Bilal Mark McDowell Bomani

Glenn Research Center, Cleveland, Ohio

Dirk Link, Brian Kail, and Bryan Morreale

Department of Energy, National Energy Technical Laboratory, Pittsburgh, Pennsylvania

Eric S. Lee, Bethany M. Gigante, and Robert C. Hendricks

Glenn Research Center, Cleveland, Ohio

The Effect of Growth Environment and Salinity 

on Lipid Production and Composition of 

Salicornia virginica

NASA/TM—2014-216645

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Bilal Mark McDowell Bomani

Glenn Research Center, Cleveland, Ohio

Dirk Link, Brian Kail, and Bryan Morreale

Department of Energy, National Energy Technical Laboratory, Pittsburgh, Pennsylvania

Eric S. Lee, Bethany M. Gigante, and Robert C. Hendricks

Glenn Research Center, Cleveland, Ohio

The Effect of Growth Environment and Salinity 

on Lipid Production and Composition of 

Salicornia virginica

NASA/TM—2014-216645

March 2014


	



	


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NASA/TM—2014-216645 1 

The Effect of Growth Environment and Salinity on Lipid 

Production and Composition of Salicornia virginica 

Bilal Mark McDowell Bomani 

National Aeronautics and Space Administration 

Glenn Research Center 

Cleveland, Ohio 44135 

Dirk Link, Brian Kail, and Bryan Morreale 

Department of Energy 

National Energy Technical Laboratory 

Pittsburgh, Pennsylvania 15236 

Eric S. Lee,* Bethany M. Gigante, and Robert C. Hendricks 

National Aeronautics and Space Administration 

Glenn Research Center 

Cleveland, Ohio 44135 

Abstract 

Finding a viable and sustainable source of renewable energy is a global task. Biofuels as a renewable 

energy source can potentially be a viable option for sustaining long-term energy needs. Biodiesel from 

halophytes shows great promise due to their ability to serve not only as a fuel source, but a food source as 

well. Halophytes are one of the few biomass plant species that can tolerate a wide range of saline 

conditions. We investigate the feasibility of using the halophyte, Salicornia virginica as a biofuel source 

by conducting a series of experiments utilizing various growth and salinity conditions. The goal is to 

determine if the saline content of Salicornia virginica in our indoor growth versus outdoor growth 

conditions has an influence on lipid recovery and total biomass composition. We focused on using 

standard lipid extraction protocols and characterization methods to evaluate 12 Salicornia virginica

samples under six saline values ranging from freshwater to seawater and two growth conditions. The 

overall goal is to develop an optimal lipid extraction protocol for Salicornia virginica and potentially 

apply this protocol to halophytes in general. 

Introduction 

Accepting the fact that conventional sources of inexpensive fuel and energy are rapidly depleting and 

cannot be renewed, identifying renewable energy sources is a global task that will require innovative 

thinking and methods. Biofuels are a viable option for sustaining long-term renewable energy needs 

because they satisfy the criteria to be considered green ecologically friendly energy sources—halophytes 

are alternative, renewable and sustainable (Refs. 1, 2, 3, and 4). More specifically, developing alternative 

energy needs that do not rely on freshwater, compete with food crops or use arable land are increasingly 

important due to increased world demand on these resources. Halophytes are a viable option for 

renewable energy biomass because they can potentially be used as a fuel or food source and do not rely 

on freshwater or compete with existing land that conventional food crops utilize (Refs. 5, 6, 7, and 8). 

A halophyte is any plant (macro or micro) that is able to grow and thrive in salt-water environments 

or brackish-water such as salt marshes, seas coasts and deserts. The world flora lists approximately 

885 species of halophytic angiosperms distributed over 250 genera (Ref. 8). Less than 2 percent of plant 

species are halophytes. Halophytes have a variety of advantages over traditional crops used for biofuel. 

