Vikram Kumar for the award of the degree of Doctor of Philosophy is an authentic account of the results obtained from the research work carried out under my supervision at the Center for Energy, Indian Institute of Technology Guwahati, Guwahati, India. The oil quality in terms of fatty acid compositions of the biomass obtained under different culture conditions was also assessed by gas chromatography (GC).
Development of direct transesterification (DT) method for accurate quantification of microalgal lipid content
Physicochemical characterization of the strain and media optimization for maximization of growth
6 Biochemical characterization of the strain under different cultivation conditions
121 6.2.2 Evaluation of the strain in different trophic modes 122 6.2.3 Analysis of growth, use of substrates and biomass.
7 Process development for high cell density lipid rich microalgae cultivation and enhanced lipid productivity via
7 Development of a process for the cultivation of lipid-rich microalgae with high cell density and increased lipid productivity via.
8 Development of electrochemical dewatering process for efficient harvesting of microalgae
List of Publications Vitae
L ist of Figures
5.6 (A) Effect of different concentrations of sodium acetate on stem growth under heterotrophic conditions. (B) Effect of different concentrations of glucose on growth under mixotrophic culture. For statistical analysis, the lowest concentration of the respective lipid inducer was taken as the control group.
L ist of Tables
Introduction
- Background and motivation
- Objectives of the study
- Approach
- Organization of the thesis
- References
Further improvement in net lipid productivity of the strain was achieved by designing process engineering strategies to achieve synchronized growth and neutral lipid accumulation in batch and chemostat conditions under nutrient-sufficient conditions. Thus, the final result of this study is the creation of a sustainable bioprocess for the production of biodiesel from microalgae (Figure 1.1).
Review of literature
Global energy scenarios and alternate renewable energy resources
Nuclear energy Hydroelectricity Renewables
Biofuels
However, second-generation biofuels have low conversion rates and the conversion processes are currently not economically feasible (Alam et al., 2012; Adenle et al., 2013). The data in the table shows that microalgae appear very promising as a renewable raw material for the production of biodiesel as an alternative to fossil fuels (Milano et al., 2016).
Microalgae are emerging renewable feedstock for biodiesel production
Microalgae can be considered as model cell factories, as their storage and accumulation capacity can be measured based on different cultivation modes and conditions presented, making them easy to manipulate according to the requirements and direction of research (Muthuraj et al., 2014a). During the stationary growth phase, their ability to store energy as neutral lipids (TAG) depends on the species and the culture conditions, although diacylglycerols (DAG) and monoacylglycerols (MAG) are also identified (Riekhof et al., 2005; Barsanti and Gualtieri, 2006).
Classification and biology of microalgae
Phospholipids are the main type of lipids found in microalgae, which act as structural lipids for the cell, being a major component of the cell wall, and these are found in great abundance during the exponential cell growth phase. The cell wall of eukaryotic cells may or may not have mucilage layers in their extracellular matrix and varies from species to species.
Biosynthesis of fatty acids and triacylglycerols
Acetyl-CoA carboxylase (ACC) initiates lipid biosynthesis, catalyzing an important binding step of the fatty acid synthetic pathway, the biotin-dependent carboxylation of acetyl-CoA to form malonyl-CoA. Fatty acid chains formed in chloroplasts are transferred from CoA to positions 1 and 2 of glycerol-3-phosphate, resulting in the formation of lysophosphatidic and phosphatidic acids, respectively.
Microalgae cultivation: mode of nutrition and reactor types
The lipid productivity ranges from 0.7 to 1.8 g L-1 day-1 which is significantly higher than that obtained in photoautotrophic conditions (Chen et al., 2011). Where 𝐼𝑑 represents the light intensity at depth d, 𝐼0 is the original incident intensity, 𝛾 is the turbidity (Chen et al., 2011).
Process engineering strategies for improved biomass and lipid productivity
However, the most efficient strategy to achieve maximum lipid productivity will be through synchronized growth and neutral lipid accumulation (Tevetia et al., 2014; Klok et al., 2013). Therefore, net lipid productivity, which considers both lipid content and biomass productivity, is used as a performance index to select the best productive strains and develop a biodiesel production process (Chen et al., 2011).
