Renewable Energy 50 (2013) 833e839

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Renewable Energy

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Seashore mallow ( pentacarpos) as a salt-tolerant feedstock for production of biodiesel and ethanol

Bryan R. Moser a,*, Bruce S. Dien b, Denise M. Seliskar c, John L. Gallagher c a Bio-Oils Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA b Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA c Halophyte Biotechnology Center, College of Earth, Ocean, and Environment, University of Delaware, Lewes, DE 19958, USA article info abstract

Article history: Seashore mallow (Kosteletzkya pentacarpos) is a non-invasive perennial nonclonal halophytic oilseed- Received 27 March 2012 producing dicot that was investigated as a feedstock for production of biodiesel from seeds and Accepted 6 August 2012 ethanol from residual stem biomass. Seashore mallow seeds contained 19.3 mass % oil, which after Available online xxx extraction with hexane and pretreatment with catalytic sulfuric acid was converted into methyl esters in 94 mass % yield utilizing homogenous base catalysis. The principal components identified were methyl Keywords: linoleate (48.9%), palmitate (24.4%) and oleate (18.3%). Fuel properties were characterized and compared Biodiesel to biodiesel standards ASTM D6751 and EN 14214. Also investigated were blends with petrodiesel. Lastly, Ethanol Kosteletzkya pentacarpos seashore mallow stems were rich in neutral carbohydrates (51.8 mass %). After simultaneous sacchari- fi Saccharomyces cerevisiae cation and fermentation employing a native Saccharomyces cerevisiae yeast strain, the stems provided Seashore mallow ethanol and xylose yields of 104 g/kg and 47.8 g/kg, respectively. Of the four pretreatment methodologies explored, dilute ammonium hydroxide provided the highest yield of sugars. Published by Elsevier Ltd.

1. Introduction up to 1500 kg/ha of seeds [1,5]. The maximum yield of SM oil (SMO) is calculated to be 330 kg/ha. Aside from oilseed production, utili- Kosteletzkya pentacarpos (L.) Ledebour, colloquially known as zation of residual biomass for bioethanol fermentation is possible, seashore mallow (SM), is a non-invasive perennial nonclonal as SM produces up to 44 stems annually once maturity (3 years) is halophytic dicot that is native from the Gulf to the Atlantic coasts of reached [1]. The mean per crown in a five year old 1.2 ha field was the U.S [1]. Blanchard [2] synonymized K. pentacarpos (found in 13 [6]. Production of cellulosic bioethanol to enhance domestic Eurasia) with (described from the U.S., Cuba biofuels production from low-value, high-volume residual biomass and Bermuda). With a lifespan of approximately 11 years, SM rea- is of interest [7]. Other positive attributes of SM include compati- ches a height of 0.6e1.3 m and is characterized by pink blooms bility with existing farm infrastructure, seeds that do not shatter (2.5e5 cm) that emerge in July to August [3]. Tolerant of saline soil readily, non-susceptibility to disease, and tolerance to water- and brackish water, SM produces seeds with high protein (32%) and logging [1]. SM may be cultivated on saline or dry land that can oil (22%) content, thus rendering it attractive as animal feed and be irrigated with brackish or seawater, thus liberating fresh water biomass production in coastal areas not suitable for traditional and high quality soil for traditional agriculture while utilizing agriculture [4]. SM shows promise as an oilseed crop, as it produces fallow land [1,8]. Biodiesel is defined as monoalkyl esters of long-chain fatty acids (FAs) prepared from lipids [9] and must meet the requirements of

Abbreviations: AV, acid value; CFPP, cold filter plugging point; CP, cloud point; fuel standards such as ASTM D6751 or EN 14214 (Table 1) before its DCN, derived cetane number; FA, fatty acid; FFA, free fatty acid; HHV, higher commercial use is approved. Although it may be used in modern heating value; IP, induction period; IV, iodine value; KV, kinematic viscosity; OT, unmodified diesel engines, biodiesel is normally encountered as onset temperature; PP, pour point; PV, peroxide value; SSF, simultaneous sacchar- a blend component in petrodiesel. Currently, blends up to B5 (5 vol ification and fermentation; SM, seashore mallow; SMME, seashore mallow methyl %) and B7 are permitted in ASTM D975 and EN 590 (Table 2). esters; SMO, seashore mallow oil. * Corresponding author. Tel.: þ1 309 681 6511; fax: þ1 309 681 6524. Additionally, blends from B6 to B20 are regulated by ASTM D7467 E-mail address: [email protected] (B.R. Moser). (Table 2). Rapeseed/canola oil is principally used for production of

0960-1481/$ e see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.renene.2012.08.016 834 B.R. Moser et al. / Renewable Energy 50 (2013) 833e839