 

*

 NASA Intern with Lewis Educational and Research Collaborative Internship Project 
NASA/TM—2014-216645 2 

They minimally impact the scarce and rapidly depleting resources of fresh water and arable land and are 

capable of thriving in a variety of environments. Unlike corn or soybeans, halophytes are not traditionally 

used as a food crop and their use as a source of biofuel will have minimal economic or sociological 

consequences. Approximately 50 species of seed-bearing halophytes are potential sources of lipids, which 

are the plant product extracted for biofuel. Glenn (Ref. 5) and Hodges (Ref. 9) have tested the feasibility 

of using seawater agriculture to grow halophytes and found that halophytes grow well under these 

conditions in desert or sandy soil environments. It is likely that most new halophyte applications will be 

inland as most halophytes have reduced productivity at very high salinities (Refs. 10 and 11), along with 

other considerations, such as more traditional farming practices and population distribution. 

Halophytes are applicable in developing subtropical desert countries with limited freshwater and 

growing populations. One of the most urgent global problems is finding enough fresh-water and arableland to support the world’s food needs. The United Nations Food and Agriculture Organization (Ref. 12) 

estimates that an additional 200 million hectares of new cropland will be needed over the next 30 years 

just to feed the burgeoning populations of the tropics and subtropics. However, only 93 million hectares 

are currently available and easily accessible. Halophytes do not require traditional arable land and are 

potentially a valuable cash crop and an elegant solution to increasing demand for food and livestock feed. 

An analysis (Ref. 13) performed at NASA Glenn Research Center (GRC) projects that advanced 

halophyte agriculture that utilizes available arid and semi-arid land can provide for the projected world 

energy demand. Additionally, halophyte seeds have higher lipid percentages than traditionally used 

oilseeds and potentially the maximum amount of oil produced per acre is much greater for halophytic 

plots (Refs. 1 and 14) with comparable total biomass. 

The halophyte Salicornia virginica naturally occurs in a wide distribution and is adaptable to a variety 

of salinities. Salicornia virginica (also known as Salicornia depressa) is a perennial, coastal, salt-marsh 

halophyte native to Florida and California and found as far north as Alaska. Salicornia virginica is the 

dominant mid-elevation plant species in salt-marshes and is a typical first-colonizer. An appealing aspect 

in maintaining an area of Salicornia virginica is that possible competitors cannot tolerate salt ions (i.e., 

high salinities) as effectively as Salicornia virginica and unwanted or invasive species can be managed 

through controlled soil salinity (Ref. 14). Salicornia virginica has the ability and is known to respond to 

increased salinity levels by decreasing root conductivity and increasing salt uptake, but its growth rates 

are minimally affected by salinity (Ref. 15). Unlike glycophitic plants, Salicornia virginica can reversibly 

change its root characteristics to adapt to different levels of salinity. Other research supports that the CO2

uptake, stomata opening and other indicators of photosynthetic activity of Salicornia virginica are 

affected by salinity to a lesser degree than other plants (Ref. 16). Figure 1 to Figure 6 show various 

habitats of Salicornia virginica

The primary goal of this study was to identity the influence of (1) the saline content of the growth 

media and (2) controlled versus natural environment on extraction recovery, lipid content, and lipid 

distribution of Salicornia virginica, with attention to total biomass (e.g., Figure 1 to Figure 6). GRC 

provided NETL-ORD with 12 Salicornia virginica specimens representing a matrix of six salinity values 

that were either collected from a controlled indoor laboratory or an outside environment (NASAs 

GreenLab Research Facility (GreenLab)). 

NETL performed two different lipid extraction methods, chosen based on previous evaluations of 

several methods, and determined the amount of lipids produced by each plant based on gravimetric 

recovery. Each sample of lipid extract was subsequently characterized by GC-MS, with mass spec library 

matches being used to identifying the major species present in the extracts. Extracts are currently being 

further characterized for concentration of major species using GC-FID. 
NASA/TM—2014-216645 3 

 

Figure 1.—Salicornia virginica (Florida). 

 

Figure 2.—Salicornia virginica (Florida). 

Figure 3.—Salicornia virginica seedlings (GreenLab). 