Dewatering technologies for harvesting of microalgae
Autoflocculation increases the interest which results in the spontaneous sediments of the cells (Park et al., 2011). However, using ozone for flotation is not economically feasible for biodiesel production (Rawat et al., 2011).
Processing of microalgal biomass for biodiesel generation
The presence of catalysts in the biodiesel can affect the engine functions and can be corrosive to engines (Hidalgo et al., 2013). However, the use of acid catalysts requires large reaction times even at higher temperatures (Hidalgo et al., 2013).
Microalgae for Third Generation Biofuels Production, Greenhouse Gas Emission Reduction and Wastewater Treatment: Present and Future Perspectives – A Mini-Review. Electrochemical harvesting process of microalgae using non-sacrificial carbon electrode: a sustainable approach for biodiesel production. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor.
Sampling, isolation and identification of the suitable microalgal strain for biodiesel production
- Background and motivation
- Materials and methods
- Sampling and isolation of indigenous microalgal strains
- Screening of neutral lipid accumulating microalgal strain
- Identification of the microalgal strain
- Analytical techniques .1 Analysis of growth
- Results and discussion
- Sampling and isolation of indigenous microalgal strains
- Screening and selection of neutral lipid accumulating microalgal strains The main aim of screening is to identify the “Oleaginous” algal strain which can
- Selection of the suitable growth medium for selected indigenous microalgal strain
- Morphometric and molecular identification of the organism
- Fatty acid profile of the stored lipid obtained from Chlorella sorokiniana FC6 IITG
- Conclusions
- References
The strain with maximum accumulation of neutral lipids was selected for further characterization and process development. Qualitative detection of neutral lipid accumulation in the microalgae strain was performed under a confocal microscope using the Nile red-based staining method. The neutral lipid content of the organism was measured by the Red-Nile method detailed in section 3.2.5.2.
Development of direct transesterification (DT) method for accurate quantification of microalgal lipid content
Background and motivation
The total lipid content of microalgae reflects the energy yield from the biomass and therefore has the greatest influence on the overall process economics of biodiesel production (Devis et al., 2011). These methods involve large amounts of solvents such as hexane, chloroform, and methanol that cause adverse effects on health and the environment (Cheng et al., 2011). A sequential two-step DT method with higher FAME yields was reported for three different algal strains (Griffiths et al., 2010).
Materials and methods
- Algal culture and biomass preparation
- Selection of best combination of transesterification method and biomass type Eight different transesterification methods were carried out to screen the best
- Optimization of process parameters for transesterification by statistical design Preliminary screening experiments resulted in sequential two stage DT method M7
- Field emission scanning electron microscopic analysis
- FAME analysis using GC
- Fourier transform infrared spectrophotometer (FTIR) Analysis
- Statistical analysis
FC2 IITG and the evaluation of the optimized method were performed in photoautotrophic biomass of Chlorella sorokiniana FC6 IITG. A total of 24 experiments were performed to select the best combination of transesterification and biomass type using FAME yield as process response. The selection of the best transesterification method was performed by comparing the FAME yield (%, w/w DCW).
Results and discussion
- Selection of best combination of biomass type and transesterification method Eight different transesterification methods were screened for three types of biomass
- Optimization of process parameters for transesterification
- Effect of transesterification methods on hydrolysis and transesterification efficiency
- Fourier transform infrared (FTIR) spectroscopy
In the case of oven-dried biomass, the reduction in FAME yield may be due to the oxidation of fatty acids (Oehrl et al., 2001). The maximum FAME yield was obtained with the two-stage DT method M7 for lyophilized biomass compared to all other methods. A higher FAME yield was obtained for the M7 method, which used NaOH in the first stage, followed by H2SO4 in the second stage of transesterification.
Conclusions
FC2 IITG biomass via two-stage optimized DT method (B) FTIR spectra of algal biodiesel, mustard biodiesel and petro-diesel. The optimal conditions for two-phase DT have shown 462.6% and 445.4% increase in FAME yield when compared to the conventional method for Chlorella sp. namely FC2 IITG and Chlorella sorokiniana FC6 IITG, which demonstrate the efficiency of two-stage DT in obtaining the near-optimal yield of FAME.
