Direct Transformation of Fungal Biomass from Submerged Cultures into Biodiesel


Published on

Direct Transformation of Fungal Biomass from Submerged Cultures into Biodiesel

Published in: Design, Technology, Business
  • Be the first to comment

  • Be the first to like this

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Direct Transformation of Fungal Biomass from Submerged Cultures into Biodiesel

  1. 1. Energy Fuels 2010, 24, 3173–3178 : DOI:10.1021/ef9015872Published on Web 04/02/2010 Direct Transformation of Fungal Biomass from Submerged Cultures into BiodieselGemma Vicente,† L. Fernando Bautista,† Francisco J. Gutirrez,† Rosalı´ a Rodrı´ guez,† Virginia Martı´ nez,† eRosa A. Rodrı´ guez-Frmeta,‡ Rosa M. Ruiz-Vzquez,‡ Santiago Torres-Martı´ nez,‡ and Victoriano Garre*,‡ o a † Department of Chemical and Environmental Technology, ESCET, Universidad Rey Juan Carlos, C/Tulipn s/n, a 28933 Mstoles, Madrid, Spain, and ‡Departamento de Gentica y Microbiologı´a (Unidad Asociada al IQFR-CSIC), o e Facultad de Biologı´a, Universidad de Murcia, 30071 Murcia, Spain Received July 29, 2009. Revised Manuscript Received March 11, 2010 Diminishing fossil fuel reserves and the increase in their consumption indicate that strategies need to be developed to produce biofuels from renewable resources. Biodiesel offers advantages over other petro- leum-derived fuel substitutes, because it is comparatively environmentally friendly and an excellent fuel for existing diesel engines. Biodiesel, which consists of fatty acid methyl esters (FAMEs), is usually obtained from plant oils. However, its extensive production from oil crops is not sustainable because of the impact this would have on food supply and the environment. Microbial oils are postulated as an alternative to plant oils, but not all oleaginous microorganisms have ideal lipid profiles for biodiesel production. On the other hand, lipid profiles could be modified by genetic engineering in some oleaginous microorganisms, such as the fungus Mucor circinelloides, which has powerful genetic tools. We show here that the biomass from submerged cultures of the oleaginous fungus M. circinelloides can be used to produce biodiesel by acid-catalyzed direct transformation, without previous extraction of the lipids. Direct transformation, which should mean a cost savings for biodiesel production, increased lipid extraction and demonstrated that structural lipids, in addition to energy storage lipids, can be transformed into FAMEs. Moreover, the analyzed properties of the M. circinelloides-derived biodiesel using three different catalysts (BF3, H2SO4, and HCl) fulfilled the specifications established by the American standards and most of the European standard specifications. 1. Introduction crop oils should be quickly developed.6 One way to increase world oil production that would cause a low ecosystem impact Society is facing an unprecedented situation with regard is to use lipids from oleaginous microorganisms (also calledto the fundamental sources of its raw materials and energy. single-cell oils), which present many significant advanta-Petroleum, the fuel that has driven modern society for the last ges over plants. Oleaginous microorganisms, such as yeasts,century, is showing signs of scarcity.1,2 Many renewable fuel fungi, bacteria, and microalgae, can accumulate high levels ofalternatives are under study,3 but ethanol and biodiesel are lipids7-14 (Table 1) and do not require arable land, so thatalready available in petrol stations. Biodiesel, which consists they do not compete with food production. More particularly,of fatty acid methyl esters (FAMEs), has many advantages, photosynthetic microalgae have attracted attention and invest-such as high energy density, great lubricity, fast biodegrada- ment because they capture carbon dioxide in lipids using sun-tion rate, and reduced emissions of sulfur, aromatic com- light. However, their growth in bioreactor systems is proble-pounds, and particulate matter.4 However, biodiesel adoption matic because of the light supply requirement.6,15 Oleaginousis complicated because it competes with the food industry for yeasts and fungi have also been considered as potential oilthe main raw material input, plant oils, and the worldwide sources for biodiesel production because they accumulate largesupply of plant oils is limited by land and water availability.4,5 amounts of lipids. Among these microorganisms, particularMoreover, a rapid expansion in biodiesel production capacity attention has been dedicated to various oleaginous zygomyce-is being observed in not only developed countries, e.g., United tes species, such as Mortierella isabelina and CunninghamellaStates and European Union, but also developing countries.To meet the demand of this industry, oil sources other than (7) Meng, X.; Yang, J.; Xu, X.; Zhang, L.; Nie, Q.; Xian, M. Renewable Energy 2009, 34, 1–5. (8) Chisti, Y. Biotechnol. Adv. 2007, 25, 294–306. *To whom correspondence should be addressed: Departamento de (9) Illman, A. M.; Scragg, A. H.; Shales, S. W. Enzyme Microb.Gentica y Microbiologı´ a, Facultad de Biologı´ a, Universidad de Murcia, e Technol. 2000, 27, 631–635.30071 Murcia, Spain. Telephone: þ34-868887148. Fax: þ34-868883963. (10) Gouda, M. K.; Omar, S. H.; Aouad, L. M. World J. Microbiol.E-mail: Biotechnol. 2008, 24, 1703–1711. (1) Grant, L. Science 2005, 309, 52–54. (11) Papanikolaou, S.; Komaitis, M.; Aggelis, G. Bioresour. Technol. (2) Vasudevan, P. T.; Briggs, M. J. Ind. Microbiol. Biotechnol. 2008, 2004, 95, 287–291.35, 421–430. (12) Fakas, S.; Papanikolaou, S.; Galiotou-Panatoyou, M.; Komaitis, (3) Wackett, L. P. Microb. Biotechnol. 2008, 1, 211–225. M.; Aggelis, G. J. Appl. Microbiol. 2008, 105, 1062–1070. (4) Durrett, T. P.; Benning., C.; Ohlrogge, J. Plant J. 2008, 54, 593– (13) Fakas, S.; Papanikolaou, S.; Batsos, A.; Galiotou-Panatoyou,607. M.; Mallouchos, A.; Aggelis, G. Biomass Bioenergy 2009, 33, 573–580. (5) Pinzi, S.; Garcia, I. L.; Lopez-Gimenez, F. J.; Luque de Castro, (14) Vicente, G.; Bautista, L. F.; Rodrı´ guez, R.; Gutirrez, F. J.; eM. D.; Dorado, G.; Dorado, M. P. Energy Fuels 2009, 23, 2325–2341. Sdaba, I.; Ruiz-Vzquez, R. M.; Torres-Martı´ nez, S.; Garre, V. a a (6) Li, Q.; Du, W.; Liu, D. Appl. Microbiol. Biotechnol. 2008, 80, 749– Biochem. Eng. J. 2009, 48, 22–27.756. (15) Rittmann, B. E. Biotechnol. Bioeng. 2008, 100, 203–212. r 2010 American Chemical Society 3173
  2. 2. Energy Fuels 2010, 24, 3173–3178 : DOI:10.1021/ef9015872 Vicente et al. Table 1. Oleaginous Microorganisms Used for by the industry. One way to generate microorganisms with Single-Cell Oil Production ideal lipid composition for biodiesel production could be by means of genetic manipulation of key genes.4,5 However, microorganisms considered thus far as a feedstock for biodie- sel production lack appropriate genetic engineering tech- niques to improve fatty acid profiles that would produce high-quality biodiesel.16 Besides, their genomes have not been sequenced, which makes it even more difficult to improve strategies based on genetic manipulation. In contrast, the oleaginous fungus Mucor circinelloides, which was used for the first commercial production of micro- bial lipids,21 has its genome sequenced and a large collection of genetic engineering techniques for its manipulation. These techniques include the expression of genes using autoreplica- tive plasmids and inactivation of genes by disruption22 or gene silencing (RNAi).23 In addition, the regulation of lipid accu- mulation in this fungus has been extensively studied for decades,24,25 and key genes have been identified.26 Moreover, the possibility to manipulate lipid accumulation in M. circi- nelloides using genetic engineering techniques has been recen- tly proven. Thus, overexpression of malic enzyme, which has been postulated to be the rate-limiting step for fatty acid biosynthesis in M. circinelloides, led to a 2.5-fold increase in lipid accumulation.27 The M. circinelloides lipids extracted for mycelium grown in a solid medium have been suggested as a suitable feedstock to produce biodiesel.14 Biodiesel was produced by acid-catalyzed transesterification/esterification because of its high free fatty acid content (31.6 ( 1.3%) following two different app- roaches: transformation of extracted microbial lipids andechinulata, which may accumulate up to 86 and 57% of lipids in