Table 1 biodiesel in Europe, palm oil predominates in tropical countries, Properties of seashore mallow oil (SMO) and methyl esters (SMME) with and soybean oil and animal fats are primarily used in the U.S [9]. a comparison to biodiesel standards. However, the combined supply of these lipids is sufficient to ASTM D6751 EN 14214 SMME SMO displace only a small percentage of petrodiesel. For example, if all Acid value, 0.50 max 0.50 max 0.13 (0.01)a 2.72 (0.05)/ U.S. soybean production were dedicated to biodiesel, an estimated mg KOH/g 0.08 (0.02)b 6% of diesel demand would be satisfied [10]. Furthermore, acqui- Glycerol content sition of refined oils may account for more than 80% of biodiesel Free, mass % 0.020 max 0.020 max 0.009 ec Total, mass % 0.240 max 0.250 max 0.068 ec production expenses [11]. Consequently, low-cost alternatives such Cold flow properties as field pennycress (Thlaspi arvense L.) and camelina (Camelina CP, C Report ed 4.2 (0.5) 0.3 (0.5) sativa L.) seed oils have attracted attention [9,12,13]. d e c CFPP, C e Variable 4.0 (0.6) e Literature precedent relating to production of biofuels from SM PP, C ed ed 3.7 (0.6) 8.0 (0) is rather limited, with only one study reporting oil content, FA Oxidative stability IP, 110 C, h 3 min 6 min 1.7 (0.1)/ 12.1 (0.6) composition, and a limited number of fuel properties from the 4.6 (0.1)f resulting fatty acid methyl esters (FAMEs) prepared by trans- OT, C ed ed 155.3 (0.5)/ 181.5 (0.2) esterification of SMO [5]. We follow up with a more thorough f 173.1 (0.6) examination of the fuel properties of SM methyl esters (SMMEs), PV, meq/kg ed ed 15.3 (0.4) 9.3 (0.2)/ 5.2 (0.1)b along with their evaluation as blend components in ultra low sulfur Iodine value, ed 120 max 111 111 diesel (ULSD, <15 ppm S) fuel. In addition, the utility of SM biomass gI2/100 g as a bioethanol feedstock is reported. Kinematic viscosity 40 C, mm2/s 1.9e6.0 3.5e5.0 4.44 (0.01) 37.50 (0.01) Cetane number 47 min 51 min 59.9 (1.6)g ec 2. Materials and methods Sulfur, ppm 15 max 10 max 2 6 Phosphorous, 0.001 max 0.001 max N/Dh 0.32 2.1. Materials mass % Moisture content, e 500 max 373 (5) 402 (2)/ b FAMEs (>99%) were purchased from Nu-Chek Prep, Inc. (Elysian, ppm 104 (5) Wear scar, ed ed 155 (8) 180 (3) MN). ULSD was donated by a petrochemical company. Cellulase 60 C, mm Optiflow RC2 and Multifect Xylanase were donated by Genencor, Specific gravity Inc. (Rochester, NY). Novo188 b-glucosidaseo was obtained from 15.6 C ed ed 0.887 (0) 0.928 (0) ed ed Novozymes A/S (Denmark). All other reagents were purchased 40 C 0.869 (0) 0.911 (0) e HHV, MJ/kg ed ed 39.53 (0.13) ec from Sigma Aldrich Corp (St. Louis, MO). Gardner color ed ed 11 12/11b

a Values in parentheses are standard deviations from the reported means. 2.2. Seashore mallow b After acid pretreatment. c Not determined. Seeds were collected from wild in Delaware and grown d fi Not speci ed. for several generations in salt-water irrigated plots at Lewes, DE. e Variable by location and time of year. f With 1000 ppm BHT added. Seeds from those plots were planted in untilled sandy loam that g Derived cetane number. had been sprayed with gramoxone. Rows were planted19 inches h Not detected. apart using a disk planter fitted with sorghum plates. The first year one stem was produced per . Over the next five years the stems per plant increased until they averaged 13 stems per crown

Table 2 Properties of seashore mallow oil methyl esters (SMME) blended with ultra low sulfur diesel (ULSD) fuel with comparison to petrodiesel standards.

Petrodiesel standards Blends of SMEME in ULSD

ASTM D975 ASTM D7467 EN 590 B0 B5 B20 Biodiesel content, vol % 0e56e20 0e70 5 20 Acid value, mg KOH/g ea 0.30 max e N/Db N/D 0.15 (0.02)c Moisture content, ppm ee 200 max 17 (1) 60 (2) 159 (1) Cold flow properties CP, C ed ed e 17.5 (0.1) 13.6 (0.1) 11.3 (0.2) CFPP, C ed ed e 18.0 (0) 17.0 (0) 16.7 (0.6) PP, C ee e24.0 (0) 23.7 (0.6) 16.7 (0.6) Oxidative stability IP, 110 C, h e 6 min 20 min 40.1 (1.5) 7.0 (0.1)/57.5 (5.1)e 3.6 (0.2)/22.7 (1.8)e Kinematic viscosity 40 C, mm2/s 1.9e4.1 1.9e4.1 2.0e4.5 2.30 (0.01) 2.31 (0.01) 2.49 (0.01) Sulfur, ppm 15 max 15 max 10 max 9 8 7 Wear scar, 60 C, mm 520 520 460 571 (4) 296 (22) 207 (9) Specific gravity 15.6 C ee e0.837 (0) 0.850 0.855 40 C ee e0.821 (0) 0.832 0.838 HHV, MJ/kg ee e46.2 (0.10) 45.2 (0.3) 43.8 (0.6)

