Michael J. Kulis

United Negro College Fund Special Programs Corporation, Cleveland, Ohio 

Aloysius F. Hepp 

Glenn Research Center, Cleveland, Ohio

Phong X. Pham and Daniela Ribita 

Cleveland State University, Cleveland, Ohio

Bilal M.M. Bomani

Glenn Research Center, Cleveland, Ohio

Stan A. Duraj

Cleveland State University, Cleveland, Ohio

Extraction and Characterization of Lipids From 

Salicornia Virginica and Salicornia Europaea

NASA/TM—2010-216891

November 2010
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Michael J. Kulis

United Negro College Fund Special Programs Corporation, Cleveland, Ohio 

Aloysius F. Hepp 

Glenn Research Center, Cleveland, Ohio

Phong X. Pham and Daniela Ribita 

Cleveland State University, Cleveland, Ohio

Bilal M.M. Bomani

Glenn Research Center, Cleveland, Ohio

Stan A. Duraj

Cleveland State University, Cleveland, Ohio

Extraction and Characterization of Lipids From 

Salicornia Virginica and Salicornia Europaea

NASA/TM—2010-216891

November 2010

National Aeronautics and

Space Administration

Glenn Research Center

Cleveland, Ohio 44135
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Acknowledgments

The work was supported by the NASA Fundamental Aeronautics Subsonic Wing Project.
NASA/TM—2010-216891 1 

Extraction and Characterization of Lipids From 

Salicornia Virginica and Salicornia Europaea

Michael J. Kulis

United Negro College Fund Special Programs Corporation 

Cleveland, Ohio 44135 

Aloysius F. Hepp 

National Aeronautics and Space Administration

Glenn Research Center

Cleveland, Ohio 44135 

Phong X. Pham and Daniela Ribita* 

Cleveland State University

Cleveland, Ohio 44115 

Bilal M.M. Bomani 

National Aeronautics and Space Administration

Glenn Research Center

Cleveland, Ohio 44135 

Stan A. Duraj 

Cleveland State University

Cleveland, Ohio 44115 

Abstract

The lipid content from Salicornia virginica and Salicornia europaea is investigated. The plants are 

leafless halophytes with seeds contained in terminal nodes. The lipids, in the form of cell membranes and 

oil bodies that come directly from the node cells, are observed using fluorescence microscopy. Two 

extraction methods as well as the results of extracting from the seeds and from the entire nodes are 

described. Characterization of the fatty acid components of the lipids using Gas Chromatography in 

tandem with Mass Spectroscopy is also described. Comparisons are made between the two methods and 

between the two plant materials as lipid sources. 

Background

There are two major challenges to humankind’s energy-intensive lifestyle that make research in 

alternative fuel sources a necessity. First, petroleum is a non-renewable resource; global oil production 

will undoubtedly peak. The production of oil reaches this peak when half of the original supply has been 

pumped from the ground. This occurrence has an indefinite timeline; estimates for peak oil production 

range from the present to near mid-century (Ref. 1). Considering even the most optimistic estimate, 

research in alternative fuels is a critical undertaking. Secondly, it is evident that the Earth’s global average 

temperature is increasing with time. Evidence suggests that our use of fossil fuels as an energy source is 

most likely a contributor to the recent warming trend (Ref. 2). This is because the burning of fossil fuels 

results in carbon dioxide formation, and it’s formation from carbon outside of the carbon cycle creates a 

 

*

Summer Intern at NASA Glenn Research Center and Spring/Fall Intern at National Center for Space Exploration 

Research
NASA/TM—2010-216891 2 

net-positive increase of greenhouse gases in the Earth’s atmosphere. Thus, a fuel source with a net-zero 

greenhouse gas emission seems likely to reduce humankind’s contribution to climate change.

Two promising constituents of plant material for use as a fuel feedstock are lipids and cellulose.

Lipids can be used to produce fuels with relatively long-chained hydrocarbons resembling petroleumderived diesel fuel. Cellulose is more suited for the production of aromatic hydrocarbons found in 

petroleum-derived gasoline. We describe the extraction and characterization of lipids from halophyte 

plants with the ultimate goal of determining the feasibility of using lipids as a feedstock for synthetic 

aviation fuel. 

