Nitrogen-limited Growth Research Articles

Isotopic estimates of new carbon inputs into litter and soils in a four-year climate change experiment with Douglas-fir

Because soil is a major reservoir of terrestrial carbon and a potential sink for atmospheric CO2, determining plant inputs to soil carbon is critical for understanding ecosystem carbon dynamics. We present a modified method to quantify the effects of global climate change on plant inputs of carbon to soil based on 13C:12C ratio (δ13C) analyses that accounts for isotopic fractionation between inputs and newly created soil carbon. In a four-year study, the effects of elevated CO2 and temperature were determined for reconstructed Douglas-fir (Pseudotsuga mensiezii (Mirb.) Franco) ecosystems in which native soil of low nitrogen content was used. The δ13C patterns in litter and mineral soil horizons were measured and compared to δ13C patterns in live needles, fine roots, and coarse roots. From regression analyses, we calculated the isotopic enrichment in 13C of newly incorporated soil carbon relative to needle and root carbon at 4‰ and 2‰, respectively. These enrichments must be considered when using shifts in soil δ13C to calculate inputs of plant carbon into the soil, and are probably a major factor in the progressive enrichment in 13C with increasing depth in soil profiles. Relative to the total carbon in each layer, the proportion of new carbon from recent photosynthate in each soil layer was 13–15% in the A horizon, 7–9% in litter layers, and 4% in the B2 and C horizons. New carbon in the A horizon was estimated at 370 g C m−2. Carbon concentrations and new carbon in A horizons were correlated (r 2=0.78, n=12), but with a slope of 0.356, indicating that about 36% of net carbon accumulation in the A horizon was from inputs via roots, root exudates or mycorrhizal fungi and 64% of carbon was derived from surface litter decomposition. Under the nitrogen-limited growth conditions used in this study, neither elevated CO2 nor temperature affected soil carbon sequestration patterns.

Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase.

Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.

The concept of multiple-nutrient-limited growth of microorganisms and its application in biotechnological processes

The “law of the minimum” (Liebig's law) states that usually one nutrient restricts the maximum quantity of biomass that can be produced within a system, whereas all other nutrients are in excess. This general rule has been applied also to the growth of microorganisms, e.g., by adjusting the relative concentrations of the individual nutrients in growth media such that one of them, in the case of heterotrophic microbes, usually the carbon source, determines the maximum cell density that can be obtained in a culture. However, experimental data demonstrated that growth of microbial cultures can be limited simultaneously by two or more nutrients. These authors reported that during growth of bacteria and yeasts at a constant dilution rate in the chemostat, three distinct growth regimes were recognised as a function of the C:N ratio in the inflowing medium: (1) a clearly carbon-limited regime with the nitrogen source in excess, (2) a transition (“double-nutrient-limited”) growth regime where both the carbon and the nitrogen source were below the detection limit, and (3) a clearly nitrogen-limited growth regime with the carbon source in excess. Subsequent calculations suggested that the extension and position of this double-nutrient-limited zone should be strongly dependent on the imposed growth rate: Whereas it is very narrow at high growth rates it should become very broad during slow growth. This pattern as a function of growth rate has now been confirmed for a number of different organisms. In industrial processes, microbial growth is always in some way controlled by the limited availability of nutrients, and limitation of specific nutrients is frequently used to force microbial cultures into a productive physiological state. This article will discuss what the consequences of multiple-nutrient-limited growth are for industrial processes and how the concept might be applied. Specific examples will be given that demonstrate the advantages and the potential of multiple nutrient-limited growth conditions for industrial production processes.

In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint.

Isoamyl alcohol (IAA) induces a phenotype that resembles pseudohyphae in the budding yeast Saccharomyces cerevisiae. We show here that IAA causes the rapid formation of linear chains of anucleate buds, each of which is accompanied by the formation of a septin ring at its neck. This process requires the activity of Swe1 and Slt2 (Mpk1). Cdc28 is phosphorylated on tyrosine 19 in a Swe1-dependent manner, while Slt2 becomes activated by dual tyrosine/threonine phosphorylation. Tyrosine 19 phosphorylation of Cdc28 is not dependent on Slt2. However, the defective response in the slt2Delta mutant is rescued by an mih1Delta mutation. The IAA response still occurs in a cell containing a dominant non-phosphorylatable form of Cdc28, but no longer occurs in an mih1Delta slt2Delta mutant containing this form of Cdc28. These observations show that IAA induces the Swe1-dependent morphogenesis checkpoint and so the resulting pseudohyphal phenotype arises in an entirely different way from the formation of pseudohyphae induced by nitrogen-limited growth.