a Not specified. b Not detected. c Values in parentheses are standard deviations. For sulfur content, n ¼ 1. d No limits are specified, but guidance is provided. e With 1000 ppm BHT added. B.R. Moser et al. / Renewable Energy 50 (2013) 833e839 835 in 2010 [6]. Seeds were harvested each year with a combine, Table 3 cleaned and stored in refrigeration. Seeds from 2010 were used for Fatty acid composition (area%) of crude seashore mallow seed oil. this study. Fatty acida Seashore mallow C14:0 0.2 2.3. Lipid extraction from seeds C16:0 24.4 (0.2)b C16:1 9c 0.8 (0.1) Air-dried seeds were ground in a coffee grinder and oil was C18:0 2.2 (0.1) C18:1 9c 18.3 (0.1) extracted with hexane for 24 h in a Soxhlet apparatus. Hexane was C18:2 9c, 12c 48.9 (0.2) removed by rotary evaporation (10 mbar, 25 C). For determination C18:3 9c, 12c, 15c 3.3 (0.1) of total oil content, 10.0 g samples (n ¼ 4) were extracted for 24 h, C20:0 0.6 and after hexane was removed (20 mbar, 25 C) the weights of the C20:1 11c 1.2 (0.1) unknown (sum) 0.1 residual oils were measured. S saturatedc 27.4 S monounsaturatedd 20.3 2.4. Fatty acid composition S polyunsaturatede 52.2 a For example, C18:1 9c signifies an 18 carbon fatty acid chain with FAMEs were prepared as described previously [12,13] and one cis (c) double bond located at carbon 9 (methyl 9Z-octadecenoate; analyzed using an HP 5890 Series II GC (Palo Alto, CA) equipped methyl oleate). b with an FID, an HP series 7673 auto sampler/injector and an SP2380 Values in parentheses are standard deviations. Where not indi- m fi cated, the value is zero. column (30 m 0.25 0.25 mm i.d., 0.20 m lm thickness). Carrier c S ¼ þ þ þ saturated C14:0 C16:0 C18:0 C20:0. gas was He at 1.0 mL/min. The temperature program was: 170 Ce d S monounsaturated ¼ C16:1 þ C18:1 þ C20:1. 190 Cat4 C/min, 30 C/min to 265 C, hold for 2.5 min. The e S polyunsaturated ¼ C18:2 þ C18:3. injector and detector temperatures were 250 C. Peaks were identified (triplicates, means reported) by comparison to determined by extraction with hexane. Nitrogen content was reference standards. determined by combustion, and crude protein concentration was estimated as Nx6.25 [16]. 2.5. Pretreatment of seashore mallow oil 2.9. Determination of sugars and ethanol production from woody SMO [acid value (AV): 2.72 mg KOH/g] (300 g) and methanol biomass (117 mL; 35 vol %) were combined, followed by addition of concentrated sulfuric acid (H2SO4; 3.0 mL; 1.0 vol %). The contents The pretreatments were dilute acid, hot water, dilute ammo- were heated at reflux for 4 h (1200 rpm). After cooling to room nium hydroxide (NH4OH), and alkaline peroxide. The effectiveness temperature the phases were separated. The oil phase was washed of the pretreatments was evaluated by measuring glucose and with distilled H2O until the pH was neutral, followed by removal of xylose yields following enzymatic digestion. The best pretreated methanol (20 mbar; 30 C). Drying with MgSO4 provided acid- material was further evaluated for conversion to ethanol using pretreated SMO (285 g; 95% yield) with an AV of 0.08 mg KOH/g. simultaneous saccharification and fermentation (SSF) employing a native Saccharomyces cerevisiae yeast. Ground stems were used 2.6. Methanolysis for all pretreatments. Stems (1.5 g dry basis) were mixed with distilled water, H2SO4 Acid-pretreated SMO (250 g; 0.290 mol) and methanol (71 mL; (0.5e4.0% w/v), or NH4OH (8% w/v ammonia basis) in 25 mL 316 1.74 mol; 6:1 mol ratio) were combined and heated to 60 C, fol- stainless steel (0.75 inch) batch reactors. The reactors were heated lowed by addition of sodium methoxide catalyst (1.25 g; 0.50 wt % to 180 C in a sand bath. The reactions were quenched after 20 min with respect to SMO). After reacting for 1.0 h (1200 rpm) the by transferring the reactors to a water bath. Reactors required 8 min mixture was cooled and the glycerol phase removed by gravity to achieve 180 C and cooled to ambient temperature in under separation, followed by removal of methanol by rotary evaporation 2 min. Dilute acid hydrolyzates were neutralized by adding (10 mbar; 30 C). The product was washed with distilled water until Ca(OH)2. Ammonium was removed by evaporation at ambient a neutral pH was obtained and dried with MgSO4 to afford SMME in temperature. 94% yield (236 g). For the alkaline peroxide pretreatment [17], stems (3 g) were hydrated with 10 mL of distilled H2O, followed by addition of 3% 2.7. Chemical and physical properties of biodiesel H2O2 (10 mL) and 10 mL of 0.5 M NaOH in a 150 ml PyrexÔ serum bottle. The base was added gradually to control foaming. The pH Properties were measured following AOCS, ASTM, and CEN was checked to ensure it was 11.5. After gas production had ceased standard test methods (Table 3) using instrumentation described (30 min), the serum bottle was sealed and reacted at 35 C for 24 h previously [12,13]. Oxidation onset temperature (OT, C) was at 250 rpm. The pH was adjusted to 5 with HCl. measured by pressurized differential scanning calorimetry (PDSC) Cellulose and xylan digestibility was determined using a modi- using a previously described procedure [12,13]. Peroxide value (PV, fied NREL procedure [15]. The whole hydrolyzate was diluted to meq. of peroxide/kg) was determined following a ferric thiocyanate a 1% w/v glucan basis by adding distilled H2O and 1 M sodium method [14]. Precisely measured amounts of SMME were splash citrate buffer (for a final concentration of 50 mM, pH 4.8) and the blended with ULSD with continuous stirring (22e24 C) to ensure antibacterial agent thymol (500 mg/L). The digestion was initiated homogeneity. B5.0 and B20.0 samples were prepared. by adding Optiflow RC2 cellulase (30 FPU/g cellulose), Novo188 b- glucosidase (40 U/g cellulose) and (where noted) Multifect Xyla- 2.8. Composition of woody biomass nase (50 U xylanase activity/g biomass). The reaction was carried out at 50 C for 72 h at 125 rpm using an incubator/shaker. Digested Cellulose, xylan arabinan, Klason lignin, and ash were deter- samples were analyzed for soluble carbohydrates and mono- mined according to the analytical procedures of the National saccharides as described below. All enzymatic saccharification Renewable Energy Laboratory (NREL) [15]. Lipid content was reactions were conducted at least in duplicate. 836 B.R. Moser et al. / Renewable Energy 50 (2013) 833e839