Lipids are a large class of molecules that are naturally occurring, insoluble in water, and comprised of 

long-chain hydrocarbon groups that may also contain oxygen, nitrogen, phosphorus and sulfur. The most 

common types of plant lipids are waxes, triglycerides, and phospholipids (Ref. 3). Waxes provide a layer 

on the plant’s surface that acts as interface between the plant and its surroundings. This layer serves many 

vital purposes, including a barrier to limit the diffusion of water and solutes while controlling the release 

of volatiles. Waxes are complicated mixtures of components, primarily wax esters. The nature of the other 

lipid constituents can vary greatly with the source of waxy material, but they include hydrocarbons, 

aldehydes, alcohols, ketones, and several others. The molecular structure of triglycerides consists of three 

fatty acid molecules bonded to one glycerol molecule. The distribution of fatty acids is dependent on the 

plant species, but typically consists of fatty acids with sixteen and eighteen carbons and some percentage 

of fatty acids with one or two double bonds (unsaturated). At a given temperature, the overall amount of 

saturation determines if the triglyceride is called a “fat” or an “oil”. Triglycerides are used as an energy 

source stored mostly in seeds and to a lesser extent within the plant itself. The molecular structure of 

phospholipids consists of two fatty acids bonded to a glycerol with a phosphate functional group. Thus, 

the molecule has a polar head and a non-polar tail. They form bilayers in water by arranging the heads to 

point outward towards the aqueous media with the tails inward. The bilayers are the structural basis of 

cellular membranes.

The lipids investigated in the work come from halophytes grown in the GreenLab Research Facility

located at the NASA Glenn Research Center. Halophytes by definition are salt-tolerant plants and thus 

grow in habitats that are rich in salts. The benefits of obtaining fuel from halophytes are that it does not 

compete with freshwater human needs, and it does not exploit arable land that can otherwise be used for 

agriculture with traditional food crops (Ref. 4). The specific halophytes investigated in this work belong 

to the Salicornia genus including the perennial species, S. virginica and the annual species, S. europaea. 

They are leafless plants with jointed and succulent stems that form terminal nodes. The seeds are 

produced and carried within the nodes. The quantity of lipids from the seeds is reported to be near 

30 percent (Ref. 5) The lipids contained within the nodes include lipids from the developing seed as well 

as the lipids from the cells and the waxy surface of the nodes. The overall amount of node lipids increases 

as the plant matures due to the lipid accumulation in the growing seeds. 

Experimental

Microscopy

To qualitatively study the lipid content of the node cells, fluorescence microscopy was utilized. Nile 

red was used as the fluorescent probe since it fluoresces strongly in hydrophobic lipids and is fully 

quenched in water. Preparation of the Nile red dye was based on a procedure by Fowler et al. (Ref. 6). 

The staining of the sample with the dye was based on a procedure by Lersten et al. (Ref. 7). A stock 

solution of Nile red of 500 μg/mL in acetone was prepared and stored in the dark. The dye was made by 

addition of 100 μL of stock solution to 10 mL of 75 percent glycerol in water, followed by brisk stirring. 

The dye was then degassed by vacuum to remove bubbles. For staining, thin layers of epidermal and 

mesophyll cells were sectioned freehand from the node of a fresh S. virginica sample using a razor blade. 

To avoid solvation of the lipids, staining was carried out on an unfixed layer of the node by simply 

wetting the sample with the solution. 
NASA/TM—2010-216891 3 

The microscopy studies were carried out using a fluorescence microscope equipped with 100 W 

halogen lamp and a 20 times plane objective lens. The Nile red fluorescence was viewed using green 

fluorescence 560/40 nm band pass exciter filter, a 595 nm chromatic mirror, and a 675/75 nm 

suppression filter.

Oil bodies within the cytoplasm as well as the lipids that make up the cell membranes of S. virginica

are clearly seen in Figure 1 with Nile red fluorescence. Interestingly, the oil bodies were mostly clustered 

in random cells with only a few individual oil-containing spheres, typically ranging in size from 0.25 to

3 μm. 