Figure 4.—Salicornia virginica (Indoor Biofuels Lab)

 

Figure 5.—Salicornia virginica (GreenLab) 

Figure 6.—Salicornia virginica (GreenLab) 
NASA/TM—2014-216645 4 

Method 

Samples of Salicornia virginica grown both indoors and outdoors (GreenLab) under six different 

saline conditions ranging from freshwater to natural seawater were collected by GRC were delivered to 

NETL. Specimens grown in the indoor, climate-controlled laboratory were grown from seeds obtained 

from a commercial seed production company. Each plant was raised exclusively in a media of specific 

salinity for its entire life cycle. Specimens comprising the GreenLab population were gathered from a 

population of wild Salicornia in the Florida Keys and acclimated to each level of salinity in the Indoor 

lab. Biomass was gathered when plants were mature and seeds were then processed to determine lipid 

composition and content. 

The following protocol was used to process samples: 

Biomass Preprocessing 

1.1 Leaf tissues were separated from the stem of the plant and placed in sealed plastic bags at room 

temperature for storage. 

1.2 To determine the moisture content of Salicornia, samples were taken from the initial stock and 

dried to constant weight. 

1.3 To account for moisture lost during extraction, a duplicate portion from the sample being 

extracted was massed and dried to constant weight. 

Hexane-Based Extraction 

2.1 5 g of leaf tissue was massed and mixed with 1.5 mL 2M HCl. 

2.2 Samples were homogenized by hand-chopping with a sharp metal spatula for 2 min. 

2.3 The mixture was placed in a 60 °C water bath for 60 min. 

2.4 8 mL of hexane was added and the extraction tubes shaken. 

2.5 The mixture was placed in water bath at 60 °C for 15 min. and mixed periodically by shaking. 

2.6 The mixture was centrifuged and the supernatant removed using a glass pipette; 

2.7 The pellet was re-extracted twice with fresh 8-mL aliquots of hexane. 

2.8 Supernatants were combined and dried completely under a low flow of nitrogen. Lipids 

manifested as an opaque residue on the bottom of each tube. 

2.9 Residues were completely redissolved in 2:1 (v:v) mixture of chloroform:methanol 

Methanol-Based Extraction 

3.1 5 g of plant tissues were weighed and mixed with 5 mL of 0.8 percent HCl in methanol. 

3.2 Samples were homogenized by hand-chopping with a sharp metal spatula for 2 min. 

3.3 The mixture was placed in water bath at 60 °C for 15 min., and mixed periodically by shaking. 

3.4 The mixture was centrifuged and the supernatant removed using a glass pipette; 

3.5 The pellet was re-extracted twice with fresh 5-mL aliquots of 0.8% hexane 

3.5 All supernatants were combined for post-extraction workup. 

3.6 Combined supernatants were dried under a low flow of nitrogen. 

3.7 Residues were redissolved in 2:1 (v:v) mixture of chloroform:methanol 

*Note. A significant portion of extract did not redissolve in chloroform:methanol, suggesting that the 

residue contained appreciable quantities of co-extracted, nonlipid material. Recoveries based solely on 

gravimetric determination show extremely high bias due to co-extracted material. Further filtration of the 

redissolved sample was required to correct for fine insoluble particulate matter (most biomass processing 

requires precleansing of raw-oils). 
NASA/TM—2014-216645 5 

Extract Species Characterization 

Following the redissolution of the residues in 2:1 MeCl2: MeOH (CH3OH), the fractions were 

analyzed by both GC-MS (for primary characterization) and GC-FID (for quantification of selected 

species). The GC-MS analysis proceeded according to the parameters outlined in Table I. After obtaining 

an appropriate chromatogram, peak identifications were made using Chemstation (Agilent) data 

processing, by searching against a spectral library (NIST), and by evaluating the quality of the match 

identified. In some instances, pure compounds were injected to determine their retention time and to 

provide additional evidence to validate the identification made by mass spectrum library search. 

The GC-FID analysis proceeded according to the parameters outlined in Table II. Because 

preliminary characterization yielded some degree of knowledge as to the species present and their 

respective retention times, calibration standards for many of the target components (free fatty acids, 

esters, sterols, etc.) were prepared and detector response was determined at different concentrations. After 

obtaining an appropriate calibration for each individual target species, samples of redissolved residue 

were analyzed and the concentration of the species was calculated. The masses of each individual species 

were summed, and compared to the mass of the extract residue. 

