Research Article pubs.acs.org/journal/ascecg
Grindelia squarrosa: A Potential Arid Lands Biofuel Plant Bishnu P. Neupane,† David Shintani,‡ Hongfei Lin,§ Charles J. Coronella,§ and Glenn C. Miller*,† †
Department of Natural Resource and Environmental Science, University of Nevada, Reno, 1664 North Virginia Street, Reno, Nevada 89557, United States ‡ Department of Molecular Biosciences, University of Nevada, Reno, 1664 North Virginia Street, Reno, Nevada 89557, United States § Department of Chemical and Materials Engineering, University of Nevada, Reno, 1664 North Virginia Street, Reno, Nevada 89557, United States Downloaded via UNIV OF SUSSEX on June 30, 2018 at 21:37:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: Gumweed (Grindelia squarrosa) has potential as a biofuel/biomaterial crop in arid lands. Two years of biomass production data at University of Nevada, Reno (UNR) field plots varied from 6700 kg/ha up to 14 900 kg/ha with an average of 9950 kg/ha. Gumweed was planted in 4 m × 4 m plots at spacings of 15, 20, 25, 30, and 37 cm with three replications each. Acetone extraction of the ground, dried biomass yielded about 12.5% extractable hydrocarbons, called biocrude or crude resin. Approximately 52% of the biocrude extract consists of the C20 diterpene acid grindelic acid, which is also approximately 6.5% of the dried plant biomass. Additional carboxylic acids bring the total carboxylic acid fraction to approximately 68% of the biocrude. Also found in the biocrude is approximately 4.7% monoterpenes, including pinene, limonene, germacrene, elemene, and camphene. Acid-catalyzed methylation of the biocrude followed by hexane extraction and removal of the hexane and remaining methanol produced an approximately 72.5% yield of biofuel materials from the original extract. This mixture contained the methyl esters of the carboxylic acids, terpenes, and other unidentified compounds. When mixed with diesel fuel up to 20%, this blend produced a biofuel that met biofuel standards, although viscosity issues limit the percentage of gumweed biocrude that can be used in the blend. Grindelic acid methyl ester and a second ester produced during the acid-catalyzed methylation reaction (also identified as grindelic acid by GC−MS) eluted toward the end of a diesel fuel chromatogram. The Grindelia biofuel materials can be produced up to 1290 L/ha on a biennial basis, which is equivalent to 138 gal/acre, when extracted in hexane. This potential fuel can be produced on the arid lands of Nevada with minimal inputs of water, nutrients, and other agricultural services. KEYWORDS: Gumweed, Grindelic acid methyl ester, Terpenes, Biomass, Biocrude, Biofuel, Grindelia squarrosa
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INTRODUCTION The impacts of carbon-based fuels on the climate as well as the depletion of liquid fossil fuel sources have prompted a search for hydrocarbon-rich plant species as a source of bioenergy,1−6 especially in agronomic situations where the conflicts between food and fuel can be minimized.7 Particularly in the American West, where water resources are scant, finding a bioenergy crop that grows well and utilizes only minimal water would have potentially large benefits. Grindelia squarrosa is one such plant that can be grown in land that has not been cultivated and is common along roadsides and disturbed soils in the arid Intermountain West and on the Great Plains. Grindelia squarrosa, commonly known as curly top gumweed, is an erect biennial or short-lived perennial herb with one to several branching stems that lies in the Asteraceae family and is a native of the arid lands of Nevada and other western states of the USA.8−10 Genus Grindelia is widely distributed in a variety of open, predominantly xeric habitats such as grasslands, © 2016 American Chemical Society
deserts, and salt marshes as well as early successional sites such as disturbed soils, along highway shoulders, and alfalfa fields, where it is not controlled and additional water is available.11 It can be grown at elevations between 975 and 1830 m above sea level.8 It grows to between 30 and 90 cm tall, depending upon the type of soil and the availability of moisture, and flowers from midsummer to early fall with an average bloom time of 41 days.11 As a biofuel crop, gumweed can be grown on marginal arid lands, as it requires only low inputs of water and nutrients and is tolerant to drought and elevated salt.12,13 Gumweed produces abundant resin on the surfaces of the flower heads and leaves; this milky material chiefly consists of grindelic acid (Figure 1) and related grindalane diterpenoids.14,15 In addition to potentially serving as a biofuel crop, Received: September 23, 2016 Revised: November 2, 2016 Published: November 11, 2016 995
DOI: 10.1021/acssuschemeng.6b02315 ACS Sustainable Chem. Eng. 2017, 5, 995−1001
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Figure 1. (left) Structure of grindelic acid.19 (tight) Height measurement of Grindelia squarrosa grown at Valley Road Field Laboratory, UNR.