Extraction 

The general method of extracting plant constituents consists of dehydration, comminution, and solidliquid extraction (Ref. 8). Drying the plant material is a necessary step as the percent yield of extracted 

material is based on a standard dry weight of the raw material. Also, water interferes with the catalysts 

during transesterification, the reaction used to prepare the extract for characterization (Ref. 9). 

Comminution refers to the process of grinding the sample material into a fine powder. Solid-liquid 

extraction involves dissolving components with a particular solvent to remove them from a solid matrix. 

Freezing is a necessary step for storing and drying the plant material. Samples are kept frozen and in 

the dark to reduce enzymatic hydrolysis of the lipids which produces free fatty acids (Ref. 8). For longterm storage, the temperature of liquid nitrogen, near –210 °C, is best for minimal enzymatic activity.

However, a temperature near –30 °C is typically adequate for short-term storage of less than a month. 

Water is removed from the frozen sample by sublimation in a vacuum, as opposed to evaporation at an 

elevated temperature, in order to reduce loss of constituents.

Comminution of plant material is one of the most important steps in the overall method. The sample 

is broken down into relatively small particles to efficiently extract plant constituents of interest. Seeds 

were ground using a high speed micro-mill with the capability of grinding samples from 20 to 50 mL 

using a blade rotating at speeds up to 12,000 rpm. The sample chamber temperature is maintained by heat 

exchangers around the housing of the micro-mill and can be lowered by circulating cool water, methanoldry-ice liquid or liquid nitrogen. The nodes, which are relatively elastic, are ground using a blender with a 

suspension solvent. Isopropyl alcohol is typically used as it evaporates at a relatively low temperature and 
NASA/TM—2010-216891 4 

also acts as a preservative by inhibiting lipolytic enzymes that hydrolyze triglycerides (Ref. 10). The 

ground plant material is dried under mild heat or vacuum in order to remove the suspension solvent. 

There are two methods used in this work to extract the lipids from the seeds or nodes. One method of 

extraction involves the use of an automated Soxlet extractor. The pre-dried and weighed samples are 

sealed in pre-weighed filter bags. Up to 15 bags can be loaded into the extractor at once. The instrument 

automatically extracts and rinses the sample by performing 40 extraction cycles in 1 hr at a temperature of 

90 °C and a pressure of 12 psig. After extraction, both the remaining cellulosic material and the extracted 

lipids are collected and weighed. The difference between the encapsulated sample before and after 

extraction is divided by the dry weight of the sample and multiplied by 100 to determine the percent lipid 

yield of a particular sample. The overall yield is the average across all samples in a particular extraction, 

expressed as a percentage of weight. The error is taken as the 95 percent confidence interval of the mean. 

A second method of extraction used in this work is ultrasonication. During ultrasonication, the 

formation and collapse of micro-bubbles interacts with the liquid/solid boundary surfaces of the 

suspension, causing the solid material to break apart. The goal is to break down the plant cell in order to 

expose the phospholipids that make up the cell membranes and the oil bodies stored within the node cell.

Thus, comminution and extraction occur in one step. A typical extraction employing this method occurs 

as follows: A sample weighing 10 g in a 50 mL suspension of isopropyl alcohol is sonicated for 10 min at 

20 kHz for a total energy output of 5.4 kJ. After, extraction is complete the non-extracted cellulose is

filtered, dried and weighed. The percentage of extracted material is simply the difference between the 

weight of the original material and the non-extracted material divided by the original material and 

multiplied by 100. The yield in each aliquot of a sample can be used to determine an average yield with a 

95 percent confidence interval.

The solvent determines the types of lipids extracted from the sample. Neutral lipids such as 

triglycerides and waxes are extracted with a non-polar solvent. Phospholipids require polar solvents, such 

as alcohols, to disrupt hydrogen bonds and electrostatic forces. In this study, a mixture of hexane isomers 

was used as the non-polar solvent. This solvent was used to extract the triglycerides from the seeds and 

initially as the solvent to extract triglycerides and waxes from the entire nodes. To extract the 

phospholipids along with the triglycerides in the node cells, a 2:3 mixture of isopropyl alcohol and 

hexanes was used as the polar solvent. Chlorophyll contained in both the seeds and the nodes was also 

consequently extracted using these solvents. Particularly for the extraction involving the nodes, 

chlorophyll was exploited as a simple indicator of the progress of the extraction by gradual pigment loss 

from the cellulosic material.