Identification of additional TnrA‐regulated genes of Bacillus subtilis associated with a TnrA box

Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.

Nitrogen limitation of growth and nutrient dynamics in a disturbed mangrove forest, Indian River Lagoon, Florida.

The objectives of this study were to determine effects of nutrient enrichment on plant growth, nutrient dynamics, and photosynthesis in a disturbed mangrove forest in an abandoned mosquito impoundment in Florida. Impounding altered the hydrology and soil chemistry of the site. In 1997, we established a factorial experiment along a tree-height gradient with three zones, i.e., fringe, transition, dwarf, and three fertilizer treatment levels, i.e., nitrogen (N), phosphorus (P), control, in Mosquito Impoundment 23 on the eastern side of Indian River. Transects traversed the forest perpendicular to the shoreline, from a Rhizophora mangle-dominated fringe through an Avicennia germinans stand of intermediate height, and into a scrub or dwarf stand of A. germinans in the hinterland. Growth rates increased significantly in response to N fertilization. Our growth data indicated that this site is N-limited along the tree-height gradient. After 2 years of N addition, dwarf trees resembled vigorously growing saplings. Addition of N also affected internal dynamics of N and P and caused increases in rates of photosynthesis. These findings contrast with results for a R. mangle-dominated forest in Belize where the fringe is N-limited, but the dwarf zone is P-limited and the transition zone is co-limited by N and P. This study demonstrated that patterns of nutrient limitation in mangrove ecosystems are complex, that not all processes respond similarly to the same nutrient, and that similar habitats are not limited by the same nutrient when different mangrove forests are compared.

Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter.

Expression of the P3 promoter of the Bacillus subtilis ureABC operon is activated during nitrogen-limited growth by PucR, the transcriptional regulator of the purine-degradative genes. Addition of allantoic acid, a purine-degradative intermediate, to nitrogen-limited cells stimulated transcription of ure P3 twofold. Since urea is produced during purine degradation in B. subtilis, regulation of ureABC expression by PucR allows purines to be completely degraded to ammonia. The nitrogen transcription factor TnrA was found to indirectly regulate ure P3 expression by activating pucR expression. The two consensus GlnR/TnrA binding sites located in the ure P3 promoter region were shown to be required for negative regulation by GlnR. Mutational analysis indicates that a cooperative interaction occurs between GlnR dimers bound at these two sites. B. subtilis is the first example where urease expression is both nitrogen regulated and coordinately regulated with the enzymes involved in purine transport and degradation.

Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase.

Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

Lactobacillus helveticus growth and lactic acid production during pH-controlled batch cultures in whey permeate/yeast extract medium. Part II: kinetic modeling and model validation

A kinetic model was developed that describes growth and lactic acid production rates of Lactobacillus helveticus during pH-controlled batch cultures in whey permeate-yeast extract medium as a function of four variables: sugar, nitrogen substrate and lactic acid concentrations, and pH. Since available nitrogen substrate could not be measured directly, it was related to medium composition through a response surface model of maximum biomass concentration in nitrogen-limited growth cultures. The effect of pH on growth was modeled with a Gaussian equation instead of the traditional second order polynomial term. Product inhibition was modeled with a logistic term for lactate and an exponential term for undissociated lactic acid. The kinetic model accurately described the experimental data with few exceptions.

Steady-State Measurements of Lactic Acid Production in a Wild-Type and a Putative D-Lactic Acid Dehydrogenase-Negative Mutant of Zymomonas mobilis: Influence of Glycolytic Flux

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of D- and L-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of DL-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, D-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. D-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.

The nitrogen assimilation control (Nac) protein represses asnC and asnA transcription in Escherichia coli

In this work, we show that the expression of the asnA and asnC genes is regulated by the availability of ammonium in the growth medium. Our results suggest that, under nitrogen-limiting growth conditions, the nitrogen assimilation control (Nac) protein is involved in the repression of the asnC gene, whose product is required to activate the transcription of asnA. We also show that asparagine negatively affects the expression of asnA, independently of the presence of Nac. These results allow us to conclude that asnA transcription is regulated by two different mechanisms that respond to different effectors: nitrogen and asparagine availability.

