8 Table 1.3 Comparison of open and closed microalgae cultivation systems 10 Table 1.4 Biomass and lipid productivity of different microalgae 15 Table 1.5 Common fatty acid profiles of different microalgae species (%) 17 Table 1.6 Effect of of carbon dioxide concentration in accumulative lipids 31 1.8 Comparison of different techniques used for biodiesel production 40 Table 1.9 Catalytic production of biodiesel from different microalgae 44 Chapter 2.
INTRODUCTION AND
LITERATURE REVIEW
Introduction and Literature Review
General Introduction
- Chemical composition of microalgae
- Microalgal cultivation
The cell wall structure of microalgae depends on the strain and growth conditions of the microalgae, especially the chemical composition and thickness of the cell wall (Lee et al., 2017). On the other hand, light and CO2 are the source of energy and carbon for phototrophic cultures (Zhu et al., 2017).
Microalgae–derived compounds and their applications
- Lipids – For Biodiesel production
- Protein – In Food and Feed Industry
- Pigments – In Pharmaceutical and Cosmetic Industry
- Carbohydrates – For Bioethanol production
- Other biofuels from microalgae
- Other applications of microalgae
However, bioethanol still has few limitations, such as low energy density and low vapor pressure (Balat et al., 2008). Chlorella and Spirulina species are common microalgae species used for nutrient removal (Gonzales et al., 1997).
Intensification strategies for enhanced lipid (biodiesel) production
- Optimization of medium components and process parameters
- Application of sonication during fermentation
- Basic principles of ultrasound and cavitation
- Genetic modification of microalgae
However, the nutrient limitation may affect other biochemical pathways in the cells that affect lipid production indirectly (Srinuanpan et al., 2018). Such propagation causes an oscillatory motion of fluid elements in the medium (Shah et al. 1999).
Catalytic and non–catalytic microalgal biodiesel production
On the contrary, heterogeneous catalysts can be easily separated and recovered and are environmentally friendly (Daniel et al., 2017). -solvents are reported to improve the lipid extraction and facilitate the mass transfer of reactant (Park et al., 2017). Addition of a co-solvent leads to a positive effect on increasing the efficiency of lipid extraction (Abedini et al., 2015).
Thus, the decomposition temperature limit for the respective strain of microalgae must be known (Abedini et al., 2015).
Biorefinery approach for microalgal biodiesel production
However, excessive consumption of alcohol should be avoided as it may reduce the final yield (Patil et al., 2013). Major components include proteins, lipids, and carbohydrates, while minor components include vitamins, pigments, and fatty acids (Parniakov et al., 2015). Ethyllevulinate (EL) can be used as a flavor or its addition to biodiesel can improve the low-temperature properties of biodiesel (Im et al., 2015).
These pigments have gradually gained importance in the food, cosmetic and pharmaceutical industries (Chew et al., 2017).
Objectives, Approach and Scope of the present Thesis
Biodiesel production from algal oil high in free fatty acids by two-step catalytic conversion. Biodiesel production from wet microalgae feedstock using sequential wet extraction/transesterification and direct transesterification processes. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production.
Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass.
ISOLATION AND
CHARACTERIZATION OF WILD FRESH WATER
MICROALGAE
Isolation and Characterization of Wild Fresh Water Microalgae
Introduction
A summary of the literature on lipid and β-carotene production of various strains of genus Scenedesmus is given in Table 2.1. Summary of previous literature on lipid and β-carotene production of different strains belonging to genus Scenedesmus. However, we only compared the final biomass yield with lipid and β-carotene content.
The present study has demonstrated the potential of a native or wild microalgae species (isolated from a lake on the campus of the Indian Institute of Technology Guwahati) for a biorefinery that simultaneously produces lipids and β-carotene.
Materials and Methods
- Sampling, isolation and maintenance of culture
- Morphological and taxonomic investigations
- Monitoring of microalgal growth
- Influence of growth conditions on biomass and associated products
- Biochemical characterization
- Thermogravimetric analysis (TGA) and determination of structural composition
- Fatty acid methyl esters (FAME) analysis
The growth of the microalgae was observed with optical density (OD) on a UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) at 680 nm by collecting aliquots at regular intervals. Nitrogen sources: Growth experiments were carried out with seven different nitrogen sources: sodium nitrate (SN), beef extract (BF), urea (UR), peptone (PE), yeast extract (YE), ammonium nitrate (AN) and glycine (GL). The initial concentration from each source was 1.5 g/L and the rest of the growth mixture was BG-11 media with pH 7.0. Nile red staining: Staining of the lipid in microalgal cells was performed by mixing 0.05 ml of 0.1 mg/ml Nile red solution in acetone with the microalgal cells followed by incubation in the dark at 37°C for 10 min.
