concentration via photosynthesis; and that the internal CO2 concentration then controls the rate of malate decarboxylation.     

Disturbance, rainfall and contrasting species responses mediated aboveground biomass response to 11 years of CO2 enrichment in a Florida scrub-oak ecosystem

This study reports the aboveground biomass response of a fire-regenerated Florida scrub-oak ecosystem exposed to elevated CO₂ (1996–2007), from emergence after fire through canopy closure. Eleven years exposure to elevated CO₂ caused a 67% increase in aboveground shoot biomass. Growth stimulation was sustained throughout the experiment; although there was significant variability between years. The absolute stimulation of aboveground biomass generally declined over time, reflecting increasing environmental limitations to long-term growth response. Extensive defoliation caused by hurricanes in September 2004 was followed by a strong increase in shoot density in 2005 that may have resulted from reopening the canopy and relocating nitrogen from leaves to the nutrient-poor soil. Biomass response to elevated CO₂ was driven primarily by stimulation of growth of the dominant species, Quercus myrtifolia , while Quercus geminata , the other co-dominant oak, displayed no significant CO₂ response. Aboveground growth also displayed interannual variation, which was correlated with total annual rainfall. The rainfall × CO₂ interaction was partially masked at the community level by species-specific responses: elevated CO₂ had an ameliorating effect on Q. myrtifolia growth under water stress. The results of this long-term study not only show that atmospheric CO₂ concentration had a consistent stimulating effect on aboveground biomass production, but also showed that available water is the primary driver of interannual variation in shoot growth and that the long-term response to elevated CO₂ may have been caused by other factors such as nutrient limitation and disturbance.     

Effects of elevated CO2 concentration on photosynthesis, respiration and carbohydrate status of coppice Populus hybrids

To determine how increased atmospheric CO₂ will affect the physiology of coppiced plants, sprouts originating from two hybrid poplar clones ( Populus trichocarpa × P. deltoides - Beaupre and P. deltoides × P. nigra - Robusta) were grown in open-top chambers containing ambient or elevated (ambient + 360 μmol mol⁻¹) CO₂ concentration. The effects of elevated CO₂ concentration on leaf photosynthesis, stomatal conductance, dark respiration, carbohydrate concentration and nitrogen concentration were measured. Furthermore, dark respiration of leaves was partitioned into growth and maintenance components by regressing specific respiration rate vs specific growth rate. Sprouts of both clones exposed to CO₂ enrichment showed no indication of photosynthetic down-regulation. During reciprocal gas exchange measurements, CO₂ enrichment significantly increased photosynthesis of all sprouts by approximately 60% ( P < 0.01) on both an early and late season sampling date, decreased stomatal conductance of all sprouts by 10% ( P < 0.04) on the early sampling date and nonsignificantly decreased dark respiration by an average of 11%. Growth under elevated CO₂ had no consistent effect on foliar sugar concentration but significantly increased foliar starch by 80%. Respiration rate was highly correlated with both specific growth rate and percent nitrogen. Long-term CO₂ enrichment did not significantly affect the maintenance respiration coefficient or the growth respiration coefficient. Carbon dioxide enrichment affected the physiology of the sprouts the same way it affected these plants before they were coppiced.     

CO2 enrichment and development of freezing tolerance in Norway spruce

Plant growth and adaptation to cold and freezing temperatures in a CO₂-enriched atmosphere have received little attention despite their predicted effects on plant distribution and productivity. In this study we looked at the interaction between elevated CO₂ and development of freezing tolerance in Norway spruce ( Picea abies (L.) Karst.). First-year seedlings were grown under controlled conditions in an atmosphere enriched in CO₂ (70 Pa) for one simulated growth season. We measured shoot growth, registered the timing of growth cessation and bud set, measured needle net photosynthetic rate, and determined needle carbohydrate concentration (fructose+pinitol, glucose, sucrose, inositol, raffinose and starch). Freezing tolerance (LT₅₀) was determined after exposing whole seedlings to temperatures ranging from −6.5 to −36.0°C and scoring for visual needle browning. Elevated CO₂ did not affect height growth or the timing of growth cessation and bud set. The only statistically significant effects of CO₂ treatment were on seedling dry weight, percent dry matter and starch content. During the three weeks after growth cessation and bud set, freezing tolerance increased from −10 to −35°C, and there was a marked increase in all soluble sugars except inositol. However, neither freezing tolerance nor the concentration of soluble sugars was significantly influenced by elevated CO₂.     