Figure 2. (left) Gumweed plots in the experimental field at Main Station, UNR. (middle) Sticky milky white bud stage of gumweed, which is found to have the highest amount of grindelic acid. (right) Highest-yield harvesting stage of the gumweed, when the floral heads are a mix of the white milky stage and yellow flowers plus a few buds that are in the premilky stage. methylated biocrude was completed using sulfuric acid and methanol. The diesel fuel blended with the methylated product was obtained locally from an Exxon fueling station. Seeds and Germination. Seeds were collected from wild stands, primarily along roadsides in Northern Nevada and Eastern California, during September and October of 2011. The seeds were handthrashed into 35 gallon plastic containers, after which the collected material was sieved through a #16 (1.18 mm) sieve. Additional enrichment of the seed/plant mixture to approximately 50% seeds was obtained by removing the very light material (chaff) using an air stream. Germination of seeds directly in a field was accomplished only with extensive irrigation in May and June of 2012, which also resulted in extensive weed germination as well as uneven germination of the gumweed. For the agronomic data below, gumweed was germinated at Valley Road Field Station in 2012, but because of the uneven germination, it was transplanted to obtain uniform spacing. In 2013, all of the gumweed was germinated in a greenhouse (24 °C, 65% relative humidity) in March and then transplanted into the field in June−July when the plant spacing trials were initiated. Germination tubes (27 cm in length) were used to obtain the seedlings. Seedlings germinated in the greenhouse were exposed to direct sunlight for 1 week prior to transplanting. Plot Design. A total of 39 plots with dimensions of 4 m × 4 m were established for growing the gumweed at the University of Nevada, Reno (UNR) Valley Road Field Station in 2012, and 42 plots were established at the UNR Main Station Farm in 2013 (Figure 2). Each plot consisted of four rows of gumweed spaced 1 m apart. The effect of plant spacing within the plots was examined using distances of 15, 20, 25, 30, and 37 cm between each plant. Each plot had at least three randomized replications throughout the field. The plots were separated by 2 m in all directions.
the resin that gumweed produces has the potential to substitute for pine and coniferous wood rosin14 and has potential applications in naval stores, pharmaceutical applications, and bioenergy.14,15 The possibility of using gumweed as a source of fuel and feedstock for certain chemical processing industries was studied by Lemaire in the late 1970s.5 From a biofuel perspective, he observed that gumweed had the largest amount of extractable hydrocarbons of the species found in Nevada. Gumweed has also been used by Native Americans for various medicinal purposes, including remedies for asthma, bronchitis, cough, scabs, burns, migraine headaches, and disinfection.16 This study aimed to explore the agronomic characteristics17 of gumweed in semiarid lands of Nevada as well as methods for the extraction of the hydrocarbons and the conversion of the extracted hydrocarbons to usable biofuel feedstock that can be blended with diesel fuel. A plot study was used to compare the effects of differential irrigation on the production of total dry biomass and extractable hydrocarbons. The total extractable hydrocarbons were examined with a specific focus on grindelic acid, the major constituent of the acetone extract.