FRAP (Iron Reducing Antioxidant Power) Test: The FRAP test involves sample preparation, reactions, and measurement of absorbance at 700 nm spectrophotometrically.
Results and Discussion
- Preliminary analysis of biochemical characteristics of isolated strains .1 Morphological and molecular identification
- Growth of T. obliquus SGM19 and associated product synthesis .1 Influence of nitrogen sources
- TGA and Structural composition analysis
- Analysis and characterization of biodiesel
The sequencing of the 18S rRNA showed 921 and 702 bp long nucleotide sequences in the isolated microalgal strains. Based on results described in previous sections (sections 2.3.2.1 to 2.3.2.4), the final growth medium and conditions used for simultaneous lipid and β-carotene production in T. TGA of biomass samples: The thermogravimetric curves of the raw and digested alga biomass with nitrogen and air as carrier gases is shown in Fig 2.5.
In the last stage (stage 3 with temperature range 500–900C), the remaining biomass (decomposition of high molecular weight components) undergoes decomposition resulting in the formation of carbon.
Conclusions
Recycled defoliated algal biomass extract as feedstock to increase biodiesel production from Chlorella minutissima. Isolation and characterization of a Scenedesmus acutus strain to be used for urban wastewater bioremediation. Utilization of Scenedesmus obliquus biomass as feedstock for biodiesel and other industrially important by-products: An integrated paradigm for microalgal biorefinery.
Evaluation of bioethanol and biodiesel production from Scenedesmus obliquus grown in biodiesel waste glycerol: a sequential integrated pathway for improved energy recovery.
ULTRASONIC
ENHANCEMENT OF
LIPIDS AND -CAROTENE PRODUCTION BY T
Ultrasonic Enhancement of Lipids and β-carotene Production by T. obliquus SGM19
Introduction
In addition to pigmentation, carotenoids are also an important component of human nutrients, such as β-carotene, which is a precursor of vitamin A (Spolaore et al., 2006). The present study is aimed at increasing lipid and β-carotene production from wild microalgae strains of Tetradesmus obliquus SGM19. As previously mentioned, wild strains are more stable in natural (or uncontrolled) environments than genetically engineered strains (Szvjka et al., 2017).
The specific objectives of the study are as follows: (1) Statistical optimization of media and physical parameters for the natural microalgae strain, (2) Growth of the microalgae isolate by mechanical shaking for the production of lipids and β-carotene, (3) Intensification of growth of microalgae (biomass and products) with application of sonication with appropriate duty cycle, (4) Kinetic analysis of time profiles of biomass, substrate (nitrate) and products, i.e. lipid and β-carotene with the Luedeking-Piret model, (5 ) Mechanistic analysis of sonication-induced enhancement of microalgae growth with quantification of NAD(H) concentration in the cells.
Materials and methods
- Microalgae and Culture Conditions
- Analytical Determinations
- Statistical optimization
- Plackett–Burman design for media components
- Central composite design
- Validation experiments
- Ultrasound-assisted growth of T. obliquus SGM19
- Microassay of NAD(H)
- Viability assessment of microalgae post sonication
Each parameter was tested at two levels, coded as (+1) for higher level and (−1) for lower level (details in Appendix B). The results of the statistical experimental design were analyzed using response surface methodology with a quadratic model. The sonication experiment requires maintaining the bath temperature at 28.5 2C, i.e. reaction temperatures.
Propidium iodide is a fluorescent dye that binds to the DNA of the non-viable cell (Ormerod, 1990).
Kinetic analysis of experimental profiles
- Biomass
- Products
- Substrate
- Fitting of experimental profiles to kinetic model
Gaden (2000) categorized product formation into 3 classes based on its relationship with microalgae growth. Substrate consumption is mainly aimed at the growth of microalgae, the accumulation of intracellular products and the maintenance of microalgae cells. The kinetic model basically consists of 4 ordinary differential equations (ODEs) for substrate (S), biomass (X), lipid (P1) and β-carotene (P2).
The objective function is the total root mean square (RMS) error between the experimental values and the model predicted values.
Results and Discussion
- Sonication–induced intensification of lipid and β-carotene yield
- Kinetic analysis of experimental profiles
- Microassay of NAD(H)
- Viability assessment through Flow cytometry
Lipid yields at day 13 are 29% and 40% w/w DCW in control and test experiments, respectively. After the 13th day of the growth cycle, a slight reduction in lipid and β-carotene content in biomass is observed in both control and test experiments. The experimental and simulated profiles of biomass, substrate, lipids and β-carotene in the control and test experiments are shown in Figs.