The nitrogen assimilation control (Nac) protein represses asnC and asnA transcription in Escherichia coli.

In this work, we show that the expression of the asnA and asnC genes is regulated by the availability of ammonium in the growth medium. Our results suggest that, under nitrogen-limiting growth conditions, the nitrogen assimilation control (Nac) protein is involved in the repression of the asnC gene, whose product is required to activate the transcription of asnA. We also show that asparagine negatively affects the expression of asnA, independently of the presence of Nac. These results allow us to conclude that asnA transcription is regulated by two different mechanisms that respond to different effectors: nitrogen and asparagine availability.

Microbial decomposition of dissolved organic matter in a hypertrophic pond

Planktonic heterotrophic bacteria in lakes utilize the labile fraction of dissolved organic carbon (DOC), although information about seasonal changes in labile DOC in hypertrophic lakes in terms of absolute amount and relative proportion of the total DOC is still limited. We conducted DOC decomposition experiments using GF/F filtrates in water samples from hypertrophic Furuike Pond, together with monitoring of DOC concentration and bacterial abundance in water samples from the pond, to examine seasonal changes in the amount of labile DOC and growth of bacteria on labile DOC. DOC concentrations fluctuated between 2.7 and 11 mg C l−1, and bacterial abundance fluctuated between 1.5 × 106 and 1.0 × 108 cells ml−1. In the DOC decomposition experiment when grazers of bacteria were removed, small portions of DOC (18% ± 12%) were labile for decomposition by bacteria, and the growth yield of bacteria on labile DOC ranged between 3.3% and 19%. Furthermore, addition of nitrogen to water samples enhanced bacterial growth. Thus, not only labile DOC but also nitrogen limited bacterial growth in the pond. Considering the results in the present study together with those of previous studies, bacterial abundance in Furuike Pond is subjected to bottom-up control, such as by limitation of DOC and nitrogen throughout the year, although top-down control of bacterial abundance such as by grazing is seasonally important.

Nitrogen-limited growth of lettuce is associated with lower stomatal conductance.

• C assimilation (A) has been shown to limit the growth of young Lactuca sativa (lettuce) plants following an interruption in their external N supply. Further data from these plants were used to test two hypotheses: that N-limited growth of lettuce is associated with lower stomatal conductance (gs ); and that reductions in gs result from adjustments to stomatal frequency or distribution. • The photosynthetic characteristics, nitrate and organic N-concentrations, as well as epidermal and stomatal distributions, were determined in leaves of hydroponically grown lettuce plants supplied continuously with N or with N removed for up to 14d. • Although N-limited plants had lower maximum rates of A, comparisons at equivalent values of gs showed that A was not directly limited by organic-N but by gs . Reductions in gs under N-limiting conditions did not associate with adjustments to stomatal frequency or distribution. • Associations between plant N and A could arise either through stomata responding directly to signals induced by N deprivation or to increased CO2 partial pressure at the sites of carboxylation.

Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina.

The biochemical events associated with the onset of lipid accumulation in Mucor circinelloides and Mortierella alpina, under conditions of nitrogen-limited growth, have been elucidated; they differ in key aspects from those described in oleaginous yeasts. The NAD+:isocitrate dehydrogenases of Mc. circinelloides and Mort. alpina were not absolutely dependent on AMP for activity. Furthermore, changes in the cellular adenine nucleotide pools and energy charge were different from those reported for oleaginous yeasts. In Mc. circinelloides ATP, ADP and AMP concentrations all decreased by 50% after nitrogen limitation, leading to a constant energy charge at the expense of the size of the total adenylate pool. Pyruvate carboxylase in Mc. circinelloides was cytosolic, having implications for the organization of lipid synthesis in filamentous fungi. As a result of the data obtained, a revised and more concerted mechanism for the initiation of storage lipid accumulation is put forward for filamentous fungi.

Xbp1-mediated repression of CLB gene expression contributes to the modifications of yeast cell morphology and cell cycle seen during nitrogen-limited growth.