Stomata of trees growing in CO2-enriched air show reduced sensitivity to vapour pressure deficit and drought

Stomatal conductance ( g _s) and photosynthetic rate ( A ) were measured in young beech ( Fagus sylvatica ), chestnut ( Castanea sativa ) and oak ( Quercus robur ) growing in ambient or CO₂-enriched air. In oak, g _s was consistently reduced in elevated CO₂. However, in beech and chestnut, the stomata of trees growing in elevated CO₂ failed to close normally in response to increased leaf-to-air vapour pressure deficit (LAVPD). Consequently, while g _s was reduced in elevated CO₂ on days with low LAVPD, on warm sunny days (with correspondingly high LAVPD) g _s was unchanged or even slightly higher in elevated CO₂. Furthermore, during drought, g _s of beech and chestnut was unresponsive to [CO₂], over a wide range of ambient LAVPD, whereas in oak g _s was reduced by an average of 50% in elevated CO₂. Stimulation of A by elevated CO₂ in beech and chestnut was restricted to days with high irradiance, and was greatest in beech during drought. Hence, most of the additional carbon gain in elevated CO₂ was made at the expense of water economy, at precisely those times (drought, high evaporative demand) when water conservation was most important. Such effects could have serious consequences for drought tolerance, growth and, ultimately, survival as atmospheric [CO₂] increases.     

Respiration in a future, higher-CO2 world

Abstract. Apart from its impact on global warming, the annually increasing atmospheric [CO₂] is of interest to plant scientists primarily because of its direct influence on photosynthesis and photorespiration in C₃ species. But in addition, 'dark' respiration, another major component of the carbon budget of higher plants, may be affected by a change in [CO₂] independent of an increase in temperature. Literature pertaining to an impact of [CO₂] on respiration rate is reviewed. With an increase in [CO₂], respiration rate is increased in some cases, but decreased in others. The effects of [CO₂] on respiration rate may be direct or indirect. Mechanisms responsible for various observations are proposed. These proposed mechanisms relate to changes in: (1) levels of nonstructural carbohydrates, (2) growth rate and structural phytomass accumulation, (3) composition of phytomass, (4) direct chemical interactions between CO₂ and respiratory enzymes, (5) direct chemical interactions between CO₂ and other cellular components, (6) dark CO₂ fixation rate, and (7) ethylene biosynthesis rate. Because a range-of (possibly interactive) effects exist, and present knowledge is limited, the impact of future [CO₂] on respiration rate cannot be predicted. Theoretical considerations and types of experiments that can lead to an increase in the understanding of this issue are outlined.     

Increasing growth temperature reduces the stimulatory effect of elevated CO2 on photosynthesis or biomass in two perennial species

We examined how anticipated changes in CO₂ concentration and temperature interacted to alter plant growth, harvest characteristics and photosynthesis in two cold-adapted herbaceous perennials, alfalfa ( Medicago sativa L. cv. Arc) and orchard grass ( Dactylis glomerata L. cv. Potomac). Plants were grown at two CO₂ concentrations (362 [ambient] and 717 [elevated] μmol mol⁻¹ CO₂) and four constant day/night temperatures of 15, 20, 25 and 30°C in controlled environmental chambers. Elevated CO₂ significantly increased total plant biomass and protein over a wide range of temperatures in both species. Stimulation of photosynthetic rate, however, was eliminated at the highest growth temperature in M. sativa and relative stimulation of plant biomass and protein at high CO₂ declined as temperature increased in both species. Lack of a synergistic effect between temperature and CO₂ was unexpected since elevated CO₂ reduces the amount of carbon lost via photorespiration and photorespiration increases with temperature. Differences between anticipated stimulatory effects of CO₂ and temperature and whole plant single and leaf measurements are discussed. Data from this study suggest that stimulatory effects of atmospheric CO₂ on growth and photosynthesis may decline with anticipated increases in global temperature, limiting the degree of carbon storage in these two perennial species.     

Ontogeny affects response of northern red oak seedlings to elevated CO2 and water stress: II. Recent photosynthate distribution and growth

Northern red oak in the western Lake States area of the USA exists on the most xeric edge of its distribution range. Future climate-change scenarios for this area predict decreased water availability along with increased atmospheric CO₂. We examined recent photosynthate distribution and growth in seedlings as a function of CO₂ mole fraction (400, 530 and 700 μmol mol⁻¹ CO₂), water regime (well watered and water-stressed), and ontogenic stage. Water stress effects on growth were largely offset by elevated CO₂.
Water stress increased root mass ratio without concurrently increasing allocation of recent photosynthate to the roots. However, apparent sink strength of water-stressed seedlings at the completion of the third growth stage tended to be greater than that of well watered seedlings, as shown by continued high export, which may contribute carbon reserves to support preferential root growth under water-stressed conditions.
Elevated CO₂ decreased apparent shoot sink strength associated with the rapid expansion of the third flush. Carbon resources for the observed enhanced growth under elevated CO₂ could be provided by enhanced photosynthetic rate over an increased leaf area (Anderson & Tomlinson, 1998, this volume).
Increased sink strength of LG seedlings under water-stressed conditions, together with decreased apparent shoot sink strength associated with growth in elevated CO₂ provide mechanisms for offsetting water stress effects by growth in elevated CO₂.
Careful control of ontogeny was necessary to discern these changes and provides further evidence of the need for such careful control in mechanistic studies.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Sensing of atmospheric CO2 by plants

