A Study on Enhanced Lipid Accumulation by Cold Plasma Process in Chlorella sp.

Abstract

This study investigated the enhancement in lipid accumulation in Chlorella sp. using non-thermal atmospheric pressure plasma as a pretreatment strategy for the production of value-added products. The plasma treatment was optimized by varying discharge times (0–16 min) using argon gas at a flow rate of 4 L/min. Lipid productivity was assessed through gravimetric analysis and profiling of fatty acid methyl ester using gas chromatography−mass spectrometry (GC-MS). The growth rate and pH of the treated cells were monitored. The findings demonstrated that the 4-min plasma exposure maximized the efficiency of lipid recovery, achieving a 35% of the dry cell weight and a 34.6% increase over untreated control. However, longer plasma treatment times resulted in a comparative decrease in lipid yield, as the decline is possibly due to oxidative degradation. The findings highlight the role of plasma treatment, which significantly boosts lipid yield and gives complementary optimization of downstream processes to improve biodiesel production. The accumulation of lipids in terms of size and volume in the algal cells was assessed by confocal laser scanning microscopy. The GC–MS results of the control revealed that lipids comprised primarily mixed esters such as 2H Pyran 2 carboxylic acid ethyl esters, accounting for 50.97% and 20.52% of the total peak area. In contrast, the 4-min treated sample shifted to saturated triacylglycerols (dodecanoic acid, 2,3 propanetriyl ester), comprising 85% of the total lipid content, which efficiently produced biodiesel. Thus, the non-thermal plasma-based enhancement of lipids in the algal cells has been achieved.

1. Introduction

Microalgae, a highly diverse and ubiquitous group of photosynthetic microorganisms that thrive in a wide variety of aquatic habitats including freshwater lakes to saltwater oceans and wastewater treatment plants and ranging from unicellular to multicellular forms, have emerged as a promising and sustainable platform for various applications. They efficiently convert solar energy and carbon dioxide into valuable biomass and can address issues of energy security and environmental sustainability, these benefits have increased interest from both academic and industrial sectors [1,2,3]. With the growing demand for sustainable energy and the need to reduce greenhouse gas emissions, biofuels have gathered attention as a potential alternative to fossil fuels. The capacity to assimilate flue gas carbon dioxide benefits biomass production and carbon sequestration as an attempt to mitigate global climate change [4]. In biofuels, microalgae are known for their lipid content, namely triacylglycerols (TAGs), the precursors for biofuel production through a chemical process called transesterification. Some species, namely Chlorella vulgaris, Nannochloropsis sp., and Scenedesmus obliquus, have lipids ranging from 5% to 50% of their dry weight, depending on growth conditions [5].

Microalgal lipids are a heterogeneous mixture of molecules comprising neutral lipids (e.g., triglycerides, TAGs), polar lipids (e.g., phospholipids), and sterols. In contrast, the TAGs are the primary target for biodiesel production due to their high energy density and facile conversion to fatty acid methyl esters (FAMEs) [1]. Under optimal growth conditions, microalgae prefer to invest resources in cell division and biomass production, leading to a lipid content of 5–20% dry weight [5]. Under stress conditions (e.g., nutrient starvation, high salinity, or high light intensity), most species redefine their metabolism towards lipid storage, accumulating TAGs to over 50% in some instances [6,7,8,9]. The trade-off between lipid content and biomass yield is a recurring problem, as high percentages of lipids in low biomass yields do not move to economically productive outputs.

Several alternative approaches have been developed to overcome these limitations. Genetic engineering addresses lipid biosynthesis pathways, for example, overexpression of enzymes such as acetyl-CoA: diacylglycerol acyl transferase (DGAT), enabling the synthesis of TAG [10]. Abiotic stress conditions such as high light intensity, temperature fluctuations, or salinity can also trigger lipid storage, but their effects vary with different species and are hard to standardize [5]. Increasing lipid yield in microalgae has become a major area of interest, along with investigating a broad range of approaches ranging from nutrient optimization to technological processes.