acid-catalyzed direct transformation of microbial dry mass.dry biomass, respectively.11-13 These fungi are able to grow and The FAME yield was significantly higher in the direct transfor-accumulate large amounts of lipids in cultures containing raw mation than in the two-step process, with the FAME purity alsoglycerol derived from biodiesel production as a carbon source. being higher in the direct method. However, growth in a solidGlycerol is the major byproduct of the biodiesel production, and medium is unfeasible for the industry, which should use biomassits recycling to produce oleaginous microbial biomass could from submerged cultures. Therefore, we describe here thesignificantly decrease the cost of biodiesel production.13 characterization of the lipids accumulated by M. circinelloides Biodiesel is conventionally produced by transesterification mycelia grown in submerged liquid cultures and the acid-of extracted triacylglycerides with methanol, but a single-step catalyzed direct transformation of the M. circinelloides biomassmethod has been developed that transforms lipids present in into biodiesel, without previous extraction of those lipids. Indried microbial biomass into FAMEs, without previous lipid addition, we also show that the biodiesel obtained complies withextraction.16 This method combines the lipid extraction, the the current existing standards, the ASTM D6751 standard in theacid-catalyzed transesterification of the extracted saponifiable United States and most of the specifications in the EN 14213lipids, and the acid-catalyzed esterification of the extracted and 14214 standards in the European fatty acids in one step and was initially proposed becauseof the substantial reduction in both time and solvents that this 2. Experimental Sectiontechnique offers for analytical purposes.17 Similar procedures 2.1. Strains and Growth Conditions. The strain MU241,28that avoid the lipid extraction step have already been deve- derived from R7B29 after replacement of its leuA mutant alleleloped.13,18-20 However, most of them involve a previous by a wild-type allele, was used as a wild-type strain to producetransmethylation step and do not include an acid-catalyzed fungal biomass. For biomass production, 1 Â 105 spores/mLtransesterification and esterification.13,18,19 Biodiesel quality depends upon the fatty acid composition (21) Ratledge, C. Biochimie 2004, 86, 807–815. (22) Navarro, E.; Lorca-Pascual, J. M.; Quiles-Rosillo, M. D.; Nicols, aof raw materials, and consequently, not all microorganisms F. E.; Garre, V.; Torres-Martı´ nez, S.; Ruiz-Vzquez, R. M. Mol. Genet. acan be used as a feedstock for biodiesel production.4,5 Thus, a Genomics 2001, 266, 463–470.careful characterization of the lipid composition of each (23) Nicols, F. E.; Torres-Martı´ nez, S.; Ruiz-Vzquez, R. M. EMBO a amicrobial candidate should be carried out before its adoption J. 2003, 22, 3983–3991. (24) Aggelis, G.; Ratomahenina, R.; Arnaud, A.; Galzy, P.; Martin- Privat, P.; Perraud, J. P.; Pina, M.; Graille, J. Oleagineux 1988, 43, 311– (16) Liu, B.; Zhao, Z. B. J. Chem. Technol. Biotechnol. 2007, 82, 775– 317.780. (25) Aggelis, G.; Pina, M.; Graille, J. Oleagineux 1990, 45, 229–232. (17) Lewis, T.; Nichols, P. D.; McMeekin, T. A. J. Microbiol. (26) Wynn, J. P.; bin Abdul, H. A.; Ratledge, C. Microbiology 1999,Methods 2000, 43, 107–116. 145, 1911–1917. (18) Rodrı´ guez-Ruiz, J.; Belarbi, E.-H.; Garcı´ a Snchez, J. L.; Lpez a o (27) Zhang, Y.; Adams, I. P.; Ratledge, C. Microbiology 2007, 153,Alonso, D. Biotechnol. Technol. 1998, 12, 689–691. 2013–2025. (19) Weete, J. D.; Shewmaker, F.; Gandhi, S. R. J. Am. Oil Chem. Soc. (28) Silva, F.; Navarro, E.; Pe~ aranda, A.; Murcia-Flores, L.; Torres- n1998, 75, 1367–1372. Martı´ nez, S.; Garre, V. Mol. Microbiol. 2008, 70, 1026–1036. (20) Johnson, M. B.; Wen, Z. Energy Fuels 2009, 23, 5179–5183. (29) Roncero, M. I. G. Carlsberg Res. Commun. 1984, 49, 685–690. 3174
  3. 3. Energy Fuels 2010, 24, 3173–3178 : DOI:10.1021/ef9015872 Vicente et al.were inoculated in a 500 mL flask with 100 mL of YNB2XGliquid medium (20 g/L glucose, 1.5 g/L ammonium sulfate,1.5 g/L glutamic acid, 0.5 g/L yeast nitrogen base without aminoacids and ammonium sulfate, 1 mg/L nicotinic acid, and 1 mg/Lthiamine at pH 4.5) and incubated in the dark for 24, 48, 72, or96 h at 26 °C and 250 rpm. Culture pH was measured every 24 hand manually adjusted by the addition of 1 M NaOH. 2.2. Analysis of Cell Lipids. Mycelia harvested by filtrationusing Whatman Paper No. 1 were dried between paper towels,frozen in liquid nitrogen, lyophilized, weighed to estimate drymass, and ground using a mortar and pestle. Cell lipids wereextracted as previously described.30 Characterization of cell lipids was performed following stan-dard methods when possible. Free fatty acids, tri-, di-, andmonoglycerides, FAMEs, carotenoids, sterol esters, sterols andtocoferols, retinoids and polar lipids in microbial oil were identi-fied and quantified by TLC analysis. Chromatographic separation Figure 1. Kinetics of biomass production (2), lipid biosynthesis (O), and glucose consumption (b) in M. circinelloides cultures. Data arewas developed in 20 Â 20 cm silica-coated aluminum plates presented as mean values from duplicate experiments.(Alugram Sil G/UV, Macherey-Nagel GmbH, D€ren, Germany) uusing a solvent mixture of 88% (v) n-hexane, 11% (v/v) diethyl weighed to calculate the yield and then analyzed to determine itsether, and 1% (v/v) glacial acetic acid. Visualization was carried quality as biodiesel, following standard methods according toout by staining with iodine. Digital image analyses of staining European Union specifications (EN 14214).plates were performed with Un-Scan-It Gel 6.1 software (SilkScientific, Inc., Orem, UT), and the lipid compositions were 3. Results and Discussionquantified by the corresponding calibration curves. Free fatty acid content in the lipid fraction extracted from the 3.1. Biomass Production and Lipid Characterization. Tomicroorganisms was measured following a colorimetric proce- produce biodiesel, M. circinelloides biomass was obtaineddure31 based on the formation of cupric soaps and further quan- from the prototrophic strain MU241 grown in a liquid mediumtification of the chromophore complex by absorbance at 715 nm (YNB2XG) containing glucose as a carbon source (20 g/L). Inin a Cary 500 spectrophotometer (Varian, Inc., Palo Alto, CA). our experimental conditions, the fungus grew very quickly The phosphorus content in microbial oil was determined byinductively coupled plasma-optical emission spectrometry because it consumed all of the available glucose and stopped(ICP-OES) using a Vista AX model (Varian, Inc.). The analysis growing in the first 48 h after inoculation (Figure 1). Similarwas performed according to EN 14107:2003 standard. fast growth has been observed in not only M. circinelloides,26 Fatty acid profiles of microbial, rapeseed, and sunflower oils but also other Mucorales, such as M. isabellina.32 Lipid accu-were performed by gas chromatography (GC) in a CP-3800 mulation was high in the first analyzed time (24 h) and onlygas chromatograph (Varian, Inc.) fitted with a flame ioniza- increase slightly afterward. Although culture kinetic compar-tion detector (FID) and TRB-FFAP capillary column (30 m isons are difficult, particularly when different strains or culturelength, 0.32 mm internal diameter, and 0.25 μm film thickness, conditions are used, similar lipid accumulation kinetics wereTeknokroma, Barcelona, Spain). Prior to GC analysis, the oil previously observed in cultures of M. circinelloides.26 In addi-samples were transformed into their corresponding methyl tion, the fatty acid profile of the lipid extracted fromesters by saponification in 0.5 M KOH in methanol solution(30 min at 90 °C) followed by treatment with 14% boron M. circinelloides did not change significantly with the fermen-trifluoride in methanol (10 min at 90 °C) and extraction with tation time (data not shown).n-hexane/water. Finally, 3 μL of the organic phase containing After 96 h of growth, the fungus was clearly in stationaryFAMEs was injected into the capillary column, where the phase and no further increases in lipids were expected. In thatseparation was achieved using a temperature ramp (1 °C/min) time, a 4.17 ( 0.25 g/L fungal biomass with a total lipidfrom 150 to 240 °C at a flow rate of 1 mL/min (injector tempe- content of 22.9 ( 0.9% dry mass was obtained. Nonetheless,rature, 180 °C; detector temperature, 280 °C; injection mode, not all lipids obtained from microbial biomass are suitablesplitless). Identification of chromatographic peaks was per- for making biodiesel. Only saponifiable lipids and free fattyformed by a comparison to a FAME standard mixture (refe- acids (also referred to as oils) can be converted into FAMEs,rence 07131-1AM, Supelco, Bellefonte, PA) and quantification which can be used as biodiesel if they comply with the currentby means of external standards and their corresponding calibra-tion curve. The iodine number was calculated as described in EN standards (ASTM D6751 in the United States or EN 1421314214:2003 standard from the free fatty acid profile. and 14214 in the European Union). The saponifiable lipids 2.3. Direct Acid-Catalyzed Transesterification/Esterification and free fatty acids (including energy storage and structuralReactions. M. circinelloides biomass was transesterified/ester- lipids) were 98.0 ( 1.3% of the total lipids extracted fromified by stirring (900 rpm) with a solution of the catalyst M. circinelloides biomass, with the main components being(BF3, H2SO4, or HCl) in a closed container at 65 °C for 8 h. In triglycerides, polar lipids (phospholipids, sphingolipids, andthis direct process, a 10:1 methanol/chloroform (v/v) mixture saccharolipids), and free fatty acids (Table 2). In particular,was used as a reagent-solvent system, where the appropriate the quantity of sphingolipids and saccharolipids producedamount of the corresponding acid catalyst was dissolved. The by M. circinelloides was very high (around 54% of totalobtained mixture was diluted with water and then extracted with lipids). The amount of neutral lipids (mono-, di-, and trigly-hexane and diethyl ether using a centrifuge. The solvents wereremoved in a rotary evaporator, and the residue (FAMEs) was cerides) accumulated by M. circinelloides was 23.8%. Neu- tral lipids were comprised of mainly triglycerides (22.6 ( 1.3%). In addition, the proportion of phospholipids in this (30) Folch, J.; Lees, M.; Stanley, G. H. S. J. Biol. Chem. 1957, 226,497–509. (31) Lowry, R. R.; Tinsley, I. J. J. Am. Oil Chem. Soc. 1976, 53, 470– (32) Papanikolaou, S.; Galiotou-Panatoyou, M.; Fakas, S.; Komaitis,472. M.; Aggelis, G. Eur. J. Lipid Sci. Technol. 2007, 109, 1060–1070. 3175
  4. 4. Energy Fuels 2010, 24, 3173–3178 : DOI:10.1021/ef9015872 Vicente et al. 35-38 Table 2. Composition of the Lipids Extracted from esterification or transesterification reactions. Operating M. circinelloides after 96 h of Growth conditions (temperature, time, and solvent ratio) were pre- viously optimized using M. circinelloides biomass from solid medium.14 Using optimal reaction conditions (8 h at 65 °C), biodiesel yields were 18.9, 18.9, and 18.4% relative to the dry mass of M. circinelloides, using H2SO4, HCl, and BF3, respectively. These yields were even slightly higher than the corresponding theoretical yield calculated for this micro- organism (18.1%), indicating that acid-catalyzed direct tran- sesterification/esterification of fungal biomass can be app- lied to M. circinelloides biomass from submerged cultures because it improves the amount of total lipids extracted in comparison to the conventional methods for lipid extraction from microorganisms.30,39 This observation is supported by previous works describing increased recovery of fatty acids from microorganisms by direct transterification tech- niques.17,40 Interestingly, these results also indicate that saponifiable lipids other than triglycerides, such as phospho-fungus was 16%. Significantly lower proportions of struc- lipids, sphingolipids, and saccharolipids (Table 2), are trans-tural lipids (sphingolipids, saccharolipids, and phospho- formed into FAMEs by this method and should be consi-lipids) were observed in the biomass from stationary cultures dered as substrates for FAME obtention.of other oleaginous fungi, such as Cunninghamella echinulata,33 At the end of the procedure, methanol and chloro-whereas the amount of neutral lipids (storage lipids) was form were recovered and recirculated through the processhigher at this stage. The level of neutral lipids (storage lipids) (Figure 2).increased with time during the cultivation of this fungus, 3.3. Quality Analysis of the Biodiesel. The quality of thewhich means a decrease in the relative proportion of all of biodiesel produced in the one-step procedure was deter-the structural lipids with this variable. In fact, the amount of mined according to the EN 14214 specifications, and thestructural lipids in a microorganism is concrete, and there- results were compared to the corresponding specified bio-fore, it has to keep constant with time. In contrast, lipid diesel limits in standards EN 14213 (European Union), ENaccumulation in M. circinelloides was 18.9% at 24 h, increas- 14214 (European Union), and ASTM D6751 (Uniteding only slightly after this time (Figure 1). In this case, the States). Dependent upon the catalyst, the ester contentquantity of neutral lipids did not change significantly with ranged between 99.0 and 99.2% (Table 3), which is signifi-the fermentation time, which justifies the relative high pro- cantly higher than the corresponding specified minimumportion of phospholipids, sphingolipids, and saccharolipids value in the European Union standard (96.5%). These valuesat the stationary stage. Although free fatty acid levels were were higher and the reaction was faster than those repor-still high (3.6 ( 0.6%), they were substantially reduced in ted for other oleaginous microorganisms, in which an acid-comparison to those observed in biomass from solid medium catalyzed direct transformation method was also used.16(31.6 ( 1.3%).14 The non-saponifiable lipid fraction, which Futhermore, the amounts of all byproduct analyzed wereconsisted of small amounts of carotenoids, sterols, tocopher- below the maximum allowed values for American andols, and retinoids (Table 2), was also reduced in these culture European standards. Thus, the contents of individual glyce-conditions (1.96%) in comparison to the solid medium rides (mono-, di-, and triglycerides) were within the biodiesel(13.5%), probably because of the absence of light.22 These specifications, indicating that the transesterification andresults suggest that the fungal biomass from liquid cultures in esterification reactions were complete. The free glycerolthe dark shows better characteristics for biodiesel produc- content was lower than the two standard limits, indicatingtion than that from solid cultures. that the glycerol residues were eliminated during the purifi- 3.2. Biodiesel Production. The high concentration of free cation treatment. Besides, the individual glyceride and freefatty acids (3.6 ( 0.6%) in M. circinelloides determines that glycerol levels were below the established limits. The totalan acid-catalyzed process is more suitable for producing glycerol content also met all of the standards. The acidbiodiesel than an alkali one to avoid yield losses from free values, which depend upon the free fatty acid content,fatty acid neutralization.34 Methods for simultaneous lipid were also within the specifications in all reactions. In addi-extraction and transesterification involving a previous trans- tion, non-saponifiable lipids were not detected in themethylation step have been previously used with zygomy- M. circinelloides-derived biodiesel, which means that thesecetes fungi, but they were avoided because of their low types of lipids were also eliminated during the purificationyields.13 Therefore, the acid-catalyzed direct transformation stage. Nonetheless, the biodiesel obtained had small quan-method16,17 (Figure 2) was applied to dried mycelial biomass tities of polar lipids, which were lower than 0.9% in all casesusing methanol and chloroform as solvents and H2SO4, HCl, (Table 3). These compounds are residuals of nonconvertedand BF3 as acid catalysts, all of which are commonly used in polar lipids, and they are not considered in the biodiesel specifications established thus far. (33) Fakas, S.; Papanikolaou, S.; Galiotou-Panatoyou, M.; Komaitis,M.; Aggelis, G. Appl. Microbiol. Biotechnol. 2006, 73, 676–683. (37) Canakci, M.; Van Gerpen, J. Trans. ASAE 1999, 42, 1203–1210. (34) Vicente, G.; Martı´ nez, M.; Aracil, J. Energy Fuels 2006, 20, 394– (38) Canakci, M.; Van Gerpen, J. Trans ASAE 2003, 46, 945–954.398. (39) Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911– (35) Formo, M. W. J. Am. Oil Chem. Soc. 1954, 31, 548–559. 917. (36) Freedman, B.; Pryde, E. H.; Mounts, T. L. J. Am. Oil Chem. Soc. (40) Dionisi, F.; Golay, P. A.; Elli, M.; Fay, L. B. Lipids 1999, 34,1984, 61, 1638–1643. 1107. 3176