Ethanol fermentations were conducted as described previously 3.2. Preparation and chemical properties of seashore mallow oil [18]. The ammonia-pretreated sample was placed in a capped methyl esters 25 mL bottle and sterilized by heating at 121 C for 15 min using an autoclave followed by the addition of sterile distilled H2O (8.5 mL), Previous studies indicated that oils with AVs below 1.0 mg KOH/g 1 M sodium citrate buffer (0.5 mL; 50 mM final concentration; pH and moisture contents lower than 0.3 mass % are needed for high 4.8), and YP basal 10 stock (1.0 mL; final concentration of 10 g/L FAME yield employing homogenous, alkali catalyzed methanolysis yeast extract and 20 g/L peptone). For fermentations using [19]. Consequently, H2SO4-catalyzed pretreatment was performed S. cerevisiae D5A the following enzymes were added: Optiflow RC2 prior to methanolysis to reduce the AV of crude SMO (95 mass % (15 FPU/g glucan), Novo188 (40 CBU/g glucan), and Multifect yield). Homogeneous alkali catalyzed (sodium methoxide) trans- xylanase (50 U xylanase activity/g biomass). Control fermentations esterification then afforded SMME in high yield (94 mass %) without added biomass were used to determine background employing classic reaction conditions described previously [12,13,19]. glucose and ethanol, which were subtracted. The chemical properties (Table 1) of SMME were measured by Fermentations were inoculated to an OD600 of 1.0 or 0.6 g/L of determination of AV, IV, free and total glycerol as well as moisture, yeast biomass (1.0 cm path, DU 640 Spectrophotometer, Beckman, phosphorous and sulfur contents. Both ASTM D6751 and EN 14214 Brea, CA). The bottles were incubated for 72 h at 35 C at 100 rpm. specify maximum permissible limits for free and total glycerol Cultures were analyzed for ethanol, glycerol and residual sugar (Table 1). As seen in Table 1, free and total glycerol contents were content using HPLC. significantly below the prescribed maximum thresholds. Further- The inoculum was prepared by transferring a colony grown on more, ASTM D6751 and EN 14214 specify a maximum AV of solid YP2D (YP supplemented with 20 g/L dextrose and 2.0% Bacto 0.50 mg KOH/g. The AV of SMME (0.13 mg KOH/g) was below the agar) to a liquid YP2D culture followed by a YP5D culture (YP maximum limit. ASTM D6751 does not contain an IV specification, supplemented with 50 g/L dextrose). The liquid cultures were but EN 14214 limits IV to a maximum of 120 g I2/100 g. The IV of grown for 18 h at 35 C and 200 rpm, and the cells were harvested SMME (111 g I2/100 g) was within this limit due to its high content from the YP5D culture by centrifugation and concentrated to a cell of saturated FAMEs and low content of polyunsaturated FAMEs. The solution of 50 OD600 in PPB (per L: 8.5 g NaCl, 0.3 g KH2PO4, 0.6 g content of sulfur was 2 ppm, which was below the limits of 15 and Na2HPO4, 0.4 g peptone). Fermentation cultures were inoculated to 10 ppm prescribed in ASTM D6751 and EN 14214. No phosphorous an OD600 of 1.0 from the concentrated cell solution. Experiments was detected in SMME. Moisture content (373 ppm) was below the were conducted at least in duplicate. maximum limit (500 ppm) specified in EN 14214. Samples were analyzed for sugars, organic acids, and ethanol using a SpectraSYSTEMÔ liquid chromatography system (Thermo 3.3. Fuel properties of seashore mallow methyl esters Electron Corp, CA) equipped with an automatic sampler, column heater, isocratic pump, and refractive index detector. Samples Fuel properties are given in Table 1. A minimum IP (110 C) of 3 h (20 mL) were injected onto a sugar column (Aminex HPX-87H, is required for ASTM D6751, whereas a more stringent limit of 6 h or 300 7.8 mm, Bio Rad Laboratories, Inc., Hercules, CA) and greater is specified in EN 14214 for oxidative stability. An IP of 1.7 h eluted with 