Plasma is a partially or fully ionized gas, separate from solids, liquids, and neutral gases. It is a mixture of free electrons, positive ions, and neutral atoms or molecules [11]. It is formed when enough energy from heat, electric fields, or electromagnetic radiation is transferred to a gas, removing electrons from atoms and producing a conductive medium. Thermal plasma, also known as equilibrium plasma, is defined as the state in which electrons, ions, and neutral particles achieve thermal equilibrium with temperatures between 5000 and 20,000 K. Such high temperatures are achieved with high energy input like electric arcs or torches, utilized in most industrial applications such as welding, cutting, and plasma spraying.

Using non-thermal plasma in biological sciences highlights its versatility and compatibility with living organisms. Non-thermal plasma interacts with cells through its dense chemistry of reactive species (ROS and RNS) generated during gas ionization [12]. These short-lived and highly reactive species can permeate cell membranes, oxidize biomolecules and trigger a cascade of physiological responses without causing ambient temperatures to rise to intolerable levels. The non-thermal plasma treatment in microalgae uses the principle that lipid accumulation is a physical abiotic stress response. Reactive species may release and trigger lipid biosynthetic enzymes such as acetyl-CoA carboxylase or DGAT or the increment of TAG storage by stimulating the starvation cues for the nutrients without arresting the growth. Recent developments showed that plasma-based technologies can effectively increase lipid accumulation in microalgae, but mechanistic insight and reproducibility are limited. Plasma technology has recently emerged as an effective non-GMO strategy to boost lipid and PUFA accumulation in microalgae. For example, co-mutagenesis of Haematococcus pluvialis by atmospheric-pressure room-temperature plasma yielded stable mutants (AV3 and AV8) whose biomass, lipid content, and growth rate increased by ~16%, ~54.8%, and ~45%, respectively, compared to the wild type. These mutants showed higher activities of key lipid-biosynthesis enzymes (ME, PEPC, ACCase, and DGAT) and transcriptomic or metabolomic shifts in glutathione, arginine, and arachidonic-acid pathways that underlie the elevated lipid production [5]. Similarly, the treated Chlorella sp. L166 with low-temperature dielectric-barrier plasma (LTP) for 120 s, which maximized both cell biomass and α-linolenic acid (ALA) content; the 120 s LTP exposure increased ALA by 35.5% relative to untreated algae [13]. The study aims to culture green microalga under various plasma discharge times to facilitate enhanced lipid accumulation and eventually used for biodiesel production and other biotechnological products.

2. Materials and Methods

2.1. Microalgae Isolation

An unidentified green microalgal cell was found at abandoned walls in Goa (latitude: 15.4923; longitude: 73.7737). The green algal sample was washed with distilled water and cultured on an agar plate with TAP medium (NH₄Cl, CaCl₂·H₂O, MgSO₄·7H₂O, K₂HPO₄, KH₂PO₄, Na₂EDTA·2H₂O, ZnSO₄·7H₂O, H₃BO₃, MnCl₂·4H₂O, FeSO₄·7H₂O, CoCl₂·6H₂O, CuSO₄·5H₂O, (NH₄)₆Mo₇O₂₄·4H₂O, C₂H₄O₂, and C₄H₁₁NO₃) to isolate pure colonies. Pure colonies were mounted on glass slides and examined under a compound microscope to assess their purity and structural morphology. Based on the microscopy analysis, the isolate was identified as belonging to the genus Chlorella sp. and was subsequently used for further studies.

Microalgae Cultivation

The isolated microalgae were cultured in 250 mL conical flasks for different treatment times at a temperature of 25 °C ± 2 °C with manual agitation twice daily in a bright light cabinet in TAP medium. After sufficient growth, the culture was scaled up to 500 mL conical flasks to ensure adequate biomass. The microalgae demonstrated exponential growth after 25 days, reaching the stationary phase, at which they were harvested.