  5. 5. Energy Fuels 2010, 24, 3173–3178 : DOI:10.1021/ef9015872 Vicente et al.Figure 2. Schematic diagram of the process for biodiesel production from fungal biomass. Table 3. Quality Control of M. circinelloides-Derived Biodiesela catalyst property BF3 H2SO4 HCl EU standard EN 14214 U.S. standard ASTM D6751monoglyceride content (wt %) nd nd nd 0.8 maximum nsdiglyceride content (wt %) nd nd nd 0.2 maximum nstriglyceride content (wt %) nd nd nd 0.2 maximum nsfree glycerol (wt %) 0.0020 0.0032 0.0030 0.02 maximum 0.02 maximumtotal glycerol (wt %) 0.0020 0.0032 0.0030 0.25 maximum 0.24 maximumacid value (mg of KOH/g) nd 0.40 nd 0.5 maximum 0.5 maximumnon-saponifiable lipids (wt %) nd nd nd ns nspolar lipids (wt %) 0.8 0.8 0.9 ns nsester content (wt %) 99.2 99.0 99.1 96.5 minimum ns a nd, not detected; ns, not a specified limit. Table 4. Fatty Acid Composition in Biodiesel from M. circinelloides, Rapeseed, Sunflower, Palm, and Soy Oils content (wt %) fatty acid M. circinelloides oil rapeseed oil sunflower oil palm oil41 soy oil41lauric acid 12:0 nd nd nd 0.1 ndmyristic acid 14:0 1.6 0.1 nd 0.7 ndmyristoleic acid 14:1 0.6 nd nd nd ndpentadecanoic acid 15:0 2.5 nd nd nd ndpalmı´ tic acid 16:0 20.7 5.0 6.3 36.7 11.3palmitoleic acid 16:1 1.1 nd 0.2 0.1 0.1stearic acid 18:0 7.0 1.6 2.2 6.6 3.6oleic acid 18:1 28.0 36.3 20.6 46.1 24.9linoleic acid 18:2 12.7 19.8 52.8 8.6 53.0linolenic acid 18:3 22.5a 7.8b 3.5b 0.3b 6.1barachidic acid 20:0 0.3 0.1 1.6 0.4 0.3gadoleic acid 20:1 nd 9.1 0.3 0.2 0.3behenic acid 22:0 0.4 nd 7.2 0.1 nderucic acid 22:1 0.07 20.2 5.1 nd 0.3lignoceric acid 24:0 1.2 nd 0.2 0.1 0.1nervonic acid 24:1 nd nd nd nd ndother 1.3 nd nd nd ndiodine value (g of I2/100 g) 106.0 107.7 122.4 55.6 129.7 a The γ-linolenic acid isomer was obtained. b The R-linolenic acid isomer was obtained. The fatty acid profile for the FAMEs obtained from biodiesel obtained from M. circinelloides was within theM. circinelloides was compared to those produced for rapeseed, European Union specifications because the specified limitsunflower, palm,41 and soy41 oils (Table 4), which are the most (1%) only includes polyunsaturated fatty acids with four orcommonly used raw materials by the biodiesel industry in more double bonds, which are absent in M. circinelloides-Europe and the United States. Microbial oils usually differ from derived biodiesel. FAMEs from M. circinelloides containedmost vegetable oils in being quite rich in polyunsaturated 12.7 and 22.5% of linoleic (two double bonds) and linolenicfatty acids.8 However, the content of these fatty acids in the (three double bonds) acids, respectively, which would have low oxidative stability. In fact, the linolenic acid methyl ester (41) Ramos, M. J.; Fernndez, C. M.; Casas, A.; Rodrı´ guez, L.; Prez, a e content in the M. circinelloides-derived biodiesel was aboveA. Bioresour. Technol. 2008, 100, 261–268. the specified limit, 12%, in the European standards. On the 3177
  6. 6. Energy Fuels 2010, 24, 3173–3178 : DOI:10.1021/ef9015872 Vicente et al.other hand, the high degree of unsaturation inherent to cations established by the current existing standards, ASTMmethyl esters from these fatty acids would evidence excellent D6751 in the United States and EN 14213 and 14214 infuel properties at low temperatures, which is an advantage in the European Union. In addition, efficient biodiesel produc-winter operation.42 Moreover, all of these fatty acids are tion by direct transformation of fungal biomass without lipidcommon in industrial vegetable oils, and in particular, sun- extraction is technically feasible in M. circinelloides, whichflower and soy oils are also very rich in polyunsaturated fatty represents a starting point for developing this process on anacids. Thus, the calculated iodine value, which is a measure industrial scale. However, biodiesel yields should be increasedof the total unsaturation level, for the M. circinelloides- to make the industrial process economical, which could bederived biodiesel (106.0 mg of I2/g) was far below the speci- attained by the genetic manipulation of this fungus. In thisfied limit (120 mg of I2/g) in the European Union standards sense, efforts are now dedicated to overexpress genes that codeand also met the United States standards because these for enzymes postulated to be rate-limiting steps for fattyspecifications do not include the iodine value as a quality acid biosynthesis in oleaginous fungi.26 Other strategies areparameter. In comparison to the vegetable oils, the iodine focused on the generation of strains with enhanced ability tovalue was very similar to the one obtained in biodiesel from use crop residues or industrial byproduct, avoiding competi-rapeseed oil (107.7 mg of I2/g), which is the preferred raw tion with the food supply, with low linolenic acid levels ormaterial for biodiesel production in Europe. overexpressing genes involved in saponifiable lipid biosynthe- sis. Particularly interesting is the generation of strains with 4. Conclusions low free fatty acid levels because they could be used for biodiesel production by using a base-catalyzed technology, The results shown here indicate that M. circinelloides which is the common way to produce biodiesel on an indus-biomass from submerged cultures may be a suitable feedstock trial scale.for biodiesel production. Moreover, the analyzed propertiesof the M. circinelloides-derived biodiesel fulfilled the specifi- Acknowledgment. We thank J. A. Madrid for technical assis- tance. This work was funded by the D. G. de Investigacin y o (42) Vicente, G.; Martı´ nez, M.; Aracil, J. Bioresour. Technol. 2004, 92, Polı´ tica Cientı´ fica (Comunidad Autnoma de la Regin de o o297–305. Murcia, Spain), Project BIO-BMC 07/01-0005. 3178
  7. 7. 10074 Ind. Eng. Chem. Res. 2010, 49, 10074–10079Experimental Investigations into the Insecticidal, Fungicidal, and BactericidalProperties of Pyrolysis Bio-oil from Tobacco Leaves Using a Fluidized BedPilot Plant Christina J. Booker,†,‡ Rohan Bedmutha,†,§ Tiffany Vogel,†,‡ Alex Gloor,†,‡ Ran Xu,†,§ Lorenzo Ferrante,†,§ Ken K.-C. Yeung,†,‡ Ian M. Scott,| Kenneth L. Conn,| Franco Berruti,†,§ and Cedric Briens*,†,§ Institute for Chemicals and Fuels from AlternatiVe Resources (ICFAR), 22312 Wonderland Road North, RR#3, Ilderton, Ontario N0M 2A0, Canada, Faculty of Science, The UniVersity of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B9, Canada, Faculty of Engineering, The UniVersity of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B9, Canada, and Agriculture and Agri-Food Canada, 1391, Sandford Street, London, Ontario N5V 4T3, Canada Tobacco bio-oil, gases, and char were produced through pyrolysis of tobacco leaves using a fluidized bed pilot plant under varying temperature (350, 400, 450, 500, 550, and 600 °C) and residence time (5, 10, and 17 s) conditions. The optimized condition for the production of bio-oil was found to be at 500 °C at a vapor residence time of 5 s, giving a bio-oil yield of 43.4%. The Colorado Potato Beetle (CPB) Leptinotarsa decemlineata L. (Coleoptera: Chrysomelidae), a destructive pest toward potato crops, and three microorganisms (Streptomyces scabies, ClaVibacter michiganensis, and Pythium ultimum), all problematic in Canadian agriculture, were strongly affected by tobacco bio-oil generated at all pyrolysis temperatures. Nicotine-free fractions of the tobacco bio-oil were prepared through liquid-liquid extraction, and high mortality rates for the CPB and inhibited growth for the microorganisms were still observed. A potential pesticide from tobacco bio-oil adds value to the biomass as well as the pyrolysis process.1. Introduction Canada. One of the reasons this biomass was selected for anal- ysis is that tobacco farmers across the world, and in particular Pyrolysis is one of the thermo-chemical processes that is used in Canada, are suffering as demand for their crop continues toextensively worldwide to convert biomass into liquid bio-oil,char, and gases. This process is carried out in the absence of