TABLE I.—RELEVANT PARAMETERS FOR GC-MS 

CHARACTERIZATION OF REDISSOLVED EXTRACT RESIDUES. 

[Chromatography was done using Agilent 6890 GC-Agilent 5973 MSD.] 

Inlet 

Temperature = 360 °C 

Split Ratio = 25:1 

Injection volume = 1.0 μL (autosampler) 

Constant flow at 0.8 mL/min. 

Column 

Zebron Inferno ZD-1HT (Phenomenex) 

100% dimethyl polysiloxane 

30- by 0.25-mm ID, 0.25 μm film thickness 

Oven 

Initial temperature = 40 °C (hold 0.5 min.) 

Ramp from 40 to 360 °C at 6 °C/min. 

Final temperature = 360 °C (hold 10 min.) 

Detector Transfer line temperature = 360 °C 

TABLE II.—RELEVANT PARAMETERS FOR GC-FID 

CHARACTERIZATIONOF REDISSOLVED EXTRACT RESIDUES. 

[Chromatography was done using Agilent 6890 GC-FID.]

Inlet 

Temperature = 360 °C 

Split Ratio = 25:1 

Injection volume = 1.0 μL (autosampler) 

Constant flow at 1.2 mL/min 

Column 

Zebron Inferno ZD-1HT (Phenomenex) 

100% dimethyl polysiloxane 

30- by 0.25-mm ID, 0.25 μm film thickness 

Oven 

Initial temperature = 40 °C (hold 0.5 min.) 

Ramp from 40 to 360 °C at 6 °C/min. 

Final temperature = 360 °C (hold 10 min.) 

Detector Temperature = 360 °C 
NASA/TM—2014-216645 6 

Results 

Gravimetric Recoveries 

Gravimetric lipid recoveries of the Salicornia virginica specimens evaluated in this study are reported 

in Table III and illustrated in Figure 7, using the two aforementioned protocols. 

Extract Composition 

Hexane-HCl Extraction 

The extracted materials from the 12 Salicornia virginica samples were characterized in duplicate and 

averaged. Residue extracted using the hexane-HCl based extraction method qualitatively exhibited the 

same constituent lipid components, detailed in Table IV and Figure 7. The major species identified in the 

lipid extracts were hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid) and C18:1 to C18:3 

unsaturated acids (oleic, linoleic and linolenic acids). Additional species identified were ergostanol, 

stigmasterol, sitosterol, and stigmastadiene isomers. Appreciable amounts of phytol were identified as 

well as carotenoid-based degradation products. The phytols presumably result from the breakdown of 

chlorophyll in the harsh, high-temperature conditions of the GC injector (360 °C). In general, the hexaneHCl extraction yields a cleaner lipid fraction, as most of the polar co-extracted species seen with the 

MeOH-HCl extraction are excluded. 

TABLE III.—GRAVIMETRIC LIPID RECOVERYa FOR ALL SAMPLES USING THREE EXTRACTION METHODS 

Growth 

environment 

Salinity of growth media, b

TSG 

Hexane + 2M HCl Methanol + 2M HCl c Methanol + 2M HCl 

Indoor 1.000 3.2 ± 0.5 61.1 ± 0.5 26.9 ± 5.5 

Indoor 1.005 4.1 ± 0.1 52.9 ± 9.0 17.5 ± 5.3 

Indoor 1.010 3.6 ± 0.1 64.6 ± 3.4 23.5 ± 1.0 

Indoor 1.015 2.1 ± 0.1 72.9 ± 3.2 25.5 ± 3.8 

Indoor 1.020 2.4 ± 0.1 64.5 ± 2.8 21.2 ± 0.3 

Indoor 1.025 2.7 ± 0.1 92.4 ± 5.7 37.6 ± 4.4 

GreenLab 1.000 2.8 ± 0.1 62.4 ± 0.8 23.2 ± 2.7 

GreenLab 1.005 3.4 ± 0.2 63.7 ± 6.1 19.5 ± 2.1 

GreenLab 1.010 2.4 ± 0.0 49.5 ± 3.1 15.0 ± 0.8 

GreenLab 1.015 2.0 ± 0.4 55.8 ± 0.5 31.1 ± 13.0 

GreenLab 1.020 2.1 ± 0.1 91.8 ± 21.2 26.4 ± 4.0 

GreenLab 1.025 3.1 ± 0.2 57.7 ± 2.7 19.5 ± 0.6 a

 Recoveries are the average of two replicate samples, based on the entire residue remaining after extraction compared to the dry 

weight of starting material. 