Yeast cells undergo morphological transformations in response to diverse environmental signals. One such event, called pseudohyphal differentiation, occurs when diploid yeast cells are partially starved for nitrogen on a solid agar medium. The nitrogen-starved cells elongate, and a small fraction form filaments that penetrate the agar surface. The molecular basis for the changes in cell morphology and cell cycle in response to nitrogen limitation are poorly defined, in part because the heterogeneous growth states of partially starved cells on agar media are not amenable to biochemical analysis. In this work, we used chemostat cultures to study the role of cell cycle regulators with respect to yeast differentiation in response to nitrogen limitation under controlled, homogeneous culture conditions. We found that Clb1, Clb2, and Clb5 cyclin levels are reduced in nitrogen-limited chemostat cultures compared to levels in rich-medium cultures, whereas the Xbp1 transcriptional repressor is highly induced under these conditions. Furthermore, the deletion of XBP1 prevents the drop in Clb2 levels and inhibits cellular elongation in nitrogen-limited chemostat cultures as well as inhibiting pseudohyphal growth on nitrogen-limited agar media. Deletion of the CLB2 gene restores an elongated morphology and filamentation to the xbp1Delta mutant in response to nitrogen limitation. Transcriptional activation of the XBP1 gene and the subsequent repression of CLB gene expression are thus key responses of yeast cells to nitrogen limitation.

Biogeochemical modelling of the tropical Pacific Ocean. I: Seasonal and interannual variability

Abstract A coupled physical–biogeochemical model has been developed in order to study physical–biological interactions in the tropical Pacific Ocean on seasonal-to-interannual timescales. The model incorporates both iron- and nitrogen-limited phytoplankton growth, and succession of phytoplankton size classes in accordance with the “ecumenical” iron hypothesis. The model shows a strong El Nino–Southern Oscillation component to phytoplankton variability in the central equatorial Pacific over the period 1980–1998. It is possible that this mode is more dominant in the model than in nature, although the correlation of modelled and observed chlorophyll in this region is strong. The model results show that interannual variability dominates over the mean seasonal cycle for both physical and biogeochemical fields, with the exception of undercurrent transport west of the dateline. Physical and biogeochemical fields show consistent seasonal phasing among the four El Nino events simulated, although there is variability in the magnitudes and exact timing. Nutrient concentration anomalies at constant temperature in the thermocline of the central equatorial Pacific appear to be largely advected from the west, and the strong seasonal cycle of the equatorial undercurrent in the west introduces a significant annual component to the variability of nutrient concentrations at longitudes where the mean seasonal cycle of the local physical forcing is negligible. The biogeochemical model maintains realistic nutrient pools over the time scales required for interannual simulation, and responds in a realistic fashion to changing upper ocean hydrography and circulation. The model is quite sensitive to the temporal resolution of the wind forcing, which introduces additional uncertainty into the validation and prediction of biogeochemical fields.

Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages.

This is the first report on the biosynthesis of a hitherto unknown, sulfur-containing polyester and also the first report on a bacterial polymer containing sulfur in the backbone. The Gram-negative polyhydroxyalkanoate (PHA)-accumulating bacterium Ralstonia eutropha synthesized a copolymer of 3-hydroxybutyrate and 3-mercaptopropionate, poly(3HB-co-3MP), when 3-mercaptopropionic acid or 3,3'-thiodipropionic acid was provided as carbon source in addition to fructose or gluconic acid under nitrogen-limited growth conditions. The peculiarity of this polymer was the occurrence of thioester linkages derived from the thiol groups of 3MP and the carboxyl groups of 3MP or 3HB, respectively, which occurred in addition to the common oxoester bonds of PHAs. Depending on the cultivation conditions and the feeding regime, poly(3HB-co-3MP) contributed up to 19% of the cellular dry weight, with a molar fraction of 3MP of up to 43%. The chemical structure of poly(3HB-co-3MP) was confirmed by GC/MS, IR spectroscopy, (1)H- and (13)C-NMR spectroscopy, and elemental sulfur analysis. The identification of this novel biopolymer reveals a new quality regarding the substrate range of PHA synthases and their capability for the synthesis of technically interesting polymers.

Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage

Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential sigma(54)-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the sigma(54)-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced (14)CO(2) from minimal medium containing [(14)C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated (14)CO(2) production from [(14)C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.

Accumulation of Poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth with octanoate in continuous culture at different dilution rates.

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C(0)/N(0) ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH(4)(+) in excess at low C(0)/N(0) ratios, (ii) purely nitrogen-limited growth conditions at high C(0)/N(0) ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C(0)/N(0) ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C(0)/N(0) ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C(0)/N(0) ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C(0)/N(0) ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.