TOC-L with TNM-L: Simultaneous Measurement of CO2 Fixation and Nitrogen Content in Microalgal Biomass

Significance of the topic

The quantification of CO2 fixation and cellular nitrogen in microalgal biomass is essential for both process optimization and life cycle assessment (LCA) of microalgae-derived sustainable aviation fuel (SAF). Reliable, rapid analytical methods that measure organic carbon and total nitrogen in culture suspensions enable monitoring of photosynthetic carbon conversion and the nutrient status that drives lipid accumulation—two parameters that determine biomass value as a fuel feedstock and the net greenhouse gas mitigation potential of algal processes.

Objectives and overview of the study

This application note describes the use of a Shimadzu TOC-L total organic carbon analyzer equipped with a TNM-L total nitrogen unit to simultaneously measure total organic carbon (TOC) and total nitrogen (TN) in Chlamydomonas reinhardtii CC-1010 cultures. The aims were to: quantify CO2 fixation as organic carbon in biomass, monitor intracellular nitrogen as an indicator of lipid-triggering nutrient stress, and relate these metrics to cumulative light exposure, biomass concentration, and intracellular lipid content to inform cultivation strategies for SAF feedstock production.

Methodology

Sample preparation and fractionation:

• Culture broth centrifuged at 5,000×g for 5 min at room temperature to separate cell pellet and supernatant.
• Pellet washed by resuspension in ultrapure water (volume equal to original sample), centrifuged again at 5,000×g for 5 min; final pellet resuspended in ultrapure water and homogenized by sonication to lyse cells before analysis.

TOC/TN analysis and calibration:

• TOC-LCPH analyzer with TNM-L unit was used. Measurement modes: TC by combustion catalytic oxidation with NDIR detection; IC by acidification and sparging with NDIR; TN by thermal decomposition/chemiluminescence.
• Injection volumes: TC and TN: 50 µL; IC: 250 µL. TOC calculated as TC minus IC.
• Calibration standards: TC with potassium hydrogen phthalate (0–500 mg C/L, 4-point), IC with sodium bicarbonate (0–100 mg C/L, 4-point), TN with potassium nitrate (0–100 mg N/L, 4-point).

Dry cell weight (DCW):

• Determined following ASTM D5907 (ABO-recommended) by filtration on Whatman GF/B glass fiber filters, rinsing, drying at 100 °C, and weighing to compute biomass concentration (g L−1).

Lipid extraction and FAME analysis:

• Lyophilized cells disrupted with 0.1-mm zirconia beads in CHCl3/MeOH (2:1) with tripentadecanoin internal standard; bead-beating cycles: 1 min at 2,500 rpm with 2-min intervals, repeated 8 cycles.
• Liquid–liquid extraction with Tris buffer and repeated solvent washes; pooled organic extracts dried under N2.
• Methyl esterification with 5% H2SO4 in MeOH at 70 °C for 3 h; FAMEs recovered into hexane and dried; analyzed by GCMS-TQ 8040 NX.

Used Instrumentation

• Shimadzu TOC-LCPH total organic carbon analyzer with TNM-L total nitrogen unit (TOC/TN catalyst).
• GCMS-TQ 8040 NX for FAME (fatty acid methyl ester) analysis.
• Centrifuge capable of 5,000×g, ultrasonic homogenizer (sonicator), bead beater with 0.1-mm zirconia beads.
• Whatman GF/B glass fiber filters, oven for drying at 100 °C.

Main results and discussion

Relationship with light exposure and biomass:

• Both biomass concentration and TOC in biomass increased with cumulative light intensity, indicating photosynthetically driven carbon fixation scales with light exposure.
• TOC in biomass correlated positively with biomass concentration, supporting biomass as a proxy for culture-level carbon fixation under the tested conditions.

Effects of intracellular nitrogen on lipids and carbon fixation:

• Lipid content rose as intracellular nitrogen content decreased, consistent with nitrogen starvation-triggered lipid accumulation reported in literature.
• TOC content per culture decreased slightly with lowering nitrogen content, while it increased with cumulative light; a similar trend was observed for TOC in the culture supernatant.
• Cell concentration declined with nitrogen depletion; combined with reduced per-cell photosynthetic activity, this produced a trade-off: nitrogen starvation increased lipid fraction via redistribution of existing carbon (membrane lipid remodeling to triacylglycerol) but tended to reduce ongoing carbon fixation at both per-cell and culture levels.

Implications for cultivation strategy:

• Maintaining nitrogen availability during exponential growth maximizes biomass productivity and CO2 fixation efficiency.
• Inducing nitrogen depletion after biomass has plateaued (i.e., after achieving desired cell density) can act as a strategic trigger to enhance lipid accumulation without excessively compromising total carbon fixation during productive growth phases.

Benefits and practical applications of the method

The TOC-L/TNM-L combined measurement provides several practical advantages for microalgal process development:

• Simultaneous TOC and TN measurement on the same sample allows integrated monitoring of carbon fixation and nitrogen status, which are key for balancing biomass productivity and lipid yield.
• Analysis on aqueous suspensions avoids time-consuming drying steps for carbon quantification, enabling higher throughput and faster feedback for process control.
• Data support life cycle assessment (LCA) inventory needs by providing experimental quantification of organic carbon fixed from CO2 and nitrogen dynamics relevant to lipid production.

Future trends and potential uses

Potential directions and applications enabled by this approach include:

• Real-time or higher-frequency monitoring workflows coupling TOC/TN analytics with online culture sensors to implement dynamic nutrient control strategies that optimize both lipid productivity and net CO2 uptake.
• Scale-up studies in photobioreactors and open-pond systems to quantify how light gradients, mixing, and large-scale nutrient dynamics affect integrated carbon fixation and lipid accumulation.
• Integration of TOC/TN data into LCA and techno-economic models to better quantify greenhouse gas mitigation potential and economic viability of algal SAF at various deployment scales.
• Application to a broader range of algal strains and mixed-culture systems to identify species-specific nutrient control protocols that maximize lipid yield while preserving carbon fixation.

Conclusion

Simultaneous measurement of TOC and TN using the Shimadzu TOC-L with TNM-L provides a practical, informative analytical platform for evaluating CO2 fixation and nitrogen-driven lipid dynamics in microalgal cultures. The study with Chlamydomonas reinhardtii CC-1010 demonstrates that cumulative light exposure increases biomass and organic carbon, while nitrogen depletion promotes lipid accumulation at the cost of reduced cell proliferation and modest declines in carbon fixation. Appropriate nutrient staging—maintaining nitrogen during exponential growth and depleting it after biomass accumulation—can optimize both lipid productivity and overall CO2 conversion performance. Rapid TOC/TN analytics support process optimization and provide essential data for LCA of algal SAF production.

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