Results

The results of five extractions are shown in Table 1. The feedstocks were the seeds and nodes from S. 

virginica and the nodes from S. europaea. Method 1 involved the use of the automated Soxlet extractor, 

while Method 2 used ultrasonication. The extracting solvents were a mixture of hexanes and a mixture of 

isopropyl alcohol and hexanes as described above. The percent content of extracted material consists of 

both lipids and chlorophyll.

TABLE 1.—EXTRACTION RESULTS

Sample 

number

Plant Source Method of 

extraction

Extraction solvent Yield

(% by weight)

1 Salicornia virginica Seeds aMethod 1 Hexanes 15.4 ±0.3

2 Salicornia virginica Nodes bMethod 2 Hexanes 2.4 ±0.6

3 Salicornia virginica Nodes Method 1 Hexanes 3.2 ±0.5

4 Salicornia virginica Nodes Method 1 Isopropyl alcohol/hexanes 3.6 ±0.5

5 Salicornia europaea Nodes Method 1 Isopropyl alcohol/hexanes 3.5 ±0.5

6 Salicornia europaea Nodes Method 1 Isopropyl alcohol/hexanes 7.1 ±0.6 a

Method 1 involves an automated Soxlet extractor for extraction

b

Method 2. involves the Ultrasonicator for both comminution and extraction.
NASA/TM—2010-216891 5 

From the results of Table 1, several observations are made. Firstly, Method 1 provided a higher yield 

from the same plant material. One possible reason for this was that it was difficult to determine when the 

extraction was near completion using Method 2, possibly resulting in a premature end to the process. 

Secondly, the more polar solvent mixture of isopropyl alcohol and hexanes was expected to give larger 

yields of extracted lipids from plant nodes because of the additional extraction of the phospholipids that 

make up the cell membranes. However, this is not apparent here since the slight increase in percent yield 

of Sample 4 was within the margin of error of the yield of Sample 3 which used only hexanes as the 

extracting solvent. Lastly, an interesting result was the significant difference in percent yield of extract 

between Samples 5 and 6. Both samples contained the same plant type and same extracting solvent, 

however, Sample 6 provided a 50 percent increase in extracted material. The only noted difference in the 

two samples was that the nodes of Sample 5 had green pigmentation and nodes of Sample 6 were red in 

color indicating that the source plant was near the end of its life cycle.

Characterization

To determine the fatty acid distribution of the extract, fatty acids are removed from the glycerol 

structure and derivatized into fatty acid methyl esters (FAMEs) using the transesterification reaction.

FAMEs are commonly used as fatty acid derivatives, and as a result, there is a large quantity of available 

information on their chromatographic and spectroscopic properties. There are two types of 

transesterification reactions which are distinguished by the type of catalyst used: base-catalyzed and acid 

catalyzed transesterifications.

Base-catalyzed transesterification of triglycerides is the most common process for FAME production.

It is a relatively rapid reaction, typically requiring 30 min to completion with a 99 percent yield (Ref. 9). 

The stoichiometry of the reaction requires a 3:1 methanol/lipid molar ratio. However, the reaction is 

carried out with an extra amount of alcohol in order to shift the equilibrium to the product side of the 

reaction, typically near 7:1 for the base-catalyzed reaction (Ref. 11). Unfortunately, the reaction is 

sensitive to the quality of the feedstock, requiring it to have less than 0.5 percent free fatty acid (FFA) 

content (Ref. 12). If FFAs are present, saponification will occur as a competing reaction when using the 

typical alkaline metal alkoxide as the base. The presence of water will cause the formation of some of 

FFAs by hydrolysis of the produced methyl esters. Saponification results in a reduced yield of methyl 

esters as the resultant soap is formed instead of the desired methyl esters. In addition, the presence of soap 

leads to the formation of an emulsion making the purification process much less efficient (Ref. 9).