Abstract. Despite recent interest in the effects of high CO₂ on plant growth and physiology, very little is known about the mechanisms by which plants sense changes in the concentration of this gas. Because atmospheric CO₂ concentration is relatively constant and because the conductance of the cuticle to CO₂ is low, sensory mechanisms are likely to exist only for intercellular CO₂ concentration. Therefore, responses of plants to changes in atmospheric CO₂ will depend on the effect of these changes on intercellular CO₂ concentration. Although a variety of plant responses to atmospheric CO₂ concentration have been reported, most of these can be attributed to the effects of intercellular CO₂ on photosynthesis or stomatal conductance. Short-term and long-term effects of CO₂ on photosynthesis and stomatal conductance are discussed as sensory mechanisms for responses of plants to atmospheric CO₂. Available data suggest that plants do not fully realize the potential increases in productivity associated with increased atmospheric CO₂. This may be because of genetic and environmental limitations to productivity or because plant responses to CO₂ have evolved to cope with variations in intercellular CO₂ caused by factors other than changes in atmospheric CO₂.     

Acquisition and allocation of carbon and nitrogen by Danthonia richardsonii in response to restricted nitrogen supply and CO2 enrichment

Dry weight (DW) and nitrogen (N) accumulation and allocation were measured in isolated plants of Danthonia richardsonii (Wallaby Grass) for 37 d following seed imbibition. Plants were grown at ≈ 365 or 735 μ L L^–1 CO₂ with N supply of 0·05, 0·2 or 0·5 mg N plant^–1 d^–1. Elevated CO₂ increased DW accumulation by 28% (low-N) to 103% (high-N), following an initial stimulation of relative growth rate. Net assimilation rate and leaf nitrogen productivity were increased by elevated CO₂, while N concentration was reduced. N uptake per unit root surface area was unaffected by CO₂ enrichment. The ratio of leaf area to root surface area was decreased by CO₂ enrichment. Allometric analysis revealed a decrease in the shoot-N to root-N ratio at elevated CO₂, while the shoot-DW to root-DW ratio was unchanged. Allometric analysis showed leaf area was reduced, while root surface area was unchanged by elevated CO₂, indicating a down-regulation of total plant capacity for carbon gain rather than a stimulation of mineral nutrient acquisition capacity. Overall, growth in elevated CO₂ resulted in changes in plant morphology and nitrogen use, other than those associated simply with changing plant size and non-structural carbohydrate content.     

Response of beech (Fagus sylvatica) to elevated CO2 and N: Influence on larval performance of the gypsy moth Lymantria dispar (Lep., Lymantriidae)

Two-year-old beech seedlings were kept from germination to bioassays with Lymantria dispar under the following conditions: ambient CO₂/low N, elevated CO₂/low N, ambient CO₂/elevated N, and elevated CO₂/elevated N. The effect of these growing conditions of the trees on the performance of the defoliator L. dispar was studied 2 years after initiating the tree cultivation. The developmental success of third-instar larvae of L. dispar was characterized by the weight gained, percentage of weight gain, relative growth rate (RGR), relative consumption rate (RCR), and efficiency of conversion of ingested food into body substance (ECI). Contrary to our expectations, additional N-fertilization did not increase and elevated CO₂ did not delay larval growth rate. However, the environmental treatments of the beech seedlings were found to affect the larval performance. Larvae consumed significantly higher amounts of foliage (RCR) on beech trees under controlled conditions (ambient CO₂ and low N) compared to those under elevated CO₂ and enhanced N. The opposite was true for ECI. The lowest efficacy to convert consumed food to body substance was observed under control conditions and the highest when the larvae were kept on beech trees grown under elevated CO₂ and additional N-fertilization. These opposite effects resulted in the weight gain-based parameters (absolute growth, percentage of growth, and RGR) of the gypsy moth larvae remaining unaffected. The results indicate that the gypsy moth larvae are able to change their ECI and RCR to obtain a specific growth rate. This is discussed as an adaptation to specific food qualities.     

Elevated CO2 and nitrogen influence exudation of soluble organic compounds by ectomycorrhizal root systems

Root and mycelial exudation contributes significantly to soil carbon (C) fluxes, and is likely to be altered by an elevated atmospheric carbon dioxide (CO₂) concentration and nitrogen (N) deposition. We quantified soluble, low-molecular-weight (LMW) organic compounds exuded by ectomycorrhizal plants grown under ambient (360 p.p.m.) or elevated (710 p.p.m.) CO₂ concentrations and with different N sources. Scots pine seedlings, colonized by one of five different ectomycorrhizal or nonmycorrhizal fungi, received 70 μM N, either as NH₄Cl or as alanine, in a liquid growth medium. Exudation of LMW organic acids (LMWOAs), dissolved monosaccharides and total dissolved organic carbon were determined. Both N and CO₂ had a significant impact on exudation, especially of LMWOAs. Exudation of LMWOAs was negatively affected by inorganic N and decreased by 30–85% compared with the organic N treatment, irrespective of the CO₂ treatment. Elevated CO₂ had a clear impact on the production of individual LMWOAs, although with very contrasting effects depending on which N source was supplied.