Optimisation of the Heterotrophic Production of EPA by the Diatom Nitzschia Laevis

Background.

The therapeutic significance of ?-3 polyunsaturated fatty acids (?-3 PUFAs) has been demonstrated by recent clinical studies. Eicosapentaenoic acid (EPA), along with docosahexaenoic acid (DHA), plays an important role in the prevention of arrhythmia, cardiovascular disease, and cancer. Currently, fish oil is the commercial source of both EPA and DHA. However, the production of ?-3 PUFAs from fish oil is hindered by limitations such as stability problems, high purification costs and contamination with pesticides and heavy metals.

As fish obtain ?-3 PUFAs from zooplankton that consume microalgae, many efforts have been devoted to producing ?-3 PUFAs directly from microalgae. Several attempts have been made to develop commercial heterotrophic processes for ?-3 PUFAs production because a conventional photoautotrophic process is disadvantageous due to light limitation resulting from cells’ mutual shading. Recently, the diatom, Nitzschia laevis, has been studied and the results showed that it is a good EPA producer as the cells showed not only good growth but a relatively high EPA content under heterotrophic conditions.

Experimentation and Data Collection.
The following experimental work was carried out by the University of Hong Kong. The diatom Nitzschia laevis (UTEX 2047) was used in that work. The cells were sub cultured in LDM medium supplemented with 5 g/L glucose and 30 mg/L Na2SiO3 . 9H2O. The LDM medium consisted of (per L) 1 g tryptone, 892 mL artificial seawater containing salts, tris-buffer and chelated iron, 100 mL bristol solution including some salts, 6 mL PIV metal solution including Na2EDTA and trace elements, 1 mL stock solutions of biotin (25.0 × 10-5 g/L) and vitamin B12 (15.0 × 10-5 g/L), respectively. The cells were incubated in 500-mL -Erlenmeyer flasks containing 200 mL medium in an orbital shaker (at 160 rpm) at 22°C in darkness. In medium preparation, if the initial pH was higher than » 8.5, precipitation would occur after autoclaving. So, the following steps were taken to avoid this problem. (1) The medium in each flask was first adjusted to pH 7.5 before autoclaving; at this pH level, no precipitation occurred even at high calcium and trace element concentrations. (2) After autoclaved at 121°C for 20 min, the flasks were cooled down to room temperature. (3) For adjustment of each desired pH level, one flask was taken out and the added amounts of acid or base were recorded. (4) The same amounts of acid or base were added to the parallel identical flasks. It was found that the final pH of these flasks was rather accurate. By the above operation, no precipitation occurred.

The cell concentration of the inoculum was about 0.1 g/L (DW). Previous studies have indicated that EPA production of N. laevis was improved by an increase in initial glucose and silicate concentrations, and 20 g/L glucose and 120 mg/L Na2SiO3 . 9H2O were found to be the most appropriate. Also, additions of 1.6 g/L tryptone and 0.8 g/L yeast extract could enhance EPA production by the alga (data not shown). So these concentrations were used in all the experiments described below.
Experiment 1.

The first step was to identify whether the PI metal solution was an important variable. Thus a traditional change one variable at a time experiment was carried out. Twenty tests were carried out at a PI value of 13.5 mL/L and twenty tests were carried out at a PI value of 4.5 mL/L. In all these tests the levels for NaCl, CaCl2, temperature and pH were held fixed at 16 g/L, 0.204 g/L, 22oC and 7.5 respectively. The EPA yield levels for each of these tests are shown in Sheet1 of the Data Sheet

Experiment 2.
This was the biggest experiment consisting of twenty seven different tests. In this experiment 1 medium component was looked at (NaCl) together with 2 environmental factors (temperature and pH). The resulting EPA yield at each of these 27 different combinations of components and environmental factors was recorded. These test conditions and the yield levels for each of these tests are shown in Sheet2 of the Data Sheet.
Objectives.
You are required to write a mini project that carries out a detailed statistical investigation on the experimental data discussed above. The project should provide an answer as to what culture conditions (including both medium components and environmental factors) are required for the maximization of EPA production by the Nitzschia laevis alga.
When writing this mini project, structure it in a way that allows you to cover and address all the following questions and issues in a way that reveals a progressively greater understanding of the two experimental data sets.
1. Describe the sources of variability present in each data set of Experiment 1. Then using appropriate data displays, describe each data set in Experiment 1, highlighting any similarities or differences that may exist between the 2 levels of PI metal solution. [20 %]

2. Using the data sets collected in Experiment 1, construct an appropriate parametric and non-parametric test to assess the claim that the typical EPA yield is not the same for each level of PI metal solution. When writing up your analysis of this claim state any assumptions that need to be made in conducting these tests and if appropriate carry out tests to validate these assumptions. Discuss also the advantages and disadvantages of each test. [20 %]

3. Using the data set collected in Experiment 2 and the technique of multiple least squares, estimate the ? parameters of the following second order response surface model:
where Y is EPA yield, X1 is the pH value, X2 the NaCl level and X3 the temperature level. ? is the prediction error or residual.
When writing up your analysis of this model, describe how well this model fits the data, which variables are statistically significant (important) and what meanings can be attached to the ? parameters. State any assumptions that need to be made is assessing such statistical significance, and if appropriate carry out tests or construct scatter plots to validate these assumptions. [30 %]
4. Derive a simplified version of the above model that includes only the statistically significant variables.
When writing up your analysis, describe how well this simplified model fits the data, the meaning of the parameters, the degree of accuracy achievable when predicting the EPA yield using this simplified model (as described by a 95 confidence interval on an actual v prediction plot). Use the model to find the optimal conditions for EPA yield, making full use of any suitable 2D or 3D scatter plots to explain these conditions. [30 %]