Comparative Analysis of Biofuel Production from Spirulina Biomass and Chlorella Powder: Evaluating Growth Efficiency and Yield Optimization through Rice Water-Based Bioremediation (Wastewater)

Title: Comparative Analysis of Biofuel Production from Spirulina Biomass and Chlorella Powder: Evaluating Growth Efficiency and Yield Optimization through Rice Water-Based Bioremediation (Wastewater) Authors: Tony Royce ([email protected]) Tejaswini S.S Thaanupriya T Ramyaa M Affiliation: Department of Biotechnology, SRM Institute of Science and Technology, Ramapuram Campus, Chennai, India Keywords: Biofuel, microalgae, Spirulina, Chlorella, wastewater bioremediation, lipid extraction 1 ABSTRACT Under microalgae-based biofuels, it provides an option for the replacement of conventional fuels with an alternative energy source, rapid algal growth with the possibility of high accumulation of lipids along with capturing carbon and remediating wastewater among others. In this regard, the study comparatively analyzed the biofuel production efficiencies of Spirulina platensis and Chlorella vulgaris in a rice water-based medium (wastewater), supplemented by True water, which is an artificial water. The preparation of growth medium for Spirulina involved modification of Zarrouk's using agricultural rice water. The culture of Chlorella powdered water (3g) and Spirulina culture (100 mL per liter in two separate beakers) was carried out under controlled light and aeration conditions. Lipids were extracted by methanol and chloroform, while transesterification was done by sodium hydroxide and methanol. However, the yield obtained was very low (<1 mL) at the end of the entire optimization of the cultivation, lipid extraction, and transesterification processes. It was found that Chlorella had better growth and lipid productivity than Spirulina in wastewater cultivation. Both gases were tested for their ability to sequester CO₂ by microalgae, plus their nutrient pollutant removal (nitrate and phosphate). Improvements in biomass concentration and optimizing solvent systems and harvesting techniques would contribute to improving biofuel yields in future studies. Recommended further tests for a more extensive characterization of biodiesel quality and energy content include lipids profile analysis (GC-MS) and calorific value testing of the extracted biofuel. Keywords: Microalgae, Rice Water, Biofuel, Lipid Extraction, Wastewater Bioremediation 2 GRAPHICAL ABSTRACT 3 TABLE OF CONTENTS ABSTRACT 2 GRAPHICAL ABSTRACT 3 LIST OF ABBREVIATIONS 5 FIGURE LEGENDS 6 TABLE LEGENDS 7 INTRODUCTION 8 PROBLEM STATEMENT 9 SCOPE OF WORK 10 OBJECTIVE 11 PROPOSED SOLUTION 12 EXPERIMENTAL PROCEDURE 13 RESULTS AND DISCUSSION 16 VALIDATION OF THE PROJECT VALUES: 21 DISCUSSION AND IMPLICATIONS: 22 CONCLUSION 23 REFERENCE : 25 4 LIST OF ABBREVIATIONS ● CO₂ – Carbon Dioxide ● OD – Optical Density ● NaOH – Sodium Hydroxide ● FAME – Fatty Acid Methyl Esters ● GC-MS – Gas Chromatography-Mass Spectrometry ● PBR – Photobioreactor ● TDS – Total Dissolved Solids ● ASTM – American Society for Testing and Materials ● EN – European Norm (European Standard) ● HCl – Hydrochloric Acid ● NaHCO₃ – Sodium Bicarbonate ● NaNO₃ – Sodium Nitrate ● K₂HPO₄ – Dipotassium Hydrogen Phosphate ● K₂SO₄ – Potassium Sulfate ● MgSO₄ – Magnesium Sulfate ● CaCl₂ – Calcium Chloride ● FeSO₄ – Ferrous Sulfate 5 FIGURE LEGENDS Figure no. Title Figure 1 Spirulina vulgaris culture Figure 2 Measurement of Chlorella platensis biomass Figure 3 Media preparation using rice water and supplementation Figure 4 Cultivation of Spirulina platensis in wastewater medium Figure 5 Harvesting of microalgal biomass after cultivation Figure 6 Drying and grinding of biomass into fine powder using mortar and pestle Figure 7 Dried biomass prepared for lipid extraction Figure 8 Chemical solution setup in a water bath for transesterification Figure 9 Visible separation of glycerol and biodiesel layers Figure 10 Centrifugation of solution for separation of biomass Figure 11 Extraction of Spirulina supernatant Figure 12 Addition of chemical reagents to Chlorella biomass powder Figure13 Lipid layer obtained from Chlorella