2.2. Plasma Treatment

The non-thermal atmospheric-pressure plasma jet was coupled to an alternating current (AC) power source of 17 kV and 30 mA to generate the plasma using two electrodes, namely the live electrode and the ground electrode, operating at atmospheric pressure using argon (Ar) gas at a flow rate of 4 L/min. The gas was maintained at room temperature. In contrast, the electron temperature could reach around 10,000 to 100,000 K. The plasma plume was directed onto petri dishes containing Chlorella sp. culture. The microalgal cultures were kept in petri plates, one at a time, and were positioned directly beneath the plasma jet, allowing the luminous plume to encounter the surface of the culture, as shown in Figure 1. The time of discharge was controlled for each culture, with a time interval of 4, 8, 12, and 16 min, including untreated culture, which was taken as 0 min (control). A time interval of 4 min was kept between the plasma treatments (0–16 min), allowing responsive research to evaluate the impact of plasma exposure on the lipid content. The distance between the electrodes was set as 6 mm. Under these conditions, an intense plasma was formed between the two electrodes, and a luminous plume was created at the end, which reached the algal culture in the petri plate. After the plasma exposure at the set time intervals, the microalgal suspensions were collected for further analysis.

2.3. Confocal Laser Scanning Microscopic Investigation of Lipid Accumulation

Confocal laser scanning microscopy (CLSM) provides high-resolution, three-dimensional visualization of cellular structures. It uses focused laser beams to scan samples in an inverted position, capturing images at various depths while minimizing out-of-focus light, enhancing image clarity, and enabling detailed analysis of lipid distribution within cells [1]. CLSM was captured using a CLSM (LSM710, Carl Zeiss, Oberkochen, Germany) and a 100× oil objective lens. Nile red staining is a crucial technique in CLSM that visualizes lipid accumulation in microalgae. This fluorescent dye binds to neutral lipids, allowing for clear observation of lipid droplets and their distribution within algal cells. The stock solution of Nile red stain was prepared by dissolving 10 mg of NR powder in 10 mL of HPLC-grade acetone. An aliquot of cells was mixed with 50 μL NR stock solution and incubated in the dark. The stained cells were then placed between two microscopy coverslips for imaging. Fluorescence analysis was performed using an argon laser with an excitation filter set at 488 nm and an emission band-pass filter set at 530–600 nm.

2.4. Biomass and Lipid Extraction

The microalgal cultures were taken, and the pellet was collected by centrifugation and then shallow-dried. Then, the dried algal biomass from all the different plasma treatment times was measured, and 0.5 g from each culture was taken and powdered using mortar and pestle.

The lipid extraction was performed using the Bligh and Dyer method [14]. The powdered sample was taken in separate 15 mL centrifuge tubes and suspended in a chloroform and methanol solvent mixture (2:1, v/v), followed by agitation using a vortex mixer. The samples were homogenized by ultrasonication for 30 min at an amplitude of 30%, and pulses of 40 s and 20 s were applied, with the samples resting on an ice bath. After sonication, a monophasic system was observed, and phase separation was induced by adding a 0.75% NaCl solution. The cell debris was separated from the supernatant, and the mixture was vortexed for 2 min and centrifuged at 4500 rpm for 5 min to separate the two different layers. The upper and the middle layers containing cell debris were removed, and the lower organic phase containing lipids was transferred to sterilized petri plates and kept for drying until the solvent was evaporated. The lipid content in percentage was calculated using Formulae (1)

Total Lipid content (%) = (Weight of the lipid/Weight of biomass) × 100

(1)

2.5. FAME Profiling

The extracted lipids were trans-esterified to fatty acid methyl esters (FAMEs) using acid-catalyzed esterification and were analyzed by GC-MS (PerkinElmer Clarus 680/600, PerkinElmer, Massachusetts, USA). The peaks were identified by comparison to library spectra and the retention times. The relative area percentage of each peak was calculated to profile fatty acid composition. The fatty acid composition of the oil extracted from the algal biomass was quantified using gas chromatography. By comparing the retention times of the identified peaks with corresponding fatty acid standards, the qualitative composition was determined, and the quantitative composition was obtained by the normalization of the peak area, where the area of each identified FAME peak was expressed as a mass percentage of the total area of all the FAME peaks [4,15,16]. The analysis of fatty acid composition is essential for understanding lipid profiles, and GC-MS offers a powerful tool for both qualitative identification and quantitative assessment [5].