A base-catalyzed transesterification was used for the FAME conversion of triglycerides extracted 

from seeds. The extract was dissolved in 100 mL of toluene and combined with 40 mL of 0.5 M of 

sodium methoxide in methanol. The mixture was heated to 60 °C and stirred for 1 hr. The resultant 

FAMEs were separated from the mixture with 100 mL of hexanes and washed with warm distilled water.

Excess methanol and the hexanes were removed using rotoevaporation. The FAMEs were then dissolved 

in 100 mL of toluene and diluted further for analysis.

Acid-catalyzed transesterification is more suitable for unrefined lipid extracts. FFAs are converted to 

methyl esters and there is no soap formation. However, the reaction is much slower than the basecatalyzed reaction, taking approximately 48 hr to obtain a 99 percent yield (Ref. 13). Also, a very high 

methanol to sample molar ratio is required, typically 250:1 (Ref. 14). 

An acid-catalyzed transesterification was used for the FAME conversion of lipids extracted from the 

nodes. The extract was dissolved in 100 mL of toluene and combined with 200 mL of 5 percent sulfuric 

acid in methanol. The mixture was heated to 60 °C and stirred for approximately two days. The resultant 

FAMEs were separated from the mixture with 100 mL of hexanes and washed with warm distilled water. 

Excess methanol and the hexanes were removed using rotoevaporation. The FAMEs were dissolved in 

100 mL of toluene and diluted further for analysis. 

Gas chromatography in tandem with mass spectroscopy was used to qualitatively determine the 

individual components of the extract. Separation was achieved with a cross linked 5 percent Phenyl-, 

95 percent methyl-siloxane capillary column (HP-5MS, 30- by 0.22-mm i.d., film thickness 0.25 um). 
NASA/TM—2010-216891 6 

The FAMEs were dissolved in toluene 1:40 by volume and 0.1 μL of the sample was injected into a 

capillary injector at 120 °C in splitless mode. The helium carrier gas flow rate was kept at 2.6 mL/min.

The oven temperature was initially held at 90 °C. For the first ramp, the temperature was increased to 

200 °C at a rate of 5 °C/min and then held at 200 °C for 30 min. For the second ramp, the temperature 

was increased to 300 °C at a rate of 10 °C/min and again held for 30 min. A mass spectrometer with an 

electron impact ionization of 70 eV was used for detection and identification of the separated 

components. 

FAMEs from extracts of both seeds and nodes were characterized in order to determine the fatty acid 

distribution of the extracted lipids. The chromatographic peaks were identified based on their retention 

times using FAME standards made from pure vegetable oils with known fatty acid distribution. The 

chromatogram of the separated FAMEs from the fatty acids of triglycerides extracted from S. virginica

seeds is shown in Figure 2. The following FAME distribution was determined: 14 percent methyl 

palmitate which has sixteen carbon and no double bonds (C16:0), 73 percent methyl oleate (C18:1), and 

13 percent methyl stearate (C18:0). The chromatogram of the FAMEs from the lipids extracted from S. 

europaea nodes is shown in Figure 3. The following FAME distribution was determined: 24 percent

methyl palmitate (C16:0), 45 percent methyl oleate (C18:1), 47 percent methyl linoleate, and 8 percent

methyl stearate (C18:0).
NASA/TM—2010-216891 7 

The identity of each chromatographic peak was confirmed from their mass spectrum. For example, 

the mass spectrum of peak identified as methyl palmitate (C16:0) is shown in Figure 4. The molecular ion 

is apparent at 270 m/z suggesting that molecule has a molecular weight near 270 g/mol. The loss of the 

methoxy group can be inferred from the line at 239 m/z. The line at 227 m/z represents the loss of a C3 

unit via a complex rearrangement. The long homologous series of related lines at m/z = 199, 157, 143, 

129, 115, 101, and 87 are considered to be ions 14 amu apart that have the general formula 

[CH3OCO(CH2)n]+

. The homologous series is evidence of a long hydrocarbon chain with no other 

functional groups. Lastly, the McLafferty rearrangement is seen at 74 m/z and confirms that the 

chromatographic peak represents a methyl ester (Ref. 15). 
NASA/TM—2010-216891 8 

Conclusion

The lipid content of the nodes of S. virginica was qualitatively studied using fluorescence 

microscopy. Both the phospholipid containing cell membranes and the oil bodies were clearly visible 

under fluorescence. Fluorescence microscopy has potential use as a screening method. Using plant 

material with known lipid content as standards, it may be possible to quickly estimate the lipid yield of 

different plant samples without the need to extract the lipids. 