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EXPERIMENTAL SECTION
Materials. Air-dried plant biomass of gumweed was obtained following cultivation at the University of Nevada Agricultural Experiment Station in Reno, NV. Acetone, pentane, hexane, diethyl ether, and methanol were ACS reagent grade and obtained from Fisher Scientific. Diazald was purchased from Sigma-Aldrich, converted to diazomethane, and used for methylation of the carboxylic acid components. Acid-catalyzed methylation for larger quantities of 996
DOI: 10.1021/acssuschemeng.6b02315 ACS Sustainable Chem. Eng. 2017, 5, 995−1001
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Figure 3. (left) Average biomass of gumweed per plot from the 2013 and 2014 harvests. The 2014 harvest gave about 1.5 times higher biomass production than the 2013 harvest. (right) Bar diagram showing the effects of irrigation and fertilizer input on biomass production in the 2014 harvest. There was no significant difference in the biomass production among the plots with fertilizer and irrigation supplied vs not supplied as assessed using the Kruskal−Wallis statistical test at the 5% confidence level with three degrees of freedom. However, a significant difference was observed in biomass production among the plots with different plant spacing using the Kruskal−Wallis statistical test at the 5% confidence level with three degrees of freedom. Irrigation and Fertilizer Supply. Once the transplanted stocks were established in June−August, irrigation was ceased for the year. During the second year of growth, approximately 10−15 cm of water was applied by sprinkler irrigation twice during the last week of May and the third week of June in all of the plots of the 2012 planting. For the second harvest, it was once again irrigated with 10−15 cm of water after a few weeks following the first harvest. For the 2013 planting, controlled drip irrigation was applied in randomly selected plots supplying 1 gal of water per plant each time in the last week of May and the third week of June. For the second harvest of 2014, it was once again irrigated with 1 gal of water after a few weeks of the first harvest. No additional fertilizer was applied to the 2012 planting, but selected plots were supplied with 2.0−3.0 g of NPK (16−4−8) chemical fertilizer around the base of each plant for the 2013 planting. Removal of weeds was accomplished by rototilling, hand-weeding, and selective treatment with glyphosate. In the 2012 planting, weed growth was aggravated as a result of whole-field irrigation by sprinkler, but in the 2013 planting, it was better controlled using drip irrigation. Harvesting. Harvesting was accomplished during the last week of July to the first week of August, following the development of the buds and the flowers. The highest yield of grindelic acid was obtained when most of the flowers had turned yellow from the white sticky buds. However, since not all of the flowers develop at the same time, there was some variability in the stage of the plant at harvest, although visually the plots were generally consistent. The entire plant mass was harvested in both 2013 and 2014, leaving about 15 cm stems at the base. The plant mass was dried in the sun for 5−7 days, and the weight was measured. A second harvest of new growth was cut and dried in 8 to 10 weeks following the first harvest. Biomass Processing. The dry biomass was ground in a hammer mill (Colorado Mill Equipment, LLC) to less than 3 mm in size (1/8″ sieve). Extraction of gumweed was conducted using acetone in a 25 gal pilot-scale refluxing extractor (Eden Laboratories, Seattle, WA), which is similar to a Soxhlet extractor. Following a 4 h extraction, the extract was removed, and the acetone was recovered using a rotatory evaporator to give a viscous, dark-colored tarlike material, termed biocrude. The extraction yield was determined to be near complete in this system, since a second extraction of the same material produced 0.5−1.0% additional biocrude, compared with the first extraction of approximately 12.5%. Isolation and Derivatization of the Carboxylic Acid Fraction. The carboxylic acid fraction of the biocrude (containing the primary constituent, grindelic acid) was isolated by dissolving the biocrude in methanol and then adding this mixture to water containing sufficient sodium hydroxide to maintain the pH above 9. The neutral nonpolar compounds were removed by extraction with hexane. Following the
hexane extraction, the aqueous extract was acidified to pH 2, and the grindelic acid (and other organic acids) were extracted back into diethyl ether. The diethyl ether was removed by rotary evaporation, leaving a dark-colored viscous gum that was treated with diazomethane to form grindelic acid methyl ester (GAME). Acid-catalyzed methylation was used for methylation of larger volumes of biocrude and carboxylic acid fractions.18 Equal weights of biocrude and methanol (an approximate molar ratio of 10:1 methanol to grindelic acid) were heated at 50 °C for 4 h in the presence of 0.5−1.0% concentrated sulfuric acid. GAME, other carboxylic acid methyl esters, and other extractable materials were extracted by hexane. Hexane was removed later by rotary evaporation, and the extracted materials were blended with petroleum diesel. Preparation of a Reference Standard. The dark, viscous carboxylic acid fraction was further purified by column chromatography using a column that was 45 cm tall and 5.6 cm in diameter and filled with 30 cm of silica gel (particle size 40−63 μm), eluting with a mixture of 95% hexane and 5% ethyl acetate, to obtain a purified (>98%) grindelic acid fraction, which was crystallized in methanol at room temperature. The observed melting point of the crystal was 106 °C (lit. 101−102 °C).19 Crystals were dissolved in methanol and methylated using diazomethane, and the resulting material was used as the gas chromatographic standard for determining grindelic acid in a variety of samples. Gas chromatography−mass spectroscopy (GC−MS) on an Agilent 5973 instrument with gas chromatography flame ionization detection (GC-FID) (Agilent 6890 GC) was used for the analysis of the sample with reference to the standards prepared. The capillary column used in the GC was a DB-5 column (30 m × 0.53 mm × 0.088 μm film thickness), whereas in the GC−MS, a DB-5 column (30 m × 0.25 mm (i.d.) × 0.25 μm film thickness) was used. The injector and detector temperatures were both set at 290 °C. The initial temperature of the oven was set at 50 °C, with a ramp of 10 °C/min to 300 °C, which was held for 5 min. The injection volume was 1 μL in each case.