3. Results

3.1. Microalgal Growth

The isolated strain was identified based on morphological characteristics under a light microscope using standard monographs (Figure 2a). The obtained microalga, Chlorella sp., based on the features mentioned in the standard monographs [17,18] and 18S rRNA might provide additional confirmation. The species was identified as Chlorella sp. and used in this study. The collected microalgal sample was streaked on TAP agar plates for isolation (Figure 2b). After incubation, distinct colonies were observed, and individual colonies were transferred into a liquid TAP medium for further cultivation. The colony was transferred to liquid TAP medium (broth) and grown in 500 mL Erlenmeyer flasks with manual agitation twice daily (Figure 2c). The cultures were illuminated at 25 ± 2 °C.

The pH of the culture medium of Chlorella sp. increased slightly from 8.852 (control) to 9.212 (16 min) after plasma treatment, as seen in Figure 3. The generation of reactive species by plasma might have led to the alkalinity of the culture. The pH increase could have impacted nutrient availability and cellular metabolic pathways, which could have resulted in the observed growth. The plasma treatment produced ammonia (NH₃) and similar nitrogenous compounds, which raised the pH of the culture medium. Organic compound degradation by the reactive species produced by plasma also liberated alkaline compounds, further increasing the pH. The studies indicate that plasma can alter pH in microalgal cultures, although the direction varies depending on the plasma type and conditions. For example, some studies showed plasma pH was lowered due to acidic species, but the results aligned with a peer study where pH increased, possibly due to NTAPP effects [18].

The optical density (OD) at 600 nm was measured to assess microalgal growth at different plasma exposure times, as shown in Figure 4. From the absorbance values, the short-term plasma treatment (4 min) showed a slight decrease in the microalgal growth, but this was not a significant decrease, whereas an 8 min exposure showed a significant decrease. However, prolonged plasma exposure (12 and 16 min) showed increased OD, which is due to the cell aggregation, oxidative stress, and cell damage caused by the plasma. Moreover, growth estimation by absorbance was not ideal for the plasma treatment after 8 min based on the presented data.

3.2. CLSM Imaging of Lipid Bodies

The investigation into lipid accumulation in Chlorella sp. following NTAPP revealed a significant enhancement, particularly in samples exposed to plasma for 4 min. The lipid accumulation in Chlorella sp. was assessed using Nile red staining. The fluorescence intensity was observed under a confocal laser scanning microscope at an excitation wavelength of 488 nm, and the emission filter was set at a wavelength between 530 and 600 nm, which showed clear lipid bodies of bright yellow color and microalgal cells of red-colored bodies.

In Figure 5, the CLSM images show the presence of bright orange-colored lipid bodies in the Chlorella cells, while the other samples with higher plasma treatment times from 8 min to 16 min showed low to no lipid content. The images show autofluorescence of cells (red) and Nile red-stained lipid vesicles (green), but the yellow color appears in some cells due to the overlap with chloroplasts.

The results confirmed that lipid bodies were highly accumulated in the 4-min plasma-treated sample and less at higher plasma treatment times when compared to the control culture. Nile red is known for its sensitivity to neutral lipids and its ability to fluoresce in hydrophobic environments, allowing for a reliable, semi-quantitative comparison of lipid content. Nile red staining conditions for Nannochloropsis salina under thermal stress reported an excitation wavelength of 488 nm and an emission range of 530–600 nm and was ideal for detecting lipids via CLSM [19]. In this study, 4-min of NTAPP treatment was optimal, while the longer durations showed lower fluorescence intensity, possibly due to oxidative degradation of accumulated lipids. The results confirmed that the use of NTAPP accumulated lipids in Chlorella sp., confirmed with CLSM and Nile red staining for visualization and analysis.