Accurate and reliable quantification of the total microalgal fuel potential as fatty acid methyl esters by in situ transesterification. Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron fluoride-methanol. Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria and wild mixed cultures.
Physicochemical characterization of the strain and media optimization for maximization of growth
Background and motivation
Mixotrophic microalgae have the ability to use both the organic and inorganic carbon sources for cellular growth in the presence of light as the energy source (Chen et al., 2011). There are a large number of reports on the effects of micro- and macronutrients on growth and lipid productivity of microalgae (Chen et al., 2011). An increase in lipid productivity up to 0.19 g L-1 day-1 was also observed when Botryococcus braunii was grown in the optimized lipid production media designed using response surface methodology (Tran et al., 2010).
Materials and methods
- Microalgal strain, media composition and inoculum preparation
- Characterization of the strain under different physicochemical conditions The effect of medium pH, temperature, nitrogen and carbon sources on growth of
- Media optimization for biomass maximization using statistical tools
- Analytical methods
- Quality of biodiesel generated from Chlorella sorokiniana FC6 IITG
Therefore, changes in the growth conditions will have significant influence on biomass and lipid productivity which illustrates the need for characterization of the potential strains under different conditions. Furthermore, BG11 media components involved in the growth of the microalgae FC6 were optimized with the aim of maximizing the biomass formation. After reaching absorption (A v/v) of the mid log phase culture was used as inoculum for all the experiments in the present study.
Results and discussion
- Effect of pH and temperature on biomass titer and intracellular lipid content The effects of temperature and pH on biomass titer and intracellular lipid content of
- Effect of nitrogen sources on biomass titer and intracellular lipid content Further screening of growth supporting and/or lipid inducing nitrogen sources were carried
- Effect of carbon sources on biomass titer and intracellular lipid content of FC6 The strain was further studied under heterotrophic and mixotrophic condition to evaluate its
- Media optimization for growth of FC6 under different cultivation conditions .1 Optimization of BG11 media for photoautotrophic growth
- Effect of physicochemical parameters on FAME composition and quality of biodiesel
To this end, the effect of physicochemical parameters on the fatty acid composition and properties of biodiesel obtained from the FC6 strain was investigated. The effect of initial average pH on fatty acid composition was negligible in the FC6 strain. A similar effect of different carbon sources on fatty acid composition was evaluated under heterotrophic and mixotrophic mode, as shown in Table 5.7.
Conclusions
Regardless of different growth conditions, the viscosity and cetane number of biodiesel obtained from FC6 varied in the range of 4.12 to 4.61 mm2 s-1 and 53.72 to 63.43, resp. Among the different nitrogen sources, sodium nitrate was selected as the best growth, and lipid-inducing nitrogen sources and biodiesel obtained from the FC6 strain grown on this nitrogen source were also in accordance with ASTM and EN standards. Similarly, glucose and sodium acetate were selected as the best growth-supporting carbon source under mixotrophic and heterotrophic conditions, respectively, and biodiesel obtained from FC6 strain grown on this carbon source was found to be in accordance with ASTM and EN standards.
Optimization of carbon and nitrogen sources for biomass and lipid production by Chlorella saccharophila under heterotrophic conditions and development of Nile red fluorescence-based method for quantification of its neutral lipid content. Prediction of cetane number, kinematic viscosity, density and higher heating value of biodiesel from its fatty acid methyl ester composition. Physiological evaluation of a new Chlorella sorkoniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures.
Biochemical characterization of the strain under different cultivation conditions
Background and motivation
14 and ~1.5 times higher lipid productivity compared to photoautotrophic and heterotrophic cultivation conditions respectively (Liang et al., 2009). Interestingly, it is not only the quantity but also the quality of lipids that varies significantly under different cultivation conditions (Liu et al., 2011). Polyunsaturated fatty acids (PUFA) were found to be abundant when cells were grown under photoautotrophic conditions, while monounsaturated fatty acids (MUFA) dominate in heterotrophic cultivation ( Liu et al., 2011 ).