decline. It is well-known that smoking tobacco has a significant,oxygen.1 However, the pyrolysis oil normally contains a high negative impact on human health. Transitioning out of tobaccoproportion of oxygenates, reflecting the oxygen content of the farming, however, is difficult due to the specified nature of theoriginal substrates. With the current focus on environmentally equipment used, and therefore many farmers are left with excessfriendly energy prospects and renewable energy resources, crop every year, which currently goes to waste. Thus, findingsignificant research is being directed toward bio-oils. Bio-oil is alternative, healthy, high value applications to this highlyconsidered a CO2 neutral alternative to fossil fuels with low abundant product is an important research area. Already, tobaccoemissions of the undesirable components SO2, NOx, and soot.2 biomass is being investigated for unique, high value applications,Despite these advantages, bio-oil has several undesirable proper- such as for medical or industrial proteins,4-6 and in the case ofties as a fuel, including high viscosity, low heating value, poor this research, as a natural pesticide. Because tobacco’s pesticidevolatility, and coking. Refining bio-oil to a satisfactory level properties are well-known, converting tobacco leaves to naturalfor commercial use has been performed, but currently uses too pesticides in the form of bio-oil could provide additional incomemuch energy and occurs at too high a cost to be economically to farmers.viable.3 Tobacco biomass has been characterized,7-9 but very limited An additional, potentially lucrative prospect for bio-oil is as work has been published on the pyrolysis of tobacco for thea source for valuable chemicals. These chemicals could be found production of bio-oil. One study concentrated on the productionin the original biomass, such as nicotine in tobacco bio-oil, orcould be created during the pyrolysis process, such as phenols of fuel gases but did not perform liquid analysis,10 while anotheror new chemicals yet to be identified. One of the many potential study performed liquid analysis but failed to analyze the bio-applications of these chemicals is as a pesticide. The search for oil for nicotine.11effective and safe pesticides is a continuing challenge as species The potential pesticide activity of bio-oil is an excitingquickly adapt to most pesticides that are applied. research area that has yet to be fully explored. Recently, bio- In this Article, tobacco bio-oil is generated through pyrolysis oil has been studied for its wood preservative qualities12 andunder a wide range of operating conditions and analyzed for specifically for its antifungal properties.13 Two species of fungipesticide properties toward a variety of species of concern in were tested and found to have inhibited growth patterns in the * To whom correspondence should be addressed. E-mail: cbriens@ presence to bio-oil from wood biomass. In contrast, this Article investigates the pesticide characteristics of bio-oil from † ICFAR. tobacco biomass, not only for antifungal activity, but also for ‡ Faculty of Science, The University of Western Ontario. § Faculty of Engineering, The University of Western Ontario. antibacterial and insecticidal activity. The pyrolysis of this | Agriculture and Agri-Food Canada. tobacco biomass is also investigated. 10.1021/ie100329z  2010 American Chemical Society Published on Web 09/14/2010
  8. 8. Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010 100752. Experimental Section series II gas chromatograph (GC) was used. A RESTEK Shin Carbon ST (micro packed), 2 m length column with 1 mm i.d. 2.1. Materials. Finely ground tobacco leaves were provided and 1.58 mm o.d., was used to separate the gas mixture. Aby Agriculture and Agri-Food Canada, London, ON. Tobacco thermal conductive detector (TCD) was used to detect theleaves were obtained from tobacco crops in 2006 and dried at composition of the gas mixture, which consisted of N2, H2, CO,60 °C. Dried tobacco leaves were then ground using a blender/ CO2, and CH4. To measure product gas yields accurately, N2mixing mill and sieved. The Sauter mean diameter of the tobacco was selected as an internal standard gas. Argon was selected asparticles used for pyrolysis was 60 µm. the GC carrier gas. A standard gas mixture with a fixed com- 2.2. Methods. 2.2.1. Pilot Plant Design for Pyrolysis. All position of H2, CO, CO2, and CH4 was used to calibrate thepyrolysis experiments were carried out using a fluidized bed system. The injector was maintained at 150 °C, and the TCDpilot plant14 (Figure S1, Supporting Information). The heart of was maintained at 275 °C. A gas sample volume of 0.5 µL wasthe plant was an atmospheric fluid bed reactor, 0.078 m in injected with a 100 µL Hamilton syringe. Upon injection, thediameter, with a 0.52 m long cylindrical section, and equipped oven temperature was held at 35 °C for 180 s, then increasedwith an expanded section made up of a 0.065 m long truncated at 10 °C/min to 150 °C, and finally increased at 20 °C/min tocone with an upper diameter of 0.168 m, topped by a second, 250 °C. The temperature was then held constant at 250 °C for0.124 m long, cylindrical section. The total volume of this 330 s.configuration was 6.09 × 10-3 m3. This assembly provided the 2.2.4. Characterization of Char. The differential pressurelowest vapor residence time (5 s). Two different freeboard drop across the fluidized bed was measured at minimumextensions were used to increase the vapor residence time to fluidization conditions before and after each experiment. The10 and 17 s. A filter capable of withstanding high temperatureswas installed at the gas exit of each of the extensions. Each increase in the reading of the differential pressure drop wasfilter was made up of a perforated pipe connected to the gas proportional to the increase in bed weight. This system wasexit covered by a fiberglass pad and wrapped inside a fine calibrated for very accurate measurement of the char yields.stainless steel mesh cover. The resulting filter was, in all cases, 2.2.5. Characterization of Bio-oil. The bio-oil was charac-0.076 m in diameter and 0.178 m long. Although not ideal, these terized through GC-MS analysis of the various fractionshot filters have been used in the initial phase of the project with examined for biological assays (see below). A HP 6890 Seriesthe objective of avoiding the use of a hot cyclone for the char gas chromatography system with a mass selective detector wasseparation, which would be impossible to properly size due to used to analyze the bio-oil fractions. All experiments werethe variety of physical characteristics of the chars expected from performed on an HP-5MS, 30 m column obtained from Agilentthe different feedstocks. Technologies with an i.d. of 0.25 mm and a film of 0.25 µm. The fluidizing nitrogen was injected through a perforated copper The injector temperature and auxiliary temperature were main-distributor plate with 33 holes, 0.5 mm in diameter, equally spaced tained at 300 °C. The oven temperature began at 60 °C for 2across the cross section. The reactor was equipped with 18 min, and then increased at 10 °C/min to 280 °C and was heldthermowells for temperature measurements and control (type K for 6 min. A threshold of 150 was used, with a mass to chargethermocouples). scan range of 50-300 at a rate of 2.98 scans/s. An innovative pulsating automatic feeder was used for 2.2.6. Bio-oil Pesticide Characterization. Pesticide activitybiomass injection to the reactor. It quickly dispersed the injected tests with the bio-oil were performed on a variety of problematicbiomass into the core of the fluidized bed. species of microorganisms and one insect. All tobacco bio-oil 2.2.2. Bio-oil Production. Tobacco, when injected into the samples used for the biological tests were produced at a vaporreactor, produced vapors that exited at the top of the reactor residence time of 5 s and at different pyrolysis temperatures,through the hot filter section and flowed into three condensers as specified for each series through a line traced with Raychem Chemelex heating Bio-oil Sample Preparation for Pesticide Analysis.cable to avoid early, undesirable condensation (as shown in To initially determine which microorganisms were negativelyFigure S1). Persistent aerosols were then separated in a cylin- affected