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RESULTS AND DISCUSSION Cultivation and Biomass Yield. Test plots were established in the springs of 2012 and 2013 and harvested the following year to determine the production of biomass. These test plots were located at different sites for these two years: the first was at the UNR Valley Road Field Laboratory, and the second was at the UNR Main Station Farm. The heights and widths of individual gumweed plants at harvest averaged 70 cm (range 40 cm to 120 cm) and 60 cm (45 cm to 90 cm), respectively, depending upon the spacing of the plants 997
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Table 1. Comparison of Gumweed Biomass and Products Obtained from the Harvests of 2013 and 2014; The 2014 Harvest Had Almost 1.5 Times More Biomass Production than the 2013 Harvest biomass production (kg/ha)
biocrude production (kg/ha)
harvest year
mean
range
mean
range
carboxylic acid fraction (L/ha) mean
range
mean
grindelic acid (kg/ha) range
2013 2014 mean
8100 11800 9950
5780−12400 8880−14900
1050 1470 1260
751−1610 1100−1860
680 952 816
487−1040 712−1200
527 731 629
376−806 577−924
Figure 4. (left) Total ion chromatogram (TIC) of the underivatized biocrude of gumweed. The major peak is grindelic acid. Besides the peak shown, there are minor peaks of retinoic acid, hexadecanoic acid, isopimaric acid, and few long-chain alkanes. (right) TIC of methylated (with diazomethane) biocrude of gumweed. The major peak is grindelic acid methyl ester, and the other peaks to the right of major peak are (1) retinoic acid methyl ester with 29% probability, (2) isopimaric acid methyl ester with 35% probability, (3) methyl triosporate B with 40% probability, and a few long-chain alkanes.
and the availability of water. Following a week of field drying of the harvested gumweed, the plants lost 55% of the weight, which was assumed to be primarily water (Table S3). The total field-dried plant biomass was found to be higher in the plots with lower plant spacing and higher plant number than in the plots with higher plant spacing and lower plant number. For example, plots with 84 plants and 20 cm plant spacing had more biomass per plot but lower biomass per plant than the plots with 68 plants and 25 cm plant spacing (Table S1). The highest production came from the plots with plant spacings of 15 and 20 cm in the 2013 and 2014 harvests, respectively (Figure 3 (left) and Table S1). Plant spacings less than 15 cm were not examined but will be in future planting. The average plant biomass production was 8100 kg/ha (range 5780−12400 kg/ha) in the 2013 harvest and 11 800 kg/ha (range 8880− 14900 kg/ha) in the 2014 harvest (Table 1). The overall plant biomass production was approximately 1.5 times greater in the 2014 harvest than in the 2013 harvest. The specific amounts of biomass produced from the floral heads, leaves, and stem plus branches in an individual plant were found to be 34%, 19%, and 47%, respectively (Table S3). The differences in the production in the two different years are likely related, at least in part, to the soil quality differences between the two plots. The soil in the UNR Main Station Farm that was used to grow the 2014 harvest is known to be more fertile for a variety of crops than the soil at Valley Road Field Laboratory that was used to grow the 2013 harvest (Paul Verburg, UNR soil scientist, 2016). Other reasons might be seed quality and somewhat different weed competition that could affect biomass production. The seeds used in the 2012 planting were collected from the wild, whereas the seeds used in the 2013 planting were collected from selected plants from the cultivated plots of the 2012 planting that had visually