vulgaris Figure 14 Chemicals added to Chlorella lipid layer and incubated at room temperature for 1 week Figure 15 Layer separation of glycerol and biofuel after 1-week incubation of Chlorella lipid layer Figure 16 Chemicals added to Spirulina lipid layer and incubated at room temperature for one week Figure 17 Result after one week of incubation Figure 18 Combustion test of Spirulina-derived biofuel Figure 19 Combustion test of Chlorella-derived biofuel 6 TABLE LEGENDS Table No. Title Table 1 Final Optical Density (OD 680 nm) and Growth Phase Duration of Spirulina and Chlorella Table 2 Biomass Productivity of Spirulina and Chlorella Cultivated in Rice Water Medium Table 3 Lipid Content and Lipid Yield per Liter of Culture for Spirulina and Chlorella Table 4 Biofuel Yield from Spirulina and Chlorella via NaOH-Catalyzed Transesterification Table 5 Estimated CO₂ Sequestration by Spirulina and Chlorella Table 6 Nitrate and Phosphate Removal Efficiency by Microalgae in Rice Wastewater Table 7 Comparative Analysis of Key Parameters for Industrial Feasibility Table 8 Validation of Experimental Results with Literature-Reported Ranges 7 INTRODUCTION The global search for sustainable energy sources has driven significant interest in microalgae-based biofuels as a renewable alternative to fossil fuels. Microalgae such as Spirulina platensis and Chlorella vulgaris are renowned for their high growth rates, biomass productivity, and lipid accumulation capabilities. However, commercial-scale production faces challenges due to the high cost of synthetic growth media and energy-intensive processing steps. To overcome these limitations, this study explores the use of rice water — an agricultural wastewater rich in essential nutrients — combined with True water, as an alternative low-cost cultivation medium. Utilizing waste rice water not only provides a cost-effective solution for microalgal growth but also contributes to environmental bioremediation by removing nitrates and phosphates from agricultural runoff. In this comparative analysis, Spirulina biomass and Chlorella powder were cultivated separately in rice water-based media. Their growth performance, biomass productivity, lipid yield, and carbon dioxide (CO₂) sequestration efficiency were evaluated. Biofuel extraction was carried out using methanol-chloroform extraction and subsequent sodium hydroxide-catalyzed transesterification. Despite optimized cultivation, the final biofuel yield observed was notably low (<1 mL), highlighting the need for further process optimization. The integrated approach of utilizing waste resources for biofuel production aims to address both energy and environmental challenges, making microalgae cultivation an attractive dual-purpose technology for a sustainable future. 8 PROBLEM STATEMENT ● The increasing reliance on fossil fuels has led to severe energy crises, environmental pollution, and accelerated climate change, necessitating the development of sustainable energy alternatives. ● Agricultural wastewater, particularly rice water, is often discharged untreated into the environment, despite being rich in nutrients that can support microalgal growth. ● Traditional biofuel production from food crops leads to land and water competition, making microalgae a more viable, sustainable feedstock for biodiesel production. ● The high cost of synthetic growth media and inefficiencies in lipid extraction methods pose major challenges to the economic feasibility of large-scale microalgal biofuel production. ● Utilizing rice water (wastewater) for microalgal cultivation offers a cost-effective solution by recycling agricultural waste and simultaneously treating nutrient-laden effluents. ● A direct comparison between Spirulina platensis and Chlorella vulgaris cultivated in rice water has not been extensively studied, especially regarding their growth, lipid accumulation, and wastewater bioremediation capabilities. ● Although lipid extraction and biodiesel production techniques are well-established, modifications to solvent systems and catalysts (using methanol-chloroform extraction and sodium hydroxide-based transesterification) require assessment under low-resource conditions. ● This study aims to optimize microalgal cultivation in agricultural rice water while evaluating the challenges encountered in achieving satisfactory biofuel yields, contributing to sustainable energy production and wastewater management. 9 fig 1 fig 2 fig 3 fig 4 fig 5 16 fig 6 fig 7 fig 8 fig 9 fig 10 fig 11 fig 12 17 fig 14 fig 13 fig 15 fig 17 fig 16 Fig 19fig 18 18 RESULTS AND DISCUSSION 1. Growth Efficiency of Spirulina and Chlorella a. Optical Density (OD 680 nm) Analysis ● Growth was monitored by measuring the optical density (OD) at 680 nm every 48 hours for both microalgae. ● Chlorella vulgaris exhibited a higher growth rate in rice water-based media, reaching an OD of 1.7 after 10 days, compared to 1.3 for Spirulina platensis. ● The exponential phase lasted approximately 7 days for Chlorella and 9 days for Spirulina before reaching the stationary phase. ● These results suggest that rice water, when combined with bottled water, serves as a viable alternative to synthetic media. Although OD values were lower than typical literature values for nutrient-rich media (~2.0–2.5 for Chlorella; Liu et al., 2017), the growth was sufficient to support biomass and lipid production in both strains. Parameter Spirulina platensis Chlorella vulgaris Final Optical Density (OD 680 nm) 1.3 1.7 Exponential Phase Duration (days) 9 7 Stationary Phase Start (day) 10 8 Literature OD Range (nutrient media) 1.5–2.5 2.0–2.5 b. Biomass Productivity ● Dry weight measurements confirmed that Chlorella yielded 3.2 g/L biomass, while Spirulina yielded 2.5g/L biomass after 10 days. ● The biomass productivity for Chlorella was calculated as 0.32 g/L/day, whereas Spirulina exhibited 0.25 g/L/day. ● The higher biomass productivity of Chlorella suggests that it is better suited for large-scale wastewater-based cultivation. 19 2. Lipid Yield and Biofuel Production a. Lipid Content Analysis ● Lipid extraction was performed on oven-dried algal biomass using a chloroform-methanol (2:1) solvent system. ● The lipid content was found to be 5.8% for Spirulina platensis and 12.5% for Chlorella vulgaris, based on dry biomass weight. ● These values align with expected lipid yields for wastewater-grown algae, although they are slightly lower than literature ranges observed in synthetic media (typically 6–12% for Spirulina and 15–30% for Chlorella; Mata et al., 2010). ● This reduction may be attributed to the non-optimized nutrient composition of rice water. Parameter Spirulina platensis Chlorella vulgaris Lipid Content (% dry biomass) 5.8% 12.5% Lipid Yield (g lipid per L) 0.104 g/L 0.337 g/L Solvent System Used Chloroform:Methanol (2:1) Chloroform:Methanol (2:1) Literature Range (Lipid Content) 6–12% 15–30% 20 b. Biofuel Yield from Transesterification ● The lipids extracted from Spirulina and Chlorella biomass were converted into biodiesel using a transesterification reaction with sodium hydroxide (NaOH) and methanol. The reaction was conducted at 60°C for 1 hour, following a 6:1 methanol-to-lipid molar ratio. ● The resulting biofuel yield was 0.7 mL/L for Spirulina platensis and 1.5 mL/L for Chlorella vulgaris. ● This lower-than-expected yield reflects both the limited lipid content and the small-scale cultivation volumes used in the study. However, the results demonstrate the feasibility of biodiesel production from wastewater-cultivated microalgae using simple, lab-scale methods. Parameter Spirulina platensis Chlorella vulgaris Biofuel Yield (mL per L culture) 0.7 mL 1.5 mL Transesterification Catalyst NaOH in Methanol NaOH in Methanol Molar Ratio (Methanol:Lipid) 6:1 6:1 Reaction Temperature 60°C 60°C Reaction Time 1 hour 1 hour 21 3. CO₂ Sequestration Efficiency ● The ability of Spirulina platensis and Chlorella vulgaris to sequester atmospheric CO₂ was evaluated by monitoring pH changes during cultivation and estimating carbonate accumulation. ● Based on biomass productivity and growth rate, CO₂ fixation was calculated using standard conversion factors. ● Chlorella demonstrated a higher CO₂ fixation efficiency of 1.7 kg CO₂ per kg of dry biomass, compared to 1.4 kg CO₂/kg for Spirulina. ● This can be attributed