b

 TSG: True Specific Gravity, as measured with a refractometer and salinity meter. 

c. Recoveries corrected for co-extracted material that did not redissolve in chloroform:methanol. 
NASA/TM—2014-216645 7 

Figure 7.—Typical GC-MS chromatograms for the extracted lipid content using the 

hexane-HCl solvent for the Salicornia Virginica samples of focus in this study. 

TABLE IV.—QUALITATIVE SPECIATION OF THE LIPID CONTENT OF 

THE SALICORNIA VIRGINICA USING A HEXANE-HCl EXTRACTION 

Retention time, 

min. Chemical name Common name Notation 

6.6 Dodecane 

10.4 2,6-di-tertbutyl-4-methylphenol BHT 

11.5 Hexadecanoic acid Palmitic acid C16:0 acid 

13.5 cis-9,cis-12-Octadecadienoic acid Linoleic acid $QI,K9,12acid 

13.6 cis,cis,cis-9,12,15-Octadecatrienoic acid Linolenic acid $QI,B9,12,15 acid 

13.7 cis-9-Octadecenoic acid Oleic acid $QI,Q9 acid 

13.8 Octadecanoic acid Stearic acid C18:0 acid 

22.4 Bis (2,2,6,6,-tetramethyl-4-piperidyl) sebacate Tinuvin 770 

22.48 Tocopherol Vitamin E sterol 

23.60 K@-Ethyl-H-cholestan-B-ol Stigmastanol sterol 

24.4 24-Ethyl-4,22E-cholestadien Stigmastadien sterol 

24.45 5--ergostan--3-ol Ergostanol sterol 

28.6 Unknown ID 

31.3 Unknown ID 

33.8 Glycolipid 

MeOH-HCl Extraction 

The extracted materials from the 12 Salicornia virginica samples were characterized in duplicate for 

chemical speciation. As expected, the MeOH-HCl extraction yields a material in which most of the lipid 

material consisted of methyl esters of C16 and C18 fatty acids, along with trace amount of free fatty acids 

5 10 15 20 25 30 35 40 Response

Retention Time [min]

NASA-1i-00-H-A

NASA-1i-25-H-A

NASA-1i-20-H-A

NASA-1i-15-H-A

NASA-1i-10-H-A

NASA-1i-05-H-A
NASA/TM—2014-216645 8 

of these carbon lengths, detailed in Table V and illustrated in Figure 8. Although more difficult to see, the 

acidic methanol extracts appear to display varying ratios of the C16 acid to C18 acids when the observed 

intensities of both the free acids and the methyl-esters of those acids are considered as shown in Figure 9. 

TABLE V.—QUALITATIVE SPECIATION OF THE LIPID CONTENT OF 

THE SALICORNIA VIRGINICA USING A MeOH-HCl EXTRACTION 

Retention time, 

min. 

Chemical name Common name 

6.6 dodecane 

10.4 2,6-di-tertbutyl-4-methylphenol BHT 

10.18 Methyl cis-9-Hexadecenoate methylpalmitoleate 

11.17 Methyl hexadecanoate methylpalmitate 

11.3 cis-9-Hexadecenoic acid Palmitoleic acid 

11.52 Hexadecanoic acid Palmitic acid 

12.86 Methyl cis-9,cis-12-Octadecadienoate Methyl linoleate 

12.95 Methyl cis,cis,cis-9,12,15-Octadecatrienoate Methyl linolenate 

13.05 Methyl cis-9-Octadecenoate Methyl oleate 

13.3 Cis-9,cis-12-Octadecadienoic acid Linoleic 

13.4 cis,cis,cis-9,12,15-Octadecatrienoic acid Linolenic 

13.45 Methyl octadecanoate methylstearate 

13.5 cis-9-Octadecenoic acid Oleic acid 

13.7 Octadecanoic acid Stearic acid 

22.48 Tocopherol Vitamin E 

23.60 K@-Ethyl-H-cholestan-B-ol Stigmastanol 

24.4 24-Ethyl-4,22E-cholestadien Stigmastadien 

24.45 5--ergostan--3-ol Ergostanol 

Figure 8.—Typical GC-MS chromatograms for the extracted lipid content using the 

MeOH-HCl solvent for Salicornia virginica samples. 