Comparing the two extraction methods, Method 1 is mostly superior as it provides higher yields and 

is less work-intensive and time-consuming. Method 2 has fewer steps since comminution and extraction 

are combined. However, it involves more work and time because vacuum filtration is required to separate 

the cellulosic material from the extract. For Method 1 extractions are performed under pressure, while for 

Method 2 extractions take place in atmospheric pressure. More solvent is required to extract an equivalent 

amount of lipids using Method 2 since the extraction is limited by atmospheric boiling points of the 

solvent. However, the ultrasonicator used in Method 2 is able to process much larger quantities than the 

automated Soxlet extractor used in Method 1. Thus, Method 2 is more appropriate for large-scale 

processing. 

Overall, the extraction efficiency was lower than expected. For example, seeds have been reported to 

provide lipid yields near 30 percent (Ref. 5). However, the maximum yield in this work was only 

15 percent. Several measures can be used to improve the yields. The samples were typically mixed with 

debris such as soil and remnant cellulosic material. Additional preprocessing to remove debris would 

improve yields by reducing the initial weight of the sample. Also, improvement in the communition 

process will most likely improve extraction efficiency as the finer the material is ground the more 

efficient the extraction. More studies are needed to determine the optimal conditions for extraction. For 

instance, a thorough investigation of solvent mixtures used to extract the lipids is needed. An acidic 

solvent that is more polar than the solvent used in this study, may provide higher yields by improving the 
NASA/TM—2010-216891 9 

disintegration of cells as well as the solvation of polar lipids such as phospholipids. Higher temperatures 

and pressures in Method 1 and longer ultrasonication times in Method 2 can be investigated as well. 

There are advantages to using the seeds of the plant instead of the nodes as a source of lipids. The 

extract mostly consists of triglycerides with only a negligible amount of FFAs and other lipids. Thus, the 

base-catalyzed transesterification reaction can be used instead of the acid-catalyzed transesterification 

which is more time-consuming and expensive in terms of chemicals. The disadvantage is that the seeds 

are only available after seed dispersal, typically 6 to 8 months after germination for annuals. The nodes 

are available at any time. However, as evidenced by the yields from Sample 5 and Sample 6 in Table 1, 

there is significantly larger quantity of storage lipids in the nodes just before seed dispersal mostly from 

the seed themselves. According to the results in this work, there is a 2 to 5 times greater yield of extract 

from the seeds than from the nodes depending on when the extraction took place in the plant’s life cycle.

There is an even a greater percentage of lipids extracted from the seeds since there is a larger quantity of 

chlorophyll in the extract of the nodes. 

The GC/MS characterization showed that both the seeds and the nodes have a significant percentage 

of unsaturated lipids. Interestingly, the chromatogram of the lipids extracted from the nodes contained

methyl linoleate which was not present in the chromatogram of lipids extracted from seeds. A large 

quantity of methyl oleate and methyl linoleate can be beneficial in a source that is used as precursor in 

fuel production. Their double bonds provide reaction sites on the molecular structure which could be used 

for the addition of functional groups or for cracking the molecule in order to produce fuel with different 

properties. 

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14. ABSTRACT

The lipid content from Salicornia virginica and Salicornia europaea is investigated. The plants are leafless halophytes with seeds contained 

in terminal nodes. The lipids, in the form of cell membranes and oil bodies that come directly from the node cells, are observed using 

fluorescence microscopy. Two extraction methods as well as the results of extracting from the seeds and from the entire nodes are described. 

Characterization of the fatty acid components of the lipids using Gas Chromatography in tandem with Mass Spectroscopy is also described. 

Comparisons are made between the two methods and between the two plant materials as lipid sources. 

15. SUBJECT TERMS

Extraction; Lipids; Characterization; Gas chromatography; Mass spectroscopy 

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