to its faster growth rate and greater biomass yield, making it more effective for carbon capture in wastewater-based systems. ● These findings are in line with previous studies reporting CO₂ fixation ranges of 1.5–2.0 kg CO₂/kg biomass (Chisti, 2007). Parameter Spirulina platensis Chlorella vulgaris CO₂ Fixation (kg CO₂/kg biomass) 1.4 1.7 Relative Growth Rate Moderate 1.8 Biomass Concentration (g/L) 1.8 2.7 Literature CO₂ Fixation Range 1.5–2.0 kg CO₂/kg biomass 1.5–2.0 kg CO₂/kg biomass 22 4. Wastewater Treatment Efficiency ● The potential of microalgae to remediate wastewater was assessed by measuring nitrate and phosphate concentrations in the rice water medium before and after cultivation. ● Both Spirulina platensis and Chlorella vulgaris contributed to nutrient removal, but Chlorella showed better performance overall. ● After 10 days of growth: ○ Spirulina removed 42% of nitrates and 37% of phosphates. ○ Chlorella removed 50% of nitrates and 48% of phosphates. 5.Comparative Analysis and Industrial Feasibility Parameter Spirulina Chlorella OD after 10 days (680 nm) 1.3 1.7 Biomass Productivity (g/L) 1.8 2.7 Lipid Content (% dry weight) 5.8% 12.5% Biofuel Yield (mL/L culture) 0.7 1.5 CO₂ Fixation (kg CO₂/kg biomass) 1.4 1.7 Nitrate Removal (%) 42% 50% Phosphate Removal (%) 37% 48% 23 Parameter Initial Concentration (mg/L) FinalSpirulina (mg/L) FinalChlorella (mg/L) ReductionSpirulina (%) ReductionChlorella (%) Nitrate (NO₃⁻) 80 mg/L 40 mg/L 35 mg/L 50 mg/L 56 mg/L Phosphate (PO₄³⁻) 30 mg/L 17 mg/L 15 mg/L 43 mg/L 50 mg/L VALIDATION OF THE PROJECT VALUES: Parameter Spirulina Chlorella Literature Range Reference OD after 10 days (680 nm) 1.3 1.7 1.5–2.5 Liu et al., 2017 Biomass Productivity (g/L) 1.8 2.7 1.5–3.5 Becker, 2007 Lipid Content (% dry weight) 5.8% 12.5% 6–12% (Spirulina), 15–30% (Chlorella) Mata et al., 2010 Biofuel Yield (mL/L culture) 0.7 1.5 0.5–2.5 (from low-scale microalgal setups) Various Lab Studies CO₂ Fixation (kg CO₂/kg biomass) 1.4 1.7 1.5–2.0 Chisti, 2007 Nitrate Removal (%) 42% 50% 40–70% Wang et al., 2010 Phosphate Removal (%) 37% 48% 30–60% Wang et al., 2010 24 DISCUSSION AND IMPLICATIONS: ● The study successfully demonstrates the potential of rice wastewater as a sustainable and cost-effective medium for microalgal cultivation. The use of agricultural effluent mixed with bottled water provided sufficient nutrients to support both biomass growth and lipid accumulation. ● Chlorella vulgaris consistently outperformed Spirulina platensis in all key parameters, including growth rate, lipid content, CO₂ sequestration, and nutrient removal. This aligns with literature reports that Chlorella has a naturally higher lipid biosynthetic capacity, making it more suitable for biodiesel production. ● The relatively low lipid content and limited biofuel yield observed in both species (1.5 mL/L for Chlorella; 0.7 mL/L for Spirulina) highlight the challenges of working with non-sterile, unoptimized wastewater media. These results indicate the need for improved nutrient balancing, supplementation, and possibly strain enhancement to boost yield. ● Despite modest fuel output, the environmental benefits were significant. Both species contributed to CO₂ mitigation (up to 1.7 kg CO₂/kg biomass) and effectively removed nitrate (up to 50%) and phosphate (up to 48%) from the medium. This supports the dual-role concept of microalgae in bioremediation and renewable fuel production. ● Compared to conventional synthetic media like BG-11 or Zarrouk's standard formula, rice water cultivation reduces operational costs significantly, making the approach more feasible for rural and semi-industrial setups. ● These findings suggest that microalgae grown on agricultural wastewater can serve not only as a sustainable energy source but also as an integrated environmental solution for wastewater treatment and greenhouse gas capture. ● For future scale-up, emphasis should be placed on: ○ Optimizing solvent extraction protocols for higher lipid recovery ○ Exploring photobioreactor designs to improve productivity ○ Incorporating enzymatic or hybrid extraction techniques ○ Investigating genetically enhanced algal strains 25