5 10 15 20 25 30 35 40 Response

Retention Time [min]

NASA-1i-00-M-A

NASA-1i-25-M-A

NASA-1i-20-M-A

NASA-1i-15-M-A

NASA-1i-10-M-A

NASA-1i-05-M-A
NASA/TM—2014-216645 9 

Figure 9.—Comparison of extract yields based on gravimetric methods as a 

function of growth environment and extraction solvent. 

Growth Environment 

Comparisons of the extract yields for the indoor and outdoor samples as a function of extraction 

solvent are illustrated in Figure 9. On average, the extracted material for the hexane-based extractions 

appeared to be slightly higher for the samples grown in the Indoor as compared to the samples grown in 

the GreenLab. There is not a large statistical significance to this difference. One important note is that the 

MeOH-based extracts were found to contain significant amounts of insoluble cellulosic residues and 

required further cleaning prior to final gravimetric yield determination. Despite our best efforts, the 

cellulosic residues, as well as possible soluble protein fractions, most likely cloud any trends in the 

MeOH-based extractions. 

Discussion 

As mentioned previously, the determination of lipid based solely on the amount of extracted material 

can lead to inaccurate assumptions about lipid production as well as lipid composition. Our initial 

attempts at calibrating the FID response for the dozens of individual species seen in the Salicornia 

Hexane HCl Lipid Extracts: NASA Samples

0

1

2

3

4

5

6

123456

Sample ID

Dry Weight Percent Lipid

Out Samples In Samples

Acid Methanol Extraction: Lipid Yield Corrected for Cellulosic 

Materials

0

5

10

15

20

25

30

35

40

45

50

1234 5 6

Sample ID

Dry Weight Percent Lipid

MeOH-Out MeOH-In
NASA/TM—2014-216645 10 

virginica extracts were met with limited success. The primary difficulty is that not only do the various 

species respond differently in the FID, but also the various chain lengths within the same species lead to 

different responses. Moreover, it is difficult to detect species such as triacylglycerides using gas 

chromatography, primarily due to poor volatility. We are currently exploring methods for improving our 

quantitative analysis of the extracts so that in the future we can provide a lipid-specific quantity, including 

species such as free fatty acids, esters, and glycerides, while excluding any extraneous materials such as 

chlorophyll, glycerol, etc. Traditional derivatization methods, which convert all species to methyl esters, 

obscure the compositional makeup of the original extracts. This compositional makeup may be critical in 

determining the optimum reaction pathways for converting the raw extract into useful fuels. Therefore, 

we continue to pursue methods whereby the original biomass extract composition can be established and 

a lipid-specific quantization of biomass oils produced can be determined. 

Conclusions 

On the surface, it appears that extraction of lipids from Salicornia virginica using a polar solvent such 

as methanol provides the most amount of material, and establishes a gravimetric lipid production at 

approximately 30 percent (dry weight). However, from the compositional analysis, it is obvious that this 

extracted material contains significant portions of species that are not typically considered to be lipids, 

and so gravimetric lipid content is artificially high. Moreover, the species that dominate this fraction are 

methyl esters of C16 and C18 fatty acids. On the other hand, hexane extraction appeared to remove 

significantly less lipid based on mass. Compositional analysis shows that acidic hexane provides a much 

cleaner fraction, composed primarily of free fatty acids. 

An important finding is that when using the extraction methods in this study, it is apparent that 

growth conditions did not influence extraction recoveries, lipid content or lipid distribution, within 

statistical error. This is significant as we move to expand our study to actual field trials around the world 

under varying salinity conditions. 

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