3.3. Biomass and Lipid Content

The dry cell weight (DCW) of cultures followed the growth trends. The 4-min treated sample produced slightly higher biomass than the 8 and 12 min treatments which yielded lower biomass. The 4-min treated sample’s higher biomass was consistent with its minimal growth inhibition. The microalgal cultures were harvested through centrifugation at 4500 rpm for 15 min at 4 °C. The biomass pellets were poured in a dry pre-weighed petri plate and then shallow-dried to remove moisture content before lipid analysis. The dry biomass weight across varying plasma treatment durations showed a gradual decrement. The untreated control culture (0 min) yielded the highest dry weight at 0.52 g L⁻¹, followed by 0.47 g L⁻¹ for the 4-min treated sample. Further treatment durations from 8 to 12 and 16 min resulted in biomass weights of 0.41, 0.42, and 0.41 g L⁻¹, respectively. These values suggested that the short-term plasma exposure (4 min) showed decent biomass retention while the higher plasma treatment times showed decreased biomass production. NTAPP was available in the TAP medium up to 45 days. After the exposure, it was kept for a week in the plasma-treated one. After that, the culture was drawn, and the dry weight was measured. Unfortunately, the live/dead cell assay was not performed for this study. A study investigating the stress conditions that affected microalgal growth and lipid production found that nitrogen-deprived conditions increased lipid content in Chlorella sp. but reduced biomass production. The DCW under stress was around 0.46 g L⁻¹, which was lower when compared to 0.60 g L⁻¹ in the control culture [19,20]. In this study, it was similar to the observed decline from 0.52 g mL⁻¹ (control) to 0.41 g L⁻¹ at 16 min of plasma exposure. Importantly, the lipid content (as % DCW) was significantly enhanced by plasma pretreatment. Untreated Chlorella contained ~26% lipids in DCW, whereas 4-min treated cells reached 35% lipids (Figure 6). The relative increase demonstrates the efficacy of NTAPP in triggering lipid accumulation. The treatments longer than 4 min showed reduced lipid percentages such as 30% at 8 min, 27% at 12 min, and 25% at 16 min. The decline at longer exposures suggests oxidative degradation of lipids or damage to biosynthetic pathways under extended plasma stress. Similar inverse effects of prolonged plasma on lipid yield were noted in cold plasma studies.

Using a non-thermal plasma combined with argon gas in atmospheric air yielded a lipid content of 35% in Chlorella sp. The lipid increment showed the benefit of the combined gases in the NTP process, and it may be related to the formation of radicals by argon ionization and the presence of atmospheric air at the gas/liquid interface. This study investigated how lipid extraction yield changed with different treatment times (0–16 min). As seen in Figure 6, the lipid yield results indicated that the 4-min treatment gave the highest lipid recovery (35%) and was higher than that of the control (26%). This suggested that the treatment time at 4 min maximized the accumulation of lipids and longer treatment durations decreased lipid biosynthesis. The decline at longer treatment times suggests potential lipid oxidation, so further research into plasma parameters such as voltage and gas composition is needed.

This study found that Chlorella sp. produced a 35% yield in 4-min plasma treatment, higher than the 19% yield for Nannochloropsis gaditana. A similar study also showed 32.8% for Scenedesmus when treated with air and argon NTAAP. This suggests that the effectiveness of NTAAP can vary, depending on the species [21]. The observation of reduced lipid yields at longer times (17% at 16 min) aligned with the research that indicates prolonged NTAAP treatment may induce lipid oxidation. This is supported by food processing studies, which show an increased TBA index with plasma treatment, indicating oxidative degradation [14,22]. The lipid content achieved from 4-min plasma treatment was higher than that obtained through the treatment process involving UV exposure, mutant selection, and chemical treatment [23]. This study showed an increment in trilaurin content, suggesting plasma-induced stress responses targeting the TAG synthesis without the complexity of mutagenesis. This study indicated a decline after 8 min due to species-specific responses or possibly the oxidative effects.