5 mM H2SO4 at 0.6 mL/min and 65 C. was observed for SMME, which was below these specifications due to its high content of methyl linoleate. IP may be improved by 3. Results and discussion addition of antioxidants [9,20]. As a result, the common antioxidant butylated hydroxytoluene (BHT) was added. The IP of SMME was 3.1. Composition and properties of seashore mallow oil improved to 4.6 h with 1000 ppm BHT, which was compliant with the specification listed in ASTM D6751. The OT (Table 1)was The FA composition of crude SMO is given in Table 3. The prop- 155.3 C, which increased to 173.1 C with addition of 1000 ppm erties of SMO are reported in Table 1. The lipid content of SM seeds BHT. The PV was 15.3 meq peroxides/kg. External factors such as from analytical extractions using hexane was 19.3% on a dry mass storage history and exposure to initiators may impact PV [21].At basis. The principal FA identified in SMO was linoleic acid (48.9%). present, neither PV nor OT is specified in the biodiesel standards. Palmitic (24.4%), oleic (18.3%), linolenic (3.3%), stearic (2.2%), and 11- Cold flow properties were measured by CP, PP and cold filter eicosenoic (1.2%) acids constituted most of the remaining FA profile. plugging point (CFPP). ASTM D6751 requires that CP be reported. The combined polyunsaturated content was thus 52.2%, with With respect to CFPP, EN 14214 set limits that vary by location and monounsaturated and saturated FAs comprising 20.2 and 27.4%, time of year. A disadvantage of biodiesel versus petrodiesel is respectively. Similar results were reported previously [5]. inferior cold flow properties, which is exacerbated by the presence The AV of crude SMO was 2.72 mg KOH/g with an IV of 111 g I2/ of higher-melting C16þ saturated FAMEs in biodiesel [22]. Previous 100 g. The Gardner color was 12 (1 is lightest, 18 is darkest). Cold studies determined that small levels of high-melting FAMEs have flow properties were determined by measurement of CP and PP, a disproportionate effect on low temperature properties of bio- and values of 0.3 and 8.0 C were obtained. Oxidative stability diesel [22]. SMME, with a relatively high content of saturated was quantified by EN 15751 and PDSC methods through measure- FAMEs, exhibited a CP of 4.2 C as well as CFPP and PP values of 4.0 ment of IP (12.1 h; EN 15751) and OT (181.5 C; PDSC). KV at 40 C and 3.7 C, respectively. These values are higher than those was 37.50 mm2/s. Densities of 0.928 and 0.911 g/cm3 were obtained observed for FAMEs prepared from soybean and canola oils [23]. at 15.6 and 40 C, respectively. The wear scar generated from ASTM The KV of SMME was 4.44 mm2/s, which was within the spec- D6079 (60 C) was 180 mm. The content of sulfur and phosphorous ified ranges for KV in ASTM D6751 (1.9e6.0 mm2/s) and EN 14214 was 6 ppm and 0.32 mass %, respectively. (3.5e5.0 mm2/s). The SG was not significantly different at 15.6 The influence of pretreatment on selected properties of SMO (0.887) and 40 C (0.869), although a higher value was obtained at was determined. A lower AV (0.08 mg KOH/g) was obtained after 40 C. The biodiesel standards do not specify limits for SG. pretreatment. Moisture content decreased to 102 ppm after The derived cetane number (DCN; ASTM D6890) of SMME was pretreatment. The initial PV of crude SMO was 9.3 meq peroxides/ 59.9, which was above the minimum limits of 47.0 and 51.0 speci- kg. After pretreatment, a PV of 5.2 meq peroxides/kg was obtained. fied in ASTM D6751 and EN 14214. The high DCN may be attributed We speculate that the conditions of pretreatment were sufficient to to high and low contents of saturated and trienoic FAMEs, respec- decompose a portion of the peroxides, thus resulting in a lower PV. tively, as these structural factors increase DCN [9]. B.R. Moser et al. / Renewable Energy 50 (2013) 833e839 837

