Experimental assessment of biochemical responses to nitrogen concentration in Ulva

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Survey of tissue δ

15N isotopes in Ulva Some studies have shown that δ 15N values in macroalgal tissues greater than about 9 ‰ (McClelland et al., 1997; Jones et al., 2001; Gartner et al., 2002; Dudley, 2005) may indicate the presence of effluent, including secondary or tertiary treated sewage as a source of nitrogen which has undergone some denitrification (Rogers, 2006). Other studies have shown that untreated wastewater sources can result in ratios approaching 2 ‰ (Rogers, 1999; Dudley, 2005) potentially due to a dominance of industrially-derived nitrates with more negative values (Rogers, 2006). Ulva from enriched urban sites in the current study showed the largest range of δ 15N values in both seasons, suggesting that these extreme values were the result of differences in nitrogen isotopic composition in the environment.

Collection of experimental algae

For the following experiments Ulva pertusa was collected from Otumoetai, Tauranga Harbour (37° 39′ 27.68 S, 176° 8′ 31.57 E). Unless otherwise specified, Ulva was returned to the Leigh Laboratory where it was kept under natural light in running seawater supplied continuously from the Leigh Laboratory’s seawater system (using natural Goat Island seawater) for at least one week prior to the commencement of any experiment to allow tissue to equilibrate to local conditions. Generally Ulva tissue was maintained in good condition while under culture, however on a few occasions sporulation occurred in Ulva tissue (particularly in non-enriched controls). On these occasions sporulated tissue was removed, and therefore excluded from growth and biochemical analysis.

Free amino acids

Amino acid samples were extracted from 1 g fresh weight of Ulva tissue as described in Chapter Two with the following modifications. All extractions were done on ice in a polystyrene cooler bin. Tissue samples were placed into a 20 ml scintillation vial to which 5 ml 1M perchloric acid was quickly added. The vial was then capped, shaken and left on ice for 10 min before neutralising with 5 ml 1M KOH/0.2 M MOPS. After 60 minutes on ice 1 ml supernatant was drawn off (while avoiding any perchlorate precipitate), using a fresh plastic eyedropper pipette, and placed into labelled 1.5 ml microcentrifuge tubes. These were stored at – 80 °C for later HPLC analysis of amino acid composition as described by Barr and Rees (2003) (See also Chapter Two). After rinsing with distilled water extracted tissue was dried to a constant weight at 65 °C.

Seawater sampling and nutrient analyses

Seawater samples were generally taken at the same time as algal tissue samples. Samples were kept chilled on ice and analysed for ammonium (NH4 + ), nitrite (NO2 – ), nitrate (NO3 – ) and phosphate (PO4 3-) with reference to standard curves containing known concentrations of nutrients. Ammonium was determined as described by Koroleff (1983a) and nitrite by Parsons et al (1984). Nitrate was reduced to nitrite by passing 20 ml of seawater sample through a cadmium column, and then determined using the method described by Parsons et al (1984). Phosphate was determined as described by Koroleff (1983b). Total inorganic nitrogen (TIN) concentrations were calculated as the sum of NH4 + , NO2 – and NO3 – . The ratio of nitrogen : phosphorus (N : P) was calculated in molar ratios as TIN : PO4 3-. All concentrations values are expressed in µM.

Nitrogen indices and seawater nitrogen concentration

Overall performance of nitrogen indices in Ulva was examined by combining the results for all four experiments. With the exception of Experiment 3 (in which no nitrogen was added) the addition of nitrogen resulted in significant changes in nearly all N-indices examined. From pooled data of these experiments, which compared responses to un-enriched natural seawater with those in 10 µM enriched seawater (irrespective of nitrogen source and light treatment), the most pronounced biochemical response to external nitrogen concentration was that of amino acids (Table 3.6). While the largest proportional change (22-fold) was shown by U29.48 the largest quantitative change was seen in asparagine (Asn) (Table 3.6).

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Nitrogen status, and light and season

While nitrogen content and other N-indices (e.g., chlorophyll) in Ulva were affected by nitrogen concentration in seawater (and water motion, Figure 3.13), these parameters were also affected by differences in light and season. From combined data the figure below (Figure 3.19) shows three important features: 1) Enriched Ulva grown in the winter tended to have the highest values of TN and chlorophyll overall, 2) There was one example of unenriched Ulva in winter that contained as much nitrogen and chlorophyll as enriched Ulva in the summer, and 3) both chlorophyll a and, to a lesser extent b, were well correlated with TN (Figure 3.19).

Table of Contents :

Abstract
Acknowledgements
Preface
Table of Contents
List of Tables
List of Figures
List of Plates
List of Appendixes
Chapter One: General introduction
- 1 General patterns of nitrogen utilisation in macroalgae
- Background
- Nitrogen and growth in seaweeds
- Sources of nitrogen in seawater
- Ammonium and nitrate uptake, and utilisation in macroalgae
- Factors affecting uptake and utilisation of nitrogen in macroalgae
- Nitrogen assimilation and incorporation in macroalgae
- Nitrogen metabolism and free protein amino acids in macroalgae
- Free amino acids as N-indices of nitrogen availability in macroalgae
- 2 Marine pollution and eutrophication
- Background
- Nutrient enrichment and eutrophication in the marine environment
- Monitoring changes in nutrients in the marine environment
- Macroalgae as indicators of nutrient enrichment and marine pollution
- Natural abundance stable nitrogen isotopes in macroalgae
- 3 Biology of Ulva (Ulvaceae, Ulotrichales, Chlorophyta)
- Ulva taxonomy
- Life history and ecology of Ulva
- 4 Aims of thesis
Chapter Two: Geographical variation in nitrogen status of New Zealand Ulva
- 2.1 Abstract
- 2.2 Introduction
- 2.3 Methods
  - 2.3.1 Survey sites and environmental categories
  - 2.3.2 General weather conditions Algal sampling
  - 2.3.3 Algal taxonomy
  - 2.3.4 Sample standardisation
  - 2.3.5 Algal tissue storage and integrity Analytical
  - 2.3.6 Amino acid extraction
  - 2.3.7 Chlorophyll
  - 2.3.8 Tissue nitrogen content
  - 2.3.9 Seawater sampling and nutrient analyses
  - 2.3.10 Statistical analysis
- 2.4 Results
  - 2.4.1 Summer and winter seawater temperature
  - 2.4.2 Long-term (2002 – 2004) monitoring of chlorophyll levels in Ulva fasciata
  - 2.4.2 Summer seawater nutrients and Ulva N-indices
  - 2.4.3 Winter seawater nutrients and Ulva N-indices
  - 2.4.4 Ordination of Ulva N-indices using cluster analysis
  - 2.4.5 Season, environment and Ulva N-status
  - 2.4.6 Ulva taxonomy and environment in relation to nitrogen status
- 2.5 Discussion
Chapter Three: Experimental assessment of biochemical responses to nitrogen concentration in Ulva
- 3.1 Abstract
- 3.2 Introduction
- 3.3 Methods
  - 3.3.1 Collection of experimental algae
  - 3.3.2 Sub-sampling Ulva thalli
  - 3.3.3 Free amino acids
  - 3.3.4 Chlorophyll
  - 3.3.5 Tissue nitrogen
  - 3.3.6 Growth
  - 3.3.7 Determination of maximum rate of ammonium assimilation
  - 3.3.8 Seawater sampling and nutrient analyses
  - 3.3.9 Statistical analysis
Chapter Four: Developing Ulva as a multi-purpose environmental test-organism
Chapter Five: General discussion Are differences in Ulva tissue-N