3.4. FAME Yield and Composition

Due to their high productivity and ability to accumulate fatty acids such as omega-3 polyunsaturated fatty acids (PUFAs), microalgae are studied for their lipid content, particularly their applications in biofuels and nutraceuticals [5]. From the results observed in Figure 7, the 4-min plasma culture showed a slightly lower FAME yield (9.48%) than the control (10.76%).

This difference in yield was related to the impact of the treatment on lipid extraction and conversion processes. The control yield of 10.76% suggests that the process is relatively effective in converting lipids into biodiesel under standard conditions. Meanwhile, the 4-min treatment resulted in a comparatively low yield of 9.48%, with no major significant difference (1.28%). This difference suggested changes in lipid class, as trilaurin has increased TAGs [22]. This was similar to the study where the stress-favored TAGs required optimization for biodiesel conversion [19]. Further, the profiling of lipids could improve yields. The reduced yield in the 4-min treatment may be due to the efficiency of the lipid extraction process. If the treatment was to break down the cell walls of the Chlorella sp. to release more lipids, it might not be sufficient. The conditions under which the transesterification reaction occurs are also vital. The process may not have allowed enough time for the lipids to fully react with methanol and the catalyst, leading to incomplete conversion of triglycerides into FAMEs.

3.5. FAME Profiling

The compositional analysis of fatty acid methyl esters derived from Chlorella sp., determined by gas chromatography−mass spectrometry, reveals a complex mixture of compounds with varying retention times and relative abundances, offering valuable insights into the lipid profile of Chlorella sp. in the control (Supplementary file Table S1). Five distinct peaks were identified as shown (Supplementary file Figure S1), each corresponding to a specific compound, with peak areas ranging from 98,968 to 907,425; representing 5.56% to 50.97% of the total area, respectively. The compound eluting at 28.366 min, identified as 2H-Pyran-2-carboxylic acid, 6-ethoxy-3,6-dihydro-, ethyl ester (C₁₀H₁₆O₄), dominated the profile, accounting for approximately 50.97% of the total peak area. The compound eluting at 29.151 min also identified as 2H-Pyran-2-carboxylic acid, 6-ethoxy-3,6-dihydro-, ethyl ester was present at a significant proportion of 20.52%, further emphasizing the prominence of this compound in the sample. Other notable compounds included 6-Cyanomethoxy-N-methoxymethyl-N’,N’-dimethyl-1,3,5-triazine-2,4-diamine (5.56%), d-Mannitol, 1-decylsulfonyl, and 2,4-Di-tert-butylthiophenol, each contributing 5.56%, 13.47%, and 9.48% to the total peak area, respectively. These compounds highlight the total number of compounds present.

Similarly, the compositional analysis of fatty acid methyl esters derived from the 4-min plasma-treated Chlorella sp. is tabulated in

Table 1. Major peaks with their retention times and other parameters in the 4-min treated sample of Chlorella sp.

Peak#	R. Time	Area (%)	Height (%)	A/H
1	16.989	14.42	14.81	5.51
2	17.390	18.82	9.33	11.42
3	17.639	23.73	18.08	7.43
4	29.159	14.41	22.55	3.62
5	29.875	28.61	35.22	4.60
		100.00	100.00

and exhibited five distinct peaks in the gas chromatograph (Supplementary Figure S2), indicating the presence of a diverse mixture of compounds with varied retention times and abundances. The total ion chromatogram revealed peak areas ranging from 168,984 to 335,267; with relative area percentages spanning 14.41% to 28.61%. As shown in

Table 2. The identified compound names with their areas (%), molecular formulae and weights (g/mol) from the 4-min treated sample of Chlorella sp.