Materials and methods
- Microalgal strain, media composition and inoculum preparation
- Evaluation of the strain under different trophic modes
- Analyses of growth, substrates utilization and biomass composition
- Quality assessment of biodiesel generated from FC6 under different cultivation conditions
After reaching absorption (A v/v) of the revived culture was used as inoculum for all the experiments in the present study. Detailed characterization of the strain was performed under photoautotrophic, heterotrophic and mixotrophic cultivation conditions in a 3L automated bioreactor (eZ Control, Applikon Biotechnology, Holland) with a working volume of 1.25 L containing optimized BG11 medium. Therefore, these properties of the biodiesel were determined based on the empirical comparisons detailed in section 5.2.5 of chapter 5.
Results and Discussion
- Characterization of Chlorella sorokiniana FC6 IITG under different trophic mode in an automated bioreactor
- Composition of FAME obtained from Chlorella sorokiniana FC6 IITG grown under different cultivation conditions
- Evaluation of biodiesel quality obtained from Chlorella sorokiniana FC6 IITG grown under different cultivation conditions
Significant changes in the fatty acid profile were observed with changes in cultivation conditions. The evaluation of the quality of biodiesel obtained from FC6 grown under different cultivation conditions was carried out through empirical equations as a function of FAME composition obtained experimentally (Su et al. The cloud point and pour point of biodiesel varied significantly depending on the conditions of cultivation.
Conclusions
The flash point of all biodiesel obtained under different cultivation conditions was found to be higher than the suggested limits in ASTM and EN standards. Regardless of the different cultivation conditions, biodiesel derived from FC6 was found to meet ASTM and EN standards. Thus, Chlorella sorokiniana FC6 IITG could be a potential cell factory for the synthesis of high-quality biodiesel under mixotrophic cultivation mode.
Mixotrophic cultivation of Chlorella vulgaris and its potential application for the accumulation of oil from non-sugar materials. Selection of Thermophysical Properties Prediction Methods for Process Modeling and Product Design of Biodiesel Manufacturing. The effect of mixotrophy on microalgal growth, lipid content and expression levels of three pathway genes in Chlorella sorokiniana.
Process development for high cell density lipid rich microalgae cultivation and enhanced lipid productivity
Background and motivation
In general, microbial fermentation in fed-batch mode was an effective technique for improved biomass and product titer and productivity (Xie et al., 2013). Two-stage cultivation strategies have been reported to achieve high cell density lipid-rich algal biomass (Karemore et al., 2013). Some of the researchers suggested simultaneous growth and neutral lipid accumulation as an effective strategy to achieve maximum lipid productivity (Tevetia et al., 2014; Klok et al., 2013).
Materials and Methods
- Generation of high cell density lipid rich biomass using single-stage two phase fed-batch mode
- Screening and optimization of elicitor molecules for lipid induction
- Analysis of growth, substrate utilization and FAME composition
Sampling was performed at regular time intervals to monitor growth and lipid accumulation in the strain. To that end, in the first experiment, the organism was grown under fed-batch cultivation with intermittent feeding of nitrate, phosphate, glucose and sodium acetate. In the second experiment, the organism was grown in a chemostat with continuous feeding of modified BG11 media supplemented with glucose and lipid-inducing molecules (sodium chloride and sodium acetate).
Results and discussion
- Generation of high cell density lipid rich biomass using single-stage two phase fed-batch mode
- Screening of elicitor molecules for improved lipid productivity of Chlorella sorokiniana FC6 IITG
- Process development for synchronized growth and neutral lipid accumulation in an automated photobioreactor
- FAME composition and biodiesel property
Use of sodium acetate resulted in an increase in neutral lipid productivity of the strain Chlorella vulgaris and Leptolyngbya sp. This combined improvement in growth and lipid content resulted in significantly improved net lipid productivity. Based on these observations, sodium acetate and sodium chloride were selected as the suitable lipid inducer for the strain FC6 and their combined effect on growth and lipid productivity was evaluated.
Conclusions
