The primary goal defined for the present study was to develop a sustainable way to produce biodiesel from microalgae. As a maiden step, potential microalgal strains were isolated based on biomass concentration and lipid yield.Among the six isolated strains, Tetradesmus obliquus KMC24 was considered the potential strain with maximum biomass (2.35 ± 0.02 g L-1) and lipid yield (29.51 ± 0.26%).Further,T. obliquus KMC24 was exposed to nutrient stress (nitrogen and/or phosphorus) for a short period via two- stage cultivation to enhance the lipid content of the biomass without compromising the biomass concentration. Nitrogen starvation (-N) and combined nitrogen and phosphorous starvation (-N-P) changed the morphology of T. obliquus KMC24 from unicell to 2 and 4 celled coenobium with multiple spines at terminal cells. Compared to various nutrient starved conditions, the photosynthetic apparatus was comparatively less affected during phosphorus starvation.The highest lipid content of 39.93 ± 0.64% was obtained in –N2 cultures, which was around 1.35 folds higher than the control. This indicated that nitrogen starvation triggered the production of intracellular lipid.The NL content in –N2 cultures was almost similar to –N4 cultures with significantly higher (P < 0.0001) DCW, suggesting that two days of nitrogen starvation is a better approach for obtaining high biomass and NL content in T. obliquus KMC24.The highest ROS fluorescence intensity (17051.49 ± 93.15 a.u.) and MDA content (3.81 ± 0.02 µM g-1 fresh weight) were observed in -N2 cultures, which indicated that a linear correlation exists between ROS level and MDA content. Moreover, Pearson's correlation analysis revealed a positive linear correlation between ROS and lipid accumulation under all nutrient starved conditions. The activities of CAT, APX, and polyphenols were consistent with lipid content suggesting a strong connection between oxidative stress and lipid accumulation.
152 | P a g e Nitrogen starvation for four days increased the degree of saturation of the total fatty acid pool by almost fourfold. Due to high SFA content in nitrogen starved cultures, biodiesel with higher CN, superior oxidative stability, and improved shelf life were obtained.Thus, it was observed thatT. obliquus KMC24 cells under two days of nitrogen starvation possess a high potential for biodiesel production.
In the next part of the study, waste eggshell derived bioflocculant was successfully applied for harvesting T. obliquus KMC24.Harvesting efficiency of 98.62 ± 0.43% was achieved within 25 minutes under optimal conditions of 120 mg L-1 flocculant dose, pH 4.0, and a temperature of 35 °C. The bioflocculant consisting of a high concentration of cationic calcium ions significantly influenced the zeta potential of the culture suspension.
Zeta potential analysis confirmed charge neutralization as the flocculation mechanism.
FESEM analysis showed no structural deformations when the cells were harvested under optimal conditions. Pseudo-second order kinetic model was a suitable fit for the data obtained from the experiments, which indicated chemisorption as the probable mechanism. The thermodynamic parameters revealed that the sorption process was spontaneous and endothermic in nature.The spent medium was successfully recycled and reused to cultivate T. obliquus KMC24, thereby reducing the water footprint.
In the final phase of the current resarch, a sustainable catalyst using de-oiled microalgal biomass as carbon support was developed. The carbon-based solid acid catalyst was synthesized at an optimum pyrolysis temperature of 600 °C, sulfonation time of 6 h, and H2SO4 concentration of 15 mL. The amorphous DMB catalyst was predominantly composed of carboxylic, phenolic, and sulfonic acid groups. TGA and NH3-TPD analysis confirmed that the DMB catalyst was thermally and chemically stable.
Hydrophobic carbon acted as potential support for sulfonated carbon catalysts.A high FAME yield with improved fatty acid composition was obtained. Moreover,FAME yield
>90% was obtained after four successive reuse cycles. Hydrophobic carbon support suppressed the leaching of −OH and –SO3H groups.Thus, the high catalytic efficiency of the DMB catalyst for the transesterification reaction process signified its potential for sustainable biodiesel production.
Therefore, the present study was able to accumulate a considerable amount of lipid in T. obliquus KMC24 under nitrogen starved conditions making it a promising
153 | P a g e candidate for biodiesel production. A waste-to-wealth approach was successfully implemented by valorizing waste eggshell-derived bioflocculant. This low-cost and energy efficient harvesting technology was developed to attain net sustainability.
Recyclability of the spent medium will certainly help to lower the water footprint of microalgal biodiesel production system. Moreover, the conversion of microalgal lipid to biodiesel and de-oiled microalgal biomass to catalyst will help to maximize the economic value of microalgae within a biorefinery concept, leading to a circular bioeconomy for biodiesel production from microalgae.
6.2 Future scope
The current research tried to address the various challenges faced during the upstream and downstream processes of biodiesel production from microalgae. However, the above study was limited to a lab-scale. Therefore, the research can be extended in the following mentioned areas to augment the work further.
Optimization of nitrogen starvation for enhanced growth and lipid accumulation in T.
obliquus KMC24 at the pilot-scale plant.
Genetic engineering of T. obliquus KMC24 for enhanced biomass and lipid production.
Process optimization and application of waste eggshell-derived bioflocculant for harvesting microalgae at the pilot-scale plant.
Process optimization and application of DMB catalyst for transesterification at the pilot-scale plant.
Leaching of active components is still an issue for the reported DMB catalyst, which needs further attention to reduce or eliminate them. It would help in enhancing the stability of the catalysts further.
Techno-economicevaluation and life cycle analysis of the overall process need to be carried out to determine the sustainability of the system.
154 | P a g e
155 | P a g e