S. No	Retention Time (min)	Area (%)	Compound Name	Molecular Formula	Molecular Weight (g/mol)
1	16.989	14.42	Dodecanoic acid, 1,2,3-propanetriyl ester (trilaurin)	C39H74O6	638
2	17.390	18.82
3	17.639	23.73
4	29.159	14.41	Ginsenol	C15H26O	222
5	29.875	28.61	Dodecanoic acid, 1,2,3-propanetriyl ester (trilaurin)	C39H74O6	638

, the compound eluting at 29.875 min, identified as Dodecanoic acid, 1,2,3-propanetriyl ester (C₃₉H₇₄O₆), was the most dominant, contributing 28.61% of the total peak area. Another significant compound at 17.639 min, also identified as Dodecanoic acid, 1,2,3-propanetriyl ester, constituted 23.73% of the profile, suggesting repeated isomeric or stereoisomeric forms of the same ester. The peaks at 17.390 and 16.989 min represented 18.82% and 14.42%, respectively, while the compound at 29.159 min, identified as Dodecanoic acid, 1,2,3-propanetriyl ester, contributed 14.41% to the total area. The data showed an accumulation of dodecanoate triesters, consistent with significant lipid methyl ester formation after treatment, resulting in an enriched medium-chain fatty acid profile in the treated sample.

Table 3. Total number of compounds identified in the 4-min treated sample of Chlorella sp.

S. No.	RT (min)	Area %	Compound Name	Molecular Formula	Molecular Weight (g/mol)
1	16.989	14.42	Dodecanoic acid, 1,2,3-propanetriyl ester	C39H74O6	638
2	16.989	14.42	Dodecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester	C27H52O5	456
3	16.989	14.42	Rac-glycerol-1,3-dilaurate	C27H52O5	456
4	17.390	18.82	Dodecanoic acid, 1,2,3-propanetriyl ester	C39H74O6	638
5	17.390	18.82	Cyclohexene-3,5-diol, cis-	C6H10O2	114
6	17.390	18.82	5-trans-Methyl-1R,3-cis-cyclohexanediol	C7H14O2	130
7	17.639	23.73	Dodecanoic acid, 1,2,3-propanetriyl ester	C39H74O6	638
8	17.639	23.73	3,5,9-Trioxa-4-phosphaheneicosan-1-aminium, 4-hydroxy-N,N,N-trimethyl-10-oxo-7-[(1-oxododecyl)oxy]-, hydroxide, inner salt, 4-oxide, (R)-	C32H64NO8P	621
9	17.639	23.73	Dodecanoyl chloride	C12H23ClO	218
10	29.159	14.41	Ginsenol	C15H26O	222
11	29.159	14.41	1,2,4-Triazol-3-amine, 5-(1,3,5-trimethyl-4-pyrazolyl) amino-	C8H13N7	207
12	29.159	14.41	3-Methoxy-2,4,5-trifluorobenzoic acid, eicosyl ester	C28H45F3O3	486
13	29.159	14.41	3-Methoxy-2,4,5-trifluorobenzoic acid, nonadecyl ester	C27H43F3O3	472
14	29.159	14.41	Dimethylmalonic acid, 2-isopropoxyphenyl nonyl ester	C23H36O5	392
15	29.875	28.61	Dodecanoic acid, 1,2,3-propanetriyl ester	C39H74O6	638
16	29.875	28.61	Dodecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester	C27H52O5	456

shows the total number of compounds present in the sample.

The 4-min treated sample was dominated (85% area) by dodecanoic acid 1,2,3-propanetriyl ester (trilaurin, a saturated triacylglycerol) (Table 2). Several minor isomers of trilaurin (with retention times of 16.989, 17.390, 17.639, and 29.159 min) collectively made up >80% of the profile (Table 2). This indicates that NTAPP treatment induced the accumulation of medium-chain saturated TAGs. The higher presence of saturated TAGs explains the efficient biodiesel properties, thereby the acid-catalyzed transesterification yield was not higher.