by the tobacco bio-oil, a cocktail of naturally separated,drical demister packed with cotton wool. The demister was organic phases and a cocktail of the aqueous phases of the bio-weighed before and after the experiment. The exact yield of oils produced from 350 to 600 °C were prepared in acetonetobacco bio-oil was obtained from the mass of oil collected in (375 mg/mL, one solution of all pyrolysis temperatures). Bio-the three condensers and the demister. oil samples from each pyrolysis temperature were then prepared Pyrolysis was initially carried out at six different temperatures separately in acetone (375 mg/mL, one solution for eachfrom 350 to 600 °C and at three different residence times (5, pyrolysis temperature). Raw tobacco bio-oil at each pyrolysis10, and 17 s). Each test was conducted with 700 g of tobacco temperature was used for the CPB tests.leaves. Fluidizing and atomizing nitrogen volumetric flow rates Two different liquid-liquid extraction techniques were usedwere precisely controlled using “Mass Trak” flow-meters from to generate nicotine-free and nicotine-containing fractions ofSierra Instruments Inc., to keep the nominal vapor residence the tobacco bio-oil. One method was used for the microorganismtime constant at all temperatures. Tobacco bio-oils produced at assays and generated six unique fractions (also analyzed throughall these temperatures separated into two separate phases, an GC-MS), while the other method was used for the insect assaysorganic and an aqueous one. and generated two distinct fractions. The reason for the two Pyrolysis of tobacco leaves was subsequently carried out methods was that two separate researchers performed theseunder the best reactor conditions for high bio-oil yield (discussed respective tests. Even so, the end result successfully allowedin Results and Discussion section and found to be at a for nicotine-free fractions to be tested on both the microorganismtemperature of 500 °C and a vapor residence time of 5 s) to and the CPB.determine the accurate liquid, gas, and char yields. The fractionation method used for the microorganism tests, 2.2.3. Characterization of Product Gases. Gases were which generated six unique fractions, is illustrated in Figure 1.sampled in plastic bags at three different time intervals. To The organic phase of the tobacco bio-oil pyrolyzed at 450 °Cmeasure the product gas composition, a Hewlett-Packard 5890 was dissolved in ether at a concentration of 175 mg/mL. This
  9. 9. 10076 Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010 organic fraction recovered was a moderately viscous brown oil, quite similar to the bio-oil itself. The aqueous fraction was orange and had low viscosity. Biological Assays for Pesticide Activity. Mi- croorganism Assays. The disk diffusion assay was used to test 11 fungi and 4 bacteria for growth inhibition in the presence of the tobacco bio-oil samples. All species are problematic microorganisms in Canada. See Table S1 for the list of species, their source, and the type of media on which they were maintained. Samples and control solutions were added to sterile, 6 mm diameter filter paper disks and allowed to air-dry before being placed onto freshly inoculated plates. For bacteria tests, the plates were inoculated by streaking the entire surface with freshly grown bacteria to generate a lawn of growth. One or three paper disks were placed into the center of the plate or in a triangular formation on the plate, depending upon the ex- periment. For fungi tests, a plug of a fresh culture was added about 1 cm away from the disks on a fresh plate. After the platesFigure 1. Bio-oil fractionation scheme for microorganism assay testing and were incubated at 24 °C for 3 days, the results were recorded.GC-MS analysis. Shaded boxes indicate fractions tested in microorganism A region of no growth around the disk indicated inhibition (withassays. a minimum measurement of inhibition being 6 mm, the diameter of the disk). Triplicate experiments were performed. Insect Assays. These tests were carried out by the leaf disk application, similar to the procedure outlined by Sengonca.18 Bio-oil fractions and control solutions were spread on both sides of a potato leaf disk with a cotton-tipped applicator. Three leaves were tested for each fraction; however, most tests were repeated on multiple dates to ensure accuracy. The potato plants (var. Cal White) were grown on site at theFigure 2. Extraction scheme for nicotine-free tobacco bio-oil fractions for Southern Crop Protection and Food Research Centre (SCPFRC),insect assays. Agriculture and Agri-Food Canada, London, Ontario, with the leaves cut to a diameter of 4 cm. The leaves were allowed to dry after sample application. After drying, the leaves werefraction was sterile filtered with a 2.5 cm diameter, 45 µm pore transferred to a Gelman Petri dish. Five, second instar insecticidesize, syringe filter with a nylon membrane (Whatman, NJ) susceptible strain Colorado Potato Beetle (CPB) larvae reared(Fraction Z). The remaining residue was dissolved in acetone at SCPFRC were then transferred to the leaf. Mortality rates(approximately 102 mg/mL) and was also sterile-filtered, giving were recorded after 24 and 48 h intervals. Adjusted percenta very dark brown solution (Fraction I). Fraction Z was then mortality values are reported, which take into account the naturalfractionated into its aqueous (Fraction A) and organic (Fraction mortality levels of the CPB in the control treatments. ControlB) components with a water/ether extraction. An additional treatments involved simply placing the beetles on leaf diskswater/ether separation was then performed with Fraction B without any oil present. If a specific test involved dilution ofwhere the water phase was acidified with HCl to a pH of 4-5. the bio-oil, the control leaf disks were coated with the solventThis step caused some components, such as the compound used.nicotine, to become charged and move into the aqueous phase.An organic, ether phase (Fraction C) and a charged, aqueous 3. Results and Discussionphase were generated. The acidic phase was then adjusted topH 9 (to move the majority of nicotine back into an organic 3.1. Tobacco Pyrolysis. The effects of pyrolysis temperaturesphase) and a final aqueous/ether extraction made an organic (350-600 °C) and residence times (5, 10, and 17 s) on the liquidphase (Fraction D) and an aqueous phase (Fraction E). Dilution yield are as shown in Figure 3. Tobacco bio-oil yields were afactors were calculated for each fraction, and the volume of strong function of temperature and residence time. The greatestsample used for the biological assays was appropriately adjusted. yield peaked at 500 °C for all residence times. It could also beEach fraction was analyzed using GC-MS (Figure S2). observed that bio-oil yield increased as the residence time To generate a nicotine-free and a nicotine-containing fraction decreased, for all temperatures. Comparable results were foundfor the insect tests, liquid-liquid extraction was performed with when this reactor was used to pyrolyze grape seeds and skins,diethyl ether and dichloromethane (DCM) (Figure 2). The for at a 5 s vapor residence time, the optimum pyrolysis temper-procedure outlined by Oasmaa et al.15 was used as it closely ature was also found to be 500 °C.14matched past literature methods for nicotine extraction from As shown in Table 1, for a residence time of 5 s and a reactiontobacco plants.16,17 A bio-oil mixture from all pyrolysis tem- temperature of 500 °C, the bio-oil yield was the highest (43.4%),peratures (15-20 g) was first passed through a filter paper followed by the char yield (29.4%) and the gas yield (22.4%).(Whatman’s #4) to remove the solid lignin residue. This residue The mass balance on the pyrolysis products was close to 95%,was washed with two, 5 mL portions of diethyl ether followed which was within the margin of error. Calculations showed thatby two, 5 mL portions of DCM. The filtrate was then extracted the heat of combustion of the gases produced was 508 J/g ofwith 20-30 mL of diethyl ether followed by 20-30 mL of biomass fed. It was assumed that the water produced by com-DCM. All organic phases were combined, and the solvent was bustion was condensed. The heat of combustion value forevaporated using a rotary evaporator (BUCHI R-114). The tobacco was on the lower side as compared to other feedstocks,