Lubricity is not specified in ASTM D6751 or EN 14214 but is Table 5 a included in the petrodiesel standards ASTM D975, D7467 and EN Enzymatic digestion results following various pretreatments of seashore mallow. 590 (Table 2) with maximum prescribed wear scars (60 C) of 520, Pretreatment Conditions Efficiencies (% max) Yields (g/kg, db) m 520 and 460 m, respectively. The wear scar generated by SMME Glucose Xylose Glucose Xylose was 155 mm, which was considerably below the maximum limits Hydrothermal 180 C, 20 m 21 (0.7) 31 (0.6) 79 (2.7) 55 (0.9) listed in the petrodiesel standards. This was in agreement with 200 C, 20 m 39 (4.2) 32 (0.4) 145 (16) 58 (0.7) previous studies indicating that biodiesel possessed inherently Ammonium 180 C, 20 m 46 (3.0) 46 (1.1) 173 (11) 77 (6.5) good lubricity [12,13,24]. For comparison, the wear scar produced hydroxide 200 C, 20 m 50 (1.2) 50 (1.1) 189 (4.3) 74 (1.7) by ULSD was 571 mm(Table 2). Dilute-acid 180 C, 20 m, 44 (1.1) 44 (2.1) 165 (7.1) 56 (2.0) 2%w/v acid Although not specified in either ASTM D6751 or EN 14214, Alkaline H2O2 35 C, 24 h 15 (0.6) 15 (1.1) 55 (2.0) 43 (3.0) energy content (higher heating value; HHV) impacts fuel efficiency a ¼ and consumption. The HHV of SMME was 39.53 MJ/kg, which was mean and standard deviation for n 3. typical for biodiesel and below ULSD (46.2 MJ/kg; Table 2) [24]. of the moisture level in neat SMME. All values were below the 3.4. Blends of seashore mallow methyl esters in petrodiesel maximum threshold listed in EN 590, although the B20 sample contained too much SMME to be covered by EN 590. 2 The fuel properties of B5 and B20 blends of SMME in ULSD along The KV (40 C) of ULSD was 2.30 mm /s. As a result, blends of with a comparison to the petrodiesel standards are presented in SMME in ULSD afforded progressively higher KVs as the percentage of biodiesel was increased from B0 to B20. Specifically, the B5 and Table 2. Fuel properties determined in the current study that were 2 affected by blending included AV, moisture content, cold flow prop- B20 blends provided values of 2.31 and 2.49 mm /s, respectively. erties, oxidative stability, KV, sulfur content, lubricity, SG, and HHV. All of the blends exhibited KVs that were within the ranges speci- fi The biodiesel-petrodiesel blend standard ASTM D7467 (B6-B20) ed in ASTM D975, EN 590 and ASTM D7467. The response of KV to SMME content was linear (R2 0.962; figure not shown). limits AV to a maximum value of 0.30 mg KOH/g. The AV of B20 m SMME (0.03 mg KOH/g) was within the range specified in ASTM The wear scar generated by ULSD was 571 m and in excess of fi D7467. The AV of the B5 blend was below the detection limit as the maximum limits speci ed in ASTM D6751, D7467 and EN 590. a result of the low AVs of SMME and ULSD (not detected). Accordingly, blends exhibited progressively shorter wear scars as The IP of ULSD was 40.1 h. Consequently, blends exhibited the content of SMME was increased from B0 to B20. The response of progressively lower IPs as the percentage of biodiesel was increased lubricity to SMME was non-linear, as a small increase in SMME fi from B0 to B20. The response of IP to SMME content was non-linear, resulted in a signi cantly shorter wear scar. The effect diminished as an increase to the B5 level resulted in a significant reduction in IP as SMME content increased beyond B5. The B5 blend was within the limits listed in ASTM D975 and EN 590 with a wear scar of but the reduction at B20 was less pronounced. The B20 blend m m (3.6 h) failed to meet the 6.0 h specification listed in ASTM D7467. 296 m. The B20 blend afforded a wear scar of 207 m, which was fi The B5 blend provided an IP of 7.0 h, which was below the well below the maximum threshold speci ed in ASTM D7467. As minimum value of 20.0 h specified in EN 590. Addition of 1000 ppm was the case in prior studies, biodiesel (SMME) proved to be an BHT improved the IPs of the B5 (57.5 h) and B20 (22.7 h) blends to excellent lubricity additive for ULSD [12,13,24]. Density at 15.6 C is limited in EN 590 to a range of 820e845 kg/ values compliant with EN 590 and ASTM D7467, respectively. 3 m and is related to SG by the density of water. As the percentage of ULSD provided CP, PP and CFPP values of 17.5, 24.0 and 18.0 C, respectively. Consequently, blends displayed higher SMME increased, SG (15.6 C) increased from 0.837 (B0) to 0.850 (B5) and 0.855 (B20) as a result of the higher SG of SMME. As ex- CPs as the percentage of SMME was increased, although PP up to B5 was essentially unaffected (Table 2). The responses of CP, CFPP and pected, lower SGs were observed at 40 C than at 15.6 C. PP to SMME content were non-linear. With regard to CP, a sharp Sulfur is limited in ASTM D975 and D7467 to a maximum of increase was noted between B0 and B5 but a less dramatic increase 15 ppm whereas a more stringent limit of 10 ppm is prescribed in was observed between B5 and B20. Neither ASTM D975, D7467 nor EN 590. As the content of SMME increased, sulfur decreased from EN 590 specify specific limits on low temperature operability and only guidance is provided in ASTM D975 and D7467. 60 5 Dissolved water is limited in EN 590 to a maximum level of Glucose Xylose 200 ppm. As the percentage of SMME in ULSD increased from B0 to 50 pH B20, the content of moisture increased linearly (R2 0.997; figure not 4 shown) from 17 ppm (B0) to 60 (B5) and 159 (B20) ppm as a result 40 3 Table 4 Chemical composition of seashore mallow seed meal and stems (g/kg, dry basis). 30

Component Seed meal Stems Bast cellsa Core cells 2

Extractable 9.0 (0.0) 14.0 (0.1) nd nd 20 Pretreatment pH Protein 254.0 (8.5) 61.5 (0.7) ndb nd Total Carbohydrates 438 518 545 565 1 10 Glucan 214 (10) 337 (6) 368 (16) 361 (2)

Xylan 145 (19) 158 (3) 147 (6) 187 (1) of Effeciency Digestion (% max) Enzyme Arabinan 16.7 (7.4) 11.0 (2.7) 17.8 (4.0) 5.8 (1.5) 0 0 Galactan 72.2 (14.4) 11.7 (1.5) 12.3 (3.3) 12.1 (0.5) 01234 Lignin 246 (10) 183 (4) 146 (3) 264 (14) Sulfuric Acid Concentration (%w/v) Ash 62.0 93.2 nd nd Total 1001 856 N/A N/A Fig. 1. Sugar yield efficiencies (% of max) following dilute acid pretreatment at 180 C Potential Ethanol (L/kg) 0.33 0.38 0.40 0.41 for 20 min, neutralization, and enzymatic digestion with cellulases for 72 h. The pHs a Bast cell accounted for 40.8% and core cells 59.2% of the whole material. were measured following pretreatment with dilute-acid. Results are the mean of b nd ¼ not determined. duplicate samples. 838 B.R. Moser et al. / Renewable Energy 50 (2013) 833e839

Table 6 Fermentation results for ammonium hydroxide pretreated of whole and fractionated seashore mallow stems.

Concentration (g/L) Yield (g/kg, db) Efficiency (% max)

Biomass Ethanol Xylose Glycerol Ethanol Xylose Ethanol Xylose Whole 14.7 (1.2) 6.8 (0.5) 0.9 (0.1) 104 (7.8) 47.8 (1.6) 54.4 (4.1) 27.0 (0.9) Outer Layer 19.5 (1.1) 6.1 (0.2) 0.7 (0.0) 144 (5.7) 45.2 (1.3) 69.2 (2.7) 27.0 (0.8) Core 1.5 (0.4) 5.3 (0.5) 0.2 (0.0) 15.7 (5.1) 55.6 (1.2) 7.7 (2.4) 26.2 (0.6)