produced larger amounts of floral and vegetative biomass than the other plants. Further seed selection might help to improve the biomass production in future years. Additionally, all of the seeds in the 2013 planting were germinated in a greenhouse, and the seedlings were later transplanted into the field. For the 2012 planting, the germination in the field was uneven, and the seedlings that were produced were transplanted to allow a uniform distribution of plants within the plots. Transplanting success was 95−98% in both years. Similarly, the 2012 planting of gumweed faced stronger weed competition resulting from sprinkler irrigation, which was controlled better by drip irrigation in the 2013 planting. Different treatments, including irrigation and fertilizer, were examined in the second year of the 2013 planting (Figure 3, right). No significant differences (Kruskal−Wallis test performed at the 5% confidence level with three degrees of freedom) were observed in biomass production among the fertilizer- and/or irrigation-supplied plots. These parameters were not examined in the 2012 planting (2013 harvest). The plant spacing for this crop was examined, and significant differences were observed in biomass production among the plots with different plant spacing/plant number (using once again the same Kruskal−Wallis statistical test at the 5% confidence level with three degrees of freedom). The first harvest was obtained during the last week of July and the first week of August each year. The length of the bloom time of the majority of plants was observed to be about 6−8 weeks from mid-June to late August. The plots were irrigated by 10−15 cm of water following the first harvest to allow regrowth from the harvested base. The second harvest was obtained 8−10 weeks after the first harvest and provided an additional 20% biomass relative to the first harvest. 998
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Figure 5. Grindelic acid contents in various parts of gumweed. Floral heads, leaves, and stems plus branches have averages of 23.2%, 4.8%, and 0.14% grindelic acid in their dry biomasses, respectively.
Figure 6. Total ion chromatogram obtained from petroleum diesel mixed with the hexane extract at a ratio of 85:15. The two large peaks (peaks 2 and 3) are grindelic acid methyl ester and an isomer of grindelic acid methyl ester. The isomer of GAME was formed only during acid-catalyzed methylation. When diazomethane was used for the methylation, only GAME was formed. Other discernible peaks were tentatively identified by GC− MS as the methyl esters of retinoic acid (1), isopimaric acid (4), and octadecanoic acid.
Biocrude Extraction and Yield. The ground material yielded an average of 12.5% (extracted in pilot-scale extractor) of the biomass as biocrude (range 9.5−14.6%) by acetone extraction. However, a laboratory-scale Soxhlet extraction using acetone gave about 15% of the biomass as biocrude. Similar results were obtained when extracting with dichloromethane. The average biocrude production was 1050 kg/ha (range 751− 1610 kg/ha) in the 2013 harvest and 1470 kg/ha (range 1100− 1860 kg/ha) in the 2014 harvest (Table 1). The floral heads, leaves, and stems/branches constituted approximately 72.5%, 18.8%, and 8.7%, respectively, as a share of the total biocrude in an individual plant (Table S3). Nonpolar and Polar Fraction Characterization in Biocrude and Their Yields. When the biocrude was extracted using hexane in a basic medium, the resulting extract, which was the neutral nonpolar fraction, contained approximately 11.4% (range 7.4−15.3%) of the biocrude by weight. This fraction primarily consists of the common terpenes, including α-pinene, β-pinene, limonene, camphene, terpinolene, γ-elemene, germacrene, and β-eudesmene, as shown in Figure 4. The total amount of identified terpenes (based on integrated area of the GC-FID peaks) was 4.7% (range 4.1−5.7%) of the biocrude
and 0.73% of the dry biomass (Table S3). This is due to the fact that all of the materials extracted in hexane do not make it through the column to the GC-FID. The polar carboxylic fraction was obtained after separation of the neutral nonpolar fraction in a basic medium followed by acid-catalyzed isolation and derivatization of the biocrude. The major constituent of the carboxylic acid fraction was grindelic acid. Other compounds tentatively identified by mass spectral library search were retinoic acid, hexadecanoic acid, isopimaric acid, and additional long-chain alkanes, as shown in Figure 4. The carboxylic acid fraction constituted about 68.0% (range 65−75%) of the biocrude by weight and about 8.5% of the dry biomass. An average carboxylic acid fraction of 816 L/ha (87 gal/acre) with a range of 487−1200 L/ha can be obtained from gumweed on a biennial basis (Table 1). The highest production of carboxylic acid from gumweed was 1200 L/ha (128 gal/ acre), which is comparable to the average biodiesel production from soybean (66 gal/acre every year).7 Grindelic Acid Yield. The average grindelic acid production potential of the 2013 and 2014 harvests was 629 kg/ha (Table 1). The acetone extract of biocrude constituted 52.0% (range 50−55%) grindelic acid by weight, and this 999