  10. 10. Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010 10077Figure 3. Effect of temperature and residence time on the liquid bio-oilyield. For experimental details, see Methods section. Figure 4. Effect of pyrolysis temperature on the diameter of inhibition for the three affected microorganism species. Error bars indicate ( standardTable 1. Pyrolysis Product Split at a Vapor Residence Time of 5 s deviation (σ) of replicate measurements within an experiment (total lengthand Pyrolysis Temperature of 500 °C 2σ). liquid yield (wt %) gas yield (wt %) char yield (wt %) interesting. This selective inhibition suggests that the active 43.4 22.4 29.4 components in the bio-oil are not destructive to all living things, H2 0.7 CO 27 which is an important quality for a potential pesticide. CH4 2.8 The Colorado Potato Beetle was also found to be negatively CO2 69.5 affected by the presence of the tobacco bio-oil. Early tests confirmed high mortality rates for the CPB, and further experimentssuch as coffee grounds and pinewood, pyrolyzed in the same were performed to investigate one of the key pyrolysis parameters:pilot plant at the same temperature. the pyrolysis temperature. The higher liquid yield at lower residence time can be at- 3.2.2. Investigation into the Effect of Pyrolysis Tempera-tributed to the fact that lower residence time minimizes ture on Pesticide Activity. Bio-oil produced at each pyrolysissecondary reactions19 such as thermal cracking, repolymeriza- temperature successfully inhibited the growth of each of thetion, and recondensation to maximize liquid yields. It is also three microorganisms (Figure 4).very well-known that higher temperature favors gasification As the pyrolysis temperature increased to 550 °C, the activity(higher gas yields and lower liquid and char yields). Thus, the of the bio-oil seemed to decrease. This could be due to the activeresults obtained are consistent with the existing literature on components being cracked into smaller, inactive componentsvarious other biomass feedstocks.20 at this high temperature. At 450 °C, the greatest inhibition was 3.2. Bio-oil Activity toward Pest Species. 3.2.1. Initial observed for all three species. For this reason, as well as thePesticide Discovery. Initial tests with tobacco bio-oil demon- fact that this temperature was close to 500 °C (the pyrolysisstrated clear pesticide activity toward a selection of microorgan- temperature with the greatest percent yield of bio-oil), the bio-ism species and the Colorado Potato Beetle. oil pyrolized at 450 °C was selected for continued investigation. To determine which microorganism species were inhibited It is important to note that, although these bio-oil samples wereby the tobacco bio-oil, naturally separated organic (375 mg/ prepared to a specific concentration, the observed variations inmL organic phase in acetone) and aqueous (used directly without the activity with pyrolysis temperature could be affected by thedilution) phase mixtures from all pyrolysis temperatures (350-550 amount of water in each bio-oil sample. The water was not°C) were assayed against 11 fungi and 4 bacteria (Table S1). removed from the sample to avoid removing other, potentiallyThese species were selected for analysis because of their important chemicals in the process. Nevertheless, each bio-oildestructive properties toward agriculture in Canada and were sample was found to successfully inhibit the growth of eachavailable for testing through Agriculture and Agri-Food Canada. species.No inhibition was found from the aqueous phases of the tobacco Similar to the microorganism pattern of inhibition, the CPBbio-oil. In contrast, the organic phases of the tobacco bio-oil was found to be strongly affected by bio-oil produced at allshowed clear inhibition for two bacteria, Streptomyces scabies pyrolysis temperatures (Figure 5). The potency of each bio-oil(S. scabies) and ClaVibacter michiganensis sub. sp. michigan- was quite strong given the high mortality levels seen. The 48 hensis (C. michiganensis), and one fungus, Pythium ultimum (P. results show that 100% of the beetles tested at each pyrolysisultimum). temperature died when in the presence of the tobacco bio-oil. Pythium ultimum is a fungus that affects plants as a seedling Although the 24 h results seem to demonstrate some changesdamping-off disease.21 Plants affected include eggplant, pepper, in toxicity with pyrolysis temperature, these changes are onlylettuce, tomato, and cucumber. ClaVibacter michiganensis kills minor.young plants and deforms fruits, primarily tomatoes.22 Strep- It was possible that the toxicity effect of the bio-oils towardtomyces scabies is a common potato scab disease that infects the CPB was caused solely by the high quantities of nicotinepotatoes and makes them unmarketable.23 Finding inhibition for in the bio-oil. Nicotine is a moderately effective insecticideS. scabies is particularly exciting because, currently, no safe against the CPB with an LD50 of 61 µg per CPB.24 Sufficientpesticide exists on the market that can control this widespread quantities of nicotine could be present in the bio-oil to accountdisease. for the observed activity. Thus, the bio-oil was separated into This discovery of tobacco bio-oil affecting only three mi- nicotine-free and nicotine-containing fractions to determine thecroorganism species (and not the remaining 12) is particularly effect of nicotine in the observed activity.
  11. 11. 10078 Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010Figure 5. Effect of pyrolysis temperature on the adjusted percent mortalityof the Colorado Potato Beetle at 24 and 48 h. Figure 7. Dilution tests comparing the aqueous (nicotine-free) fraction and the organic (nicotine containing) fraction prepared as illustrated in Figure 2. Results for both fractions were recorded at 24 and 48 h. point than 280 °C (the highest temperature in our GC program) or cannot be detected by an electron impact MS detector. A nicotine-free fraction was also found to be active in the CPB assays. The organic fraction showed greater activity over the aqueous (nicotine-free) fraction. After 24 h of testing, the organic fraction obtained 100% mortality rates, while after 48 h of testing, the aqueous fraction obtained a maximum of 80% mortality for the CPB (Figure 7). It is also worth noting that, although the aqueous phase did not result in 100% mortality to the CPB, application of the aqueous phase to the leaf resulted in a greatly reduced appetite for the beetle. Using the aqueous phase at 2% concentration or higher, the beetles would eat littleFigure 6. Measured diameters of inhibition for three microorganisms bythe six tobacco bio-oil fractions (see Figure 1 for fractionation scheme) to none of the leaf. Studies have shown that 24 h starvation ofafter 3 days of growth. Fraction C is nicotine-free. Error bars indicate ( the CPB does not prove fatal; however, starvation does causestandard deviation (σ) of replicate measurements within an experiment (total increased susceptibility to applied insecticides.28 Whether or notlength 2σ). the chemical agent that causes mortality is the same as the agent that is causing starvation is not known, but the starvation is 3.2.3. Investigation into the Activity of the Nicotine-Free aiding the insecticidal activity of the aqueous fraction.Fractions of Tobacco Bio-oil. The fractionation scheme shown Further investigation into the nicotine content of the organicin Figure 1 was used to generate the six fractions tested on the fraction was performed. Nicotine standards were tested at thethree microorganisms, as shown in Figure 6. As expected, concentration found in the organic fraction. Dilution tests ofFraction Z (the initial fraction) had high activity toward the the organic phase and the equivalent nicotine standard demon-microorganisms. However, high levels of nicotine were also strated that the potency of the samples was the same whenfound in Fraction Z (Figure S1), so much so that few other measured at 48 h. However, the 24 h results demonstrated thatchemicals could be observed in the chromatograms of this the organic fraction worked faster at causing death in the CPBfraction. than the nicotine standards. This indicates that additional, non- The fractionation scheme successfully generated a nicotine- nicotine components are acting in the organic fraction, Fraction C, which