9 (B0) to 8 (B5) and 7 (B20) ppm as a result of the lower sulfur fermented to 14.7 g/L of ethanol within 72 h, which was 54.4% of content of SMME. All samples were within the specifications listed the maximum theoretical yield (Table 6). For this experiment, for sulfur in the petrodiesel standards. a native S. cerevisiae strain was used, which was incapable of fer- The HHV of ULSD was 46.2 MJ/kg versus 39.53 MJ/kg for SMME. menting xylose. Therefore, released xylose (6.8 g/L) was not fer- As a result, blends afforded progressively lower HHVs as the mented and the ethanol yield should, thus, be considered percentage of biodiesel was increased. A linear relationship (R2 a minimum. The absence of residual glucose suggested that 0.966; figure not shown) was noted between HHV and SMME microbial inhibitors did not limit fermentation. content. Specifically, the B5 and B20 blends provided values of 45.2 Ethanol conversion efficiency was lower than observed for and 43.8 MJ/kg, respectively. pretreating other biomasses with dilute NH4OH. For examples, switchgrass, reed canary grass and alfalfa stems pretreated with dilute NH OH under similar conditions yield up to 82.9% (unpub- 3.5. Conversion of seashore mallow stems into sugars and ethanol 4 lished data), 84% [18], 95% [32] of the maximum ethanol values, respectively. The seed meal and accompanying stems were analyzed for their The stems contained two pronounce types of material, a fibrous chemical composition (Table 4). The seed meal contained signifi- outer layer (bast cells) surrounding an inner pithy core. To gain cant amounts of protein (25.4% w/w) and lignin (24.6% w/w). The further insights into the cause of the lower than expected sugar stems were rich in neutral carbohydrates (51.8%). All of the yields, the outer layer was manually fractionated from the inner components of the seed meal were accounted for and 90.2% of that material (core cells) and each fraction was then pretreated with of the stem. Part of the remaining 9.8% is expected to be pectin, NH OH and fermented to ethanol. Prior to pretreatment, each which was not assayed. The neutral carbohydrates were used to 4 fraction was analyzed for carbohydrates (Table 4) and each con- calculate the theoretical ethanol yields. The stems (0.38 L/kg) had tained similar amounts of neutral carbohydrates (545 and 565 g/kg) a significantly higher yield than the seed meal (0.33 L/kg), so and accordingly processed similar theoretical ethanol yields. Yet further work focused on the stems. The seed meal might be of value when pretreated and fermented, the fractions gave very different as a feed or solid fuel because of its high protein and lignin results (Table 6). Pretreatment and SSF of the outer layer generated contents, respectively. 19.5 g/L ethanol and a yield efficiency of 69.2% of maximum. In The biochemical process for production of ethanol from ligno- contrast, the core layer gave a very low yield efficiency of 7.7%. celluloses consists of three primary steps: pretreatment, enzymatic Similar results were obtained when this experiment was repeated digestion, and ethanol fermentation [25]. Pretreatment consists of (data not shown). a mixture of chemical and thermal conditions optimized for We suspect the low yield of the core biomass was caused by two deconstructing the cell wall to allow the cellulases access to factors. The first was that the core cells had much higher lignin cellulose microfibers for conversion into glucose [7]. Four of the content than the outer layer (Table 4) and increased lignin content most effective pretreatments for herbaceous biomass were evalu- is associated with poorer enzymatic conversion yields [28]. ated for pretreatment of SM: hydrothermal [26], dilute NH4OH [27], However, this is an insufficient explanation because the ethanol dilute acid [28], and alkaline H2O2 [29]. The conditions selected yield of the whole material was higher than the weighted average were based upon literature values from the prior citations. Their of the outer and inner fractions. We noticed that the core material effects were judged based on production of glucose and xylose readily absorbed water to the extent that it might have interfered using a modified standard enzymatic digestion assay [15]. Glucose with enzyme activity and mixing. Notably, the conversion was not efficiencies were 14e51% and the most effective were dilute NH4OH yeast-limited because no residual glucose was detected at the end and dilute acid (Table 5). The rank of xylose efficiencies mirrored of fermentation. In summary, dilute ammonium was the most that of glucose yields. efficient pretreatment method tested for conversion of SM into The reported dilute acid value is based on the highest value from ethanol. However, SM appeared more recalcitrant to conversion a range of H2SO4 concentrations (Fig. 1) reacted at 180 C. Xylose than typical herbaceous biomasses largely because of the core yield increased when the temperature was lowered to 160 C. material. However, milder temperatures resulted in a lower yield of glucose (data not shown). This observation is not unexpected and as can be observed at 180 C, the optimal for glucose occurred at a higher 4. Conclusions severity than that for xylose recovery (Fig. 1). Xylose released under acidic conditions undergoes subsequent dehydration to furfural Fuel properties of biodiesel prepared from SMO were within the [30]. In this case, we sought to optimize glucose recovery at the specifications listed in ASTM D6751 and EN 14214 with the expense of xylose because the later is not fermented by S. cerevisiae. exception of IP, which was improved after addition of 1000 ppm The dilute NH4OH pretreatment was selected for further eval- BHT. Of note was the high DCN (59.9) of SMME along with its low IV uation because it produced the highest yield and is more compat- (111 g I2/100 g), which represent advantages over soybean oil- ible with fermentation [27]. In particular, dilute-acid pretreatment derived biodiesel. The properties of SMME blended with ULSD produces side-products (e.g. furans, etc.) that greatly inhibit or even were within the ranges specified in the petrodiesel standards after stall fermentation without extensive (and expensive) cleanup addition of BHT. SM biomass (stems) provided ethanol and xylose efforts [31]. The ammonium-pretreated biomass was successfully yields of 104 g/kg and 47.8 g/kg, respectively. B.R. Moser et al. / Renewable Energy 50 (2013) 833e839 839

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