DOI: 10.1021/acssuschemeng.6b02315 ACS Sustainable Chem. Eng. 2017, 5, 995−1001
Research Article
ACS Sustainable Chemistry & Engineering compound is clearly the primary extractable organic compound in the plant (Figure 4). In the carboxylic acid fraction, on the basis of the integrated areas of the GC-FID peaks, 80% (range 75−85%) by weight is grindelic acid. These data are based on determining grindelic acid as grindelic acid methyl ester, which was produced by methylation of grindelic acid with diazomethane. However, acid-catalyzed methylation using methanol was employed when larger quantities of biocrude or the carboxylic fraction were produced. This methylation method produced an isomer of grindelic acid methyl ester, also identified as grindelic acid methyl ester by GC−MS (see Figure 6). The exact structure of this compound was not identified, although its production during acid-catalyzed methylation was consistent when acid-catalyzed methylation was used for methylation of the carboxylic acid fraction. A time series of extractions of the plant indicated that the grindelic acid content was highest in the preflowered (white milky) stage (26.8%), when the buds with sticky white materials began to transition to yellow buds. The next higher concentrations were found in the descending order of yellow bud stage (22.3%), completely developed flower (20.1%), leaves (4.8%), and stems (0.15%) (Figure 5 and Table S2). The floral parts averaged approximately 23.2% grindelic acid based on its dry biomass. Potential Biofuel from Gumweed. The initial goal for growing gumweed as an agricultural crop has been as a potential biofuel that can be grown in the arid regions of the western United States. The production rates discussed previously suggest that the biocrude can be produced at rates that become competitive with other biofuels in the diesel range. However, in order to be compatible with diesel fuel, the methylated biocrude was first extracted/partitioned with hexane and water. The hexane extract contained 72% of the biocrude by weight and consisted of methyl esters and other nonpolar compounds. This corresponds to an average of 860 L/ha (92 gal/acre). If the higher production rates of the second year harvest are used, this corresponds to 1290 L/ha (138 gal/acre) on a biennial basis. The remaining 28% of the biocrude not extracted into hexane consisted of a dark, insoluble sludge and water-soluble materials. Figure 6 shows the chromatogram obtained after mixing the diesel with the biofuel materials extracted in hexane. The major two peaks toward the end of the diesel chromatogram are GAME and an isomer of GAME, which was produced during acid-catalyzed methylation. A limited number of petroleum diesel tests were conducted on a methylated biocrude mixed with diesel fuel at 20% methylated biocrude and 80% diesel fuel by volume (Table 2). These limited tests indicated that a biofuel containing 20% methylated extract is consistent with its use as a biofuel, although further testing is required. Initial concerns were strongest regarding the kinematic viscosity, since the pure methylated biocrude is very viscous. However, although elevated, the 20% biofuel/diesel mixture does meet the ASTM standard for viscosity. Although the biocrude contains volatile terpenes, the flash point is well within the standard. As discussed previously, the primary constituent from gumweed extract is grindelic acid, a carboxylic acid, and thus has some similarity to common biodiesel, the product of transesterification of fatty acids. However, grindelic acid has a higher molecular weight than normal fatty acids in biodiesel but also is a tricyclic branched compound that potentially has a higher energy density than straight-chain hydrocarbons in
Table 2. ASTM Tests Performed on B20 Gumweed Biofuel Blend ASTM test
a
cetane number (D6890)* flash point (D93)# kinematic viscosity (cP) (D445)# sulfur, ppm (D5453)# ash (wt %) (D482)* water and sediment (vol %) (D2709)*
ASTM D975 requirement
B20 gumweed blend
min 40 min 52 °C min 1.9, max 4.1 max 15 − −
48.1 66.4 °C 3.61 13.8 0.005