was confirmed by the absence The assays performed on the CPB and the three microorgan-of a nicotine peak in the GC-MS data. This fraction was also isms clearly indicate that tobacco bio-oil contains potent, non-strongly active (as shown in Figure 6). Phenol and a variety of nicotine components with insecticidal and antibiotic activity.its derivatives were found to be in high concentration in this Multiple, active components must be present in the tobacco bio-fraction. Although phenolic compounds are known to have oil as liquid-liquid extraction produced multiple, active frac-pesticide properties,25,26 10 of the most abundant compounds tions. Some of these active compounds cannot be detected byin this fraction were quantitatively tested by chemical standards, GC-MS.and it was found that these most abundant phenolic compoundswere not present in high enough concentrations to be responsible 4. Conclusionsfor the observed activity.27 Fraction D, which contains nicotine, was also found to be Pyrolysis experiments demonstrated that the liquid bio-oilactive. However, when nicotine standards were tested to match yield was a strong function of temperature and vapor residenceand even double the concentration of nicotine found in Fraction time. The maximum bio-oil yield was found at a reactor tem-D, no inhibition was observed. It is interesting to note that perature of 500 °C and the lowest residence time, 5 s.nicotine is the most abundant and almost the only peak detected Bio-oil was found to have valuable pesticide characteris-by GC-MS in this fraction. Therefore, the active components tics toward three problematic microorganisms as well as thein Fraction D cannot be detected by our GC-MS analysis Colorado Potato Beetle, a major agricultural pest. Bio-oil pro-method. These active components either have a higher boiling duced at all pyrolysis temperatures was effective at inhibiting
  12. 12. Ind. Eng. Chem. Res., Vol. 49, No. 20, 2010 10079the growth or causing mortality in the microorganisms and (8) Shen, J. C.; Shao, X. G. Determination of tobacco alkaloids by gasColorado Potato Beetle, respectively. chromatography-mass spectrometry using cloud point extraction as a preconcentration step. Anal. Chim. Acta 2006, 561, 83–87. Nicotine was found to be active toward the Colorado Potato (9) Cai, J. B.; Liu, B. Z.; Lin, P.; Su, Q. D. Fast analysis of nicotineBeetle, but had no effect on the microorganisms. Nicotine-free related alkaloids in tobacco and cigarette smoke by megabore capillary gasfractions of tobacco bio-oil were found to be active toward the chromatography. J. Chromatogr., A 2003, 1017, 187–193.Colorado Potato Beetle and three microorganisms. Multiple (10) Encinar, J. M.; Beltran, F. J.; Gonzalez, J. F.; Moreno, M. J.components are likely responsible for this activity. These Pyrolysis of maize, sunflower, grape and tobacco residues. J. Chem. Technol. Biotechnol. 1997, 70, 400–410.components were not lethal to all of the microorganisms that (11) Demirbas, A. Analysis of liquid products from biomass via flashwere examined, demonstrating that these chemicals may only pyrolysis. Energy Sources 2002, 24, 337– toxic to selective species, which is a desirable quality in a (12) Mourant, D.; Riedl, B.; Rodrigue, D.; Yang, D. Q.; Roy, C. Phenol-potential pesticide. formaldehyde-pyrolytic oil resins for wood preservation: A rheological study. J. Appl. Polym. Sci. 2007, 106, 1087–1094. As the demand for tobacco is decreasing, the search for other (13) Mohan, D.; Shi, J.; Nicholas, D. D.; Pittman, C. U.; Steele, P. H.;valuable products from this resource is increasing. A natural Cooper, J. E. Fungicidal values of bio-oils and their lignin-rich fractionspesticide that targets problematic species is a very valuable find. obtained from wood/bark fast pyrolysis. Chemosphere 2008, 71, 456–465.Further investigation into the active components and the poten- (14) Xu, R.; Ferrante, L.; Briens, C.; Berruti, F. Flash pyrolysis of grapetial applicability of using tobacco bio-oil as a natural pesticide residues into biofuel in a bubbling fluid bed. J. Anal. Appl. Pyrolysis 2009, 86, 58–65.will continue. (15) Oasmaa, A.; Kuoppala, E. Fast pyrolysis of forestry residue. 3. Storage stability of liquid fuel. Energy Fuels 2003, 17, 1075–1084. (16) Sheen, S. J. Detection of nicotine in foods and plant materials. J.Acknowledgment Food Sci. 1988, 53, 1572–1573. (17) Shin, H. S.; Kim, J. G.; Shin, Y. J.; Jee, S. H. Sensitive and simple We wish to express our gratitude to the Ontario Centres of method for the determination of nicotine and cotinine in human urine, plasmaExcellence (OCE), the Natural Sciences and Research Council and saliva by gas chromatography-mass spectrometry. J. Chromatogr., Bof Canada (NSERC), Agri-Therm Canada, the Institute for 2002, 769, 177–183.Chemicals and Fuels from Alternative Resources (ICFAR), (18) Sengonca, C.; Liu, B.; Zhu, Y. J. Pestic. Sci. 2005, 79, 3–8. (19) Gercel, H. F.; Putun, E. Fast pyrolysis of sunflower-pressed bagasse:Agriculture and Agri-Food Canada (through ABIN), and the Effects of sweeping gas flow rate. Energy Sources 2002, 24, 451–460.University of Western Ontario for their generous support of this (20) Bridgwater, A. V. Biomass fast pyrolysis. Therm. Sci. 2004, 8, 21–research. 49. (21) Abbasi, P. A.; Lazarovits, G. Effects of AG3 phosphonate formula- Supporting Information Available: Additional figures and tions on incidence and severity of Pythium damping-off of cucumbertable. This material is available free of charge via the Internet seedlings under growth room, microplot, and field conditions. Can. J. Plant Pathol. 2005, 27, 420– (22) Gleason, M. L.; Braun, E. J.; Carlton, W. M.; Peterson, R. H. Survival and dissemination of ClaVibacter michiganensis sub. sp. michagen- ensis in tomatoes. Phytopathology 1991, 81, 1519–1523.Literature Cited (23) Wang, A. X.; Lazarovits, G. Role of seed tubers in the spread of plant pathogenic Streptomyces and initiating potato common scab disease. (1) Bridgewater, A. V.; Czernik, S.; Piskorz, J. An overview of fast Am. J. Potato Res. 2005, 82, 221–230.pyrolysis. In Progress in Thermochemical Biomass ConVersion; Bridge- (24) Mota-Sanchez, D.; Hollingworth, R. M.; Grafius, E. J.; Moyer, D. D.water, A. V., Ed.; Blackwell Science Ltd.: Oxford, 2001; pp 977-997. Resistance and cross-resistance to neonicotinoid insecticides and spinosad (2) Zhang, Q.; Chang, J.; Wang, T. J.; Xu, Y. Review of biomass in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera:pyrolysis oil properties and upgrading research. Energy ConVers. Manage. Chrysomelidae). Pest Manage. Sci. 2006, 62, 30–37.2007, 48, 87–92. (25) EI DuPont de Numours Co. Cresols, ortho-, meta-, and para-. (3) Czernik, S.; Bridgewater, A. V. Overview of applications of biomass NTIS report no. OTS0205862; EI DuPont de Numours Co., pyrolysis oil. Energy Fuels 2004, 18, 590–598. (26) Anonymous. Final report on the safety assessment of sodium (4) Joensuu, J. J.; Brown, K. D.; Conley, A. J.; Clavijo, A.; Menassa, p-chloro-m-cresol, p-chloro-m-cresol, chlorothymol, mixed cresols, m-cresol,R.; Brandle, J. E. Expression and purification of an anti-Foot-and-mouth o-cresol, p-cresol, isopropyl cresols, thymol, o-cymen-5-ol, and carvacrol.disease virus single chain variable antibody fragment in tobacco plants. Int. J. Toxicol. 2006, 25, 29–127.Transgenic Res. 2009, 18, 685–696. (27) Booker, C. J.; Bedmutha, R.; Scott, I. M.; Conn, K.; Berruti, F.; (5) Menassa, R.; Du, C. G.; Yin, Z. Q.; Ma, S. W.; Poussier, P.; Brandle, Briens, C.; Yeung, K. K.-C. Bioenergy II: Characterization of the pesticideJ.; Jevnikar, A. M. Therapeutic effectiveness of orally administered properties of tobacco bio-oil. Int. J. Chem. Reactor Eng. 2010, 8, Art. 26.transgenic low-alkaloid tobacco expressing human interleukin-10 in a mouse (28) MacQuarrie, C. J. K.; Boiteau, G. Effect of diet and feeding historymodel of colitis. Plant Biotechnol. J. 2007, 5, 50–59. on flight of Colorado potato beetle, Leptinotarsa decemlineata. Entomol. (6) Ma, S. W.; Huang, Y.; Davis, A.; Yin, Z. Q.; Mi, Q. S.; Menassa, Exp. Appl. 2003, 107, 207–213.R.; Brandle, J. E.; Jevnikar, A. M. Production of biologically active humaninterleukin-4 in transgenic tobacco and potato. Plant Biotechnol. J. 2005, ReceiVed for reView February 11, 20103, 309–318. ReVised manuscript receiVed August 15, 2010 (7) Sheng, L. Q.; Ding, L.; Tong, H. W.; Yong, G. