Helena Khatoon, Assistant Professor, Department of Aquaculture, Chattogram Veterinary and Animal Sciences University for her valuable suggestions, constructive and constant inspiration throughout the study period and in the preparation of this manuscript. Faisal, Associate Professor and Head, Department of Fisheries and Postharvest Technology; Ishrat Zahan Anka, Assistant Professor and Head, Department of Aquaculture; Dr. Ahmad-Al-Nahid, Associate Professor and Head, Department of Fisheries Resource Management, Chattogram Veterinary and Animal Sciences University for their kind cooperation, valuable suggestions and constructive criticism throughout the research period and for the thesis work.
LIST OF PLATES
LIST OF APPENDIX
ABSTRACT
CHAPTER-1 INTRODUCTION
Aims and objectives of the study
Hypothesis
Research questions
CHAPTER-2
REVIEW OF LITERATURE
- Lipids in microalgae
- Source of nitrogen
- Effects of nitrogen on lipid production of cells
- Cell growth
- Other factors affecting the growth of microalgae
- Temperature
- Salinity
- Light
The assessment of growth conditions at different concentrations of nitrogen in environments can provide valuable information about the biochemical composition of microalgae. On the contrary, the concentration of saturated fatty acids, 75.79% under nitrogen disturbance, was higher than in the case of nitrogen sufficiency, 36.63%. 2014) investigated the consequences of nitrogen limitation together with subsequent nitrogen starvation on morphological and biochemical changes in Scenedesmus sp.
Wu and Miao (2014) found that the impact of nitrogen limitation on protein content is species dependent. 2014) studied the changes in the metabolism of starch and lipid biosynthesis in the microalgae Chlorella zofingiensis under nitrogen deficiency compared to nitrogen rich conditions. They studied the effects of nitrogen deficiency on nutrient uptake, growth and gross chemical composition of Chlorella sp.
In the absence of nitrogen in culture media, the congregations of all substances varied during the growth of Chlorella sp., except lipid. They found that biomass production, total fatty acid content and fatty acid composition were influenced by the C/N ratio, but not by the agitation speed in the range studied. 2013) investigated the growth and fat content of Chlorella marina and Dunellialla salina under the influence of different concentrations of nitrogen sources to increase the fat content in microalgae.
Eg. the initial pH of the seawater was pH 8.0 in the study of growth and biochemical composition with emphasis on the fatty acids of Tetraselmis sp.
CHAPTER-3
MATERIALS AND METHOD
Culture and maintenance of Chlorella sp
Culture and maintenance of Chlorella sp. as stock 3.2 Experiments
- Purpose of the growth curve experiment
- Preparation of culture media .1 Preparation of natural seawater
- Preparation of Conway medium
- Main minerals stock solution
- Trace metals solution
- Vitamin B (thiamine) and vitamin B 12 (cyanocobalamin) solutions
- Main minerals stock solutions with modified NaNO 3 concentrations
- Experimental design for growth curve experiment
- Measurement of parameters for growth curve experiment
- Determination of optical density (OD)
- Determination of cell density
- Experimental design
11 | P a g e Corner and Laboratory of Department of Aquaculture, Faculty of Fisheries in Chattogram University of Veterinary and Animal Sciences. Before starting the main experiment to determine the consequences of different nitrogen concentrations on the growth and nutrient profile in the marine microalga Chlorella sp., it was necessary to perform the growth curve experiment for Chlorella sp. Beginning the preparation of the mineral stock master solution was the accurate weighing of all required chemicals using an analytical balance (AND, GR-200).
For the preparation of the solution, each required chemical was accurately weighed with the analytical balance (EN, GR- 200). Initially, half of the volumetric flask (50 ml) was charged with fresh Milli-Q water (Millipore Corp.). Finally, the main mineral stock solution was kept in a Schott-Duran® bottle and stored in a refrigerator (Samsung SilverNano) before later use.
The growth of the cultures was monitored daily during the growth curve experiment by determining the optical density of the culture samples using a spectrophotometer (UV-1601 UV-Visible Spectrophotometer SHIMADZU). Culture growth and densities were also monitored daily during the growth curve experiment by resolution of the cell density of the culture samples. The hemacytometer (Bright-line enhanced Neubauer hemacytometer, 0.1 mm deep chambers, Assistant, Germany) and coverslip (0.0025 mm2) were rinsed with Milli-Q water (Millipore Corp.) before filling the chambers accompanied by culture samples .
Before studying the effects of nitrogen concentrations on the growth rate and nutritional profile, the growth curve analysis was performed because it is important to know the more appropriate phase (exponential or stationary) to inoculate Chlorella sp.
Experiment setup
- Analysis of protein, lipid, and carbohydrate
18 | P a g e Growth parameters like cell density, optical density in culture were measured daily. After that, all tubes were centrifuged for 4 minutes at 1000 rpm at 4 ºC; the supernatants were transferred to clean tubes with a Pasteur pipette and placed on ice. After that, the tubes were centrifuged again under the same conditions, and the supernatants were transferred to the previous supernatant tubes.
The upper layer of methanol and chloroform was discarded, while the lower layer was transferred into previously prepared aluminum dish. Finally, the initial weight was subtracted from the final weight to obtain the lipid weight in the samples. For each sample, 5 mg of freeze-dried biomass was taken to prepare a 25 mL well-mixed (tissue homogenizer) solution using distilled water.
Then, for each sample, 1 ml was taken from the 25 ml solution, and then 1 ml of 5% phenol solution and 5 ml of concentrated sulfuric acid were added to it. After it was cooled, the absorbance of the solution was measured at a wavelength of 488 nm using a carbohydrate estimation spectrophotometer. To create a calibration graph, we prepared 1000 µg/L standard stock solution (glucose) and then a series of standards in different dilutions (20 µg/L, 40 µg/L, 60 µg/L, 100 µg/L). , and 140 µg/L) were also prepared from the stock solution.
A standard graph was drawn according to the standard absorbance results, and the carbohydrate composition of each sample was determined accordingly.
CHAPTER-4 RESULTS
RESULTS
- Growth of microalgae under different nitrogen concentrations
- Effects of different nitrogen concentrations on proximate composition
This section provides detailed information on current research work on the effects of different nitrogen concentrations on the growth and nutritional profile of Chlorella sp. The cell density (cells ml-1) and optical density (580 nm) of native marine microalgae Chlorella sp. The initial cell density (cells/ml) increased remarkably after 7 days of culture in all cultures with different nitrogen concentrations.
In addition, the corresponding N concentrations of the growth medium for each treatment were significantly (p < 0.05) different with respect to nitrate (NaNO3) (Table 1).1a. 24 | P a g e The results also revealed that the increased N concentrations in the culture media significantly (p < 0.05) complemented the growth performance where Chlorella sp. An indistinguishable mode was observed for optical density (OD) measurements by absorbance reading (580 nm) (Fig. 1b).
The protein content shows significant (p < 0.05) changes of different mode for nitrate (NaNO3), regarding sufficient or deficient N concentrations (Fig. 2a). Increasing N concentrations in the culture medium significantly (p < 0.05) reduced protein content (% dry weight). 26 | P a g e Carbohydrate content shows significant (p < 0.05) substitutions of opposite mode for nitrate (NaNO3) in terms of sufficient or deficient N concentrations (Fig. 2b).
The lipid content shows a significant (p < 0.05) difference of opposite vogue for nitrate (NaNO3), in relation to sufficient or deficient N concentrations (Fig. 2c).
CHAPTER-5 DISCUSSION
DISCUSSION
- Growth of microalgae under different nitrogen concentrations
- Effects of different nitrogen concentrations on proximate composition
This research work was intended to evaluate the growth performance and close composition of Chlorella sp. As one of the most essential nutrients, nitrogen influences the cell growth of microalgae, and the growth rate of the algal cells is significantly influenced by the nitrogen concentrations in the growth medium (Wu and Miao, 2014). In addition, the biochemical compositions of microalgae are also significantly influenced by the nitrogen concentrations in the growth medium (Kim et al., 2016; Wang et al., 2013).
The results obtained from this study are more or less consistent with those reported by Saha et al., 2013 and Gu et al., 2015 on the growth performance of Chlorella sp. grown in culture medium containing different concentrations of nitrogen. This study revealed the positive growth performance of Chlorella sp. with decreasing concentration of nitrogen. Furthermore, it is recorded by Razaghi et al., 2014 that excessive increase in the concentrations of nitrogen causes obstruction of other vitamins, trace metals and nutrients, plus negatively affects cell growth under this condition.
In this present research, Chlorella sp. within different nitrogen concentrations significantly caused protein content up to 31.90% of dry weight in relation to the decrease of nitrogen concentrations in the growth medium. The effects of preliminary nitrogen concentrations on microalgae growth as well as microalgae lipid content were investigated. In the present research, Chlorella sp. under different nitrogen concentrations significantly affected the lipid content up to 23.47% in relation to the decrease of nitrogen concentrations in the growth medium.
Research by Sudha et al. 2013) investigated the effects of different nitrogen concentrations on growth plus lipid content of Chlorella marina and Dunellialla salina to increase the lipid content of microalgae.
CHAPTER-6 CONCLUSION
Microalgae are single-celled photosynthetic algae or phytoplankton that utilize light energy plus carbon dioxide, with higher photosynthetic efficiency compared to plants for biomass production. Nitrogen is one of the most important nutrients acting on the biochemical composition and cell growth of microalgae. Increasing nitrogen concentration from 100 to 150 g/L decreased cell concentration, but at lower concentrations (50 g/L) of nitrogen, cell density increased.
The findings of this research show that both the growth rate and proximate composition of Chlorella sp. Advanced research work should be done to enhance the growth rate and proximate composition by using different nitrogen sources like Urea, KNO3, NH4NO3, etc.
CHAPTER-7
RECOMMENDATIONS AND FUTURE PERSPECTIVES
RECOMMENDATIONS AND FUTURE PERSPECTIVES
Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Growth of the marine microalga Tetraselmis suecica in batch cultures with different salinities and nutrient concentrations. Lipid accumulation and response of Chlorella pyrenoidosa oil biosynthesis genes under three nutritional stressors.
Effects of nitrogen sources on cell growth and biochemical composition of marine chlorophyte Tetraselmis sp. The influence of temperature and the initial N:P ratio on the growth of the microalgae Tetraselmis sp. Combination of nitrogen deficiency with adequate phosphorus supply for improved biodiesel productivity of Chlorella vulgaris fed with acetate.
Effects of nitrogen concentration on growth and lipid content of Chlorella marina and Dunellialla salina for biodiesel production. Combined effects of initial biomass density and nitrogen concentration on growth and astaxanthin production of Haematococcus pluvialis (Chlorophyta) in outdoor cultivation. Effects of nitrogen concentration on the growth rate and biochemical composition of the microalga, Isochrysis galbana.
Characterization of the lipid and fatty acid composition of Chlorella zofingiensis in response to nitrogen starvation.
APPENDIX
APPENDIX A
Brief Biography