The lipid profile shift under NTAPP was notable: saturated triglycerides (C12) production was advantageous for biodiesel (good cold flow and stability). A similar study that increased TAGs in Chlorella under helium plasma showed the presence of trilaurin, suggesting plasma-triggered stress metabolism favoring saturated fat synthesis, as seen in other plasma-treated algae [22].

As seen in

Table 4. A comparative study of total saturated and unsaturated fatty acids content in the control and treated samples.

Sample	Total MUFA (%)	PUFA (%)	SFA (%)
Control	~13.5	Not detected	~13.5 + minor SFAs (~20%)
NTAAP (4 min)	~14 (possibly modified)	Slightly reduced	~85

, the control sample showed the presence of 10-octadecenal (C18:1), a monounsaturated fatty acid. At the same time, the 4-min NTAAP-treated sample showed a significant shift towards saturated fatty acids. Dodecanoic acid (C12:0) triacylglycerol esters comprise approximately 85% of the lipid content. This shift indicates oxidative stress induced by NTAAP, leading to the degradation of unsaturated fatty acids and the accumulation of more stable saturated lipids. A comparative study confirmed that the major fatty acids are oleic and linoleic acid, with the most abundant saturated fatty acid being the palmitic acid. The study also highlighted the nature of these profiles during algal growth, particularly the oleic acid content. The lipid accumulation suggested that physical stressors like mild pressure or NTP exposure increase triacylglycerol accumulation in Chlorella sp. [23]. NTAAP-induced oxidative stress has been demonstrated to degrade polyunsaturated fatty acids because of peroxidation susceptibility, suggesting that the lipid profile changes were observed in the treated sample [24]. Under certain stress conditions, Chlorella vulgaris primarily produces palmitic acid (C16:0), oleic acid (C18:1, MUFA), linoleic acid (C18:2, PUFA), α-linolenic acid (C18:3, PUFA), palmitoleic acid (C16:1, MUFA), lauric acid (C12:0), and eicosapentaenoic acid (EPA, C20:5n-3, PUFA). As confirmed in this study, the major fatty acids were oleic and linoleic acids, with the most dominant saturated fatty acid being the palmitic acid. These findings also highlighted the nature of profiles observed during algal growth, specifically emphasizing the presence of unsaturated fatty acids (oleic acid and linoleic acid) and saturated fatty acids (palmitic acid) [25].

4. Conclusions

The plasma treatment was performed on Chlorella sp. using argon gas at a flow rate of 4 L/min, with exposure times of 0 (control), 4, 8, 12, and 16 min. The 4-min plasma exposure duration resulted in the highest lipid yield, reaching 35% of dry biomass, an increase of 34.6% compared to the untreated control, suggesting it is the optimal condition for lipid enhancement. The plasma exposure beyond 8 min led to a significant decrease in lipid yield, dropping to 17% at 16 min, as this decline is attributed to oxidative degradation of lipids. A gradual increment in the culture medium pH was observed after plasma treatment, ranging from 8.8 in control to 9.2 in the 16 min treatment. The Nile red staining confirmed a significant increase in neutral lipid bodies in the 4-min treated culture, with reduced fluorescence observed in higher exposure durations, indicating lipid depletion or cellular stress using CLSM. The FAME conversion efficiency was slightly reduced in the plasma-treated culture (9.48%) compared to that in the control (10.76%), suggesting that the lipid content was improved, and transesterification efficiency may have been lowered by cell disruption and release of contaminants. GC-MS analysis revealed a substantial shift in the fatty acid profile due to plasma treatment. The plasma-treated culture showed a transition to saturated fatty acids, especially dodecanoic acid, 1,2,3-propanetriyl ester, making up over 85% of the FAME content. Plasma acts as a non-lethal biochemical stressor, triggering intracellular signaling pathways that enhance lipid biosynthesis without significantly compromising cell integrity at lower exposure times to maximize lipid yield and conversion efficiency, contributing to the advancement of renewable energy technologies.