In summary, the values of kinetic parameters in control and test experiments clearly indicate the intensification of cell metabolism induced by sonication.
Conclusion
The upper right quadrant shows the percentage of damaged cells and the lower right quadrant indicates the dead cells. It can be seen that the percentages of dead cells are 0.26% and 0.86% in control and test experiments; while the percentages of damaged cells are 11.03% and 16.07% in control and test experiments, respectively. Investigations into sonication-induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans.
Microalgae for lipid and carotenoid production: a review focusing on stress regulation and adaptation.
TRANSESTERIFICATION OF T. obliquus SGM19
BIOMASS FOR BIODIESEL SYNTHESIS
- Introduction
- Materials and Methods
- Microalgal biomass preparation
- Selection of biomass type and transesterification method
- Statistical optimization of transesterification process parameters
- Ultrasound-assisted transesterification process
- Analysis of kinetic and Arrhenius parameter
- Analysis of FAME/biodiesel obtained from microalgal biomass
- Results and Discussion
- Optimization of transesterification process parameters
- Ultrasound-assisted transesterification and analysis
- FAME quantification and evaluation of biodiesel properties
- Conclusions
The maximum FAME yield was obtained in base-catalyzed single-step in-situ transesterification of the freeze-dried biomass. The activation energy of the overall process was also estimated for test and control experiments using the Arrhenius equation. The activation energies were calculated by plotting ln k vs 1/T. The Arrhenius plot is shown in Fig.
The statistical optimization of the transesterification process parameters shows the optimum conditions as catalyst loading = 1.5% (w/w biomass), methanol to biomass ratio = 30 (v/w), temperature = 50°C and time = 50 min.
CONCEPTUAL BIOREFINERY
SIMULTANEOUS SYNTHESIS OF MULTIPLE PRODUCTS
FROM T. obliquus SGM19
Conceptual Biorefinery
Simultaneous Synthesis of Multiple Products from T. obliquus SGM19
- Introduction
- Materials and Methods
- Microalgae and culture maintenance
- Extraction and estimation of β-carotene
- Extraction of lipid
- Transesterification of lipids/microalgal biomass
- FAME analysis and Estimation of Glycerol
- Biodiesel properties
- Bioethanol production
- Results and Discussion
- Transesterification of extracted lipid/whole biomass
- Biodiesel characterization
- Accumulation of various by-products .1 β-carotene production
- Microalgal biorefinery
- Conclusions
The feasibility of the produced biodiesel was evaluated by studying the composition of fatty acids containing esters with GC-MS. Comparatively, a smaller amount of glycerol (6 wt%) could be recovered during the 2-step transesterification of the biodiesel sample. From the data it can be clearly concluded that the amount of products with in-situ transesterification was higher than that of the conventional protocol.
The absence of the lipid extraction step in in-situ transesterification reduces the number of treatments the microalgae biomass undergoes, thus reducing the consumption of chemicals, energy, and time.
OVERVIEW AND SUGGESTIONS FOR
Overview and Suggestions for Future Research
Overview
The recovery and sale of valuable products from other components of microalgal cells can be a source of additional income and can boost biorefinery economics (Chew et al., 2017). In this chapter, we present an overview of the main results of the various studies carried out in this research, which have been presented in the previous chapters. Initial screening followed by 18S RNA sequence analysis of the isolated microalga helped to identify the genus Scenedesmus and the species of the microalga was named as Tetradesmus obliquus SGM19.
The kinetic analysis of the substrate and product profiles in control and test experiments revealed that both the lipid and β-carotene are growth-associated products.
Future challenges and prospects of microalgal biorefineries
The thesis also presents new direct in situ transesterification protocols for biodiesel synthesis. On exergoeconomic and exergoenvironmental evaluation and optimization of biodiesel synthesis from waste cooking oil (WCO) using a low power, high frequency ultrasonic reactor. Synthesis of biodiesel by in situ transesterification of Tetradesmus obliquus SGM19 microalgae biomass and its intensification with Tetradesmus obliquus SGM19 microalgae biomass and its intensification with ultrasound.
Tetradesmus obliquus SGM19 biomass as feedstock for the production of biodiesel and other value-added by-products: a microalgal biorefinery for the production of biodiesel and other value-added by-products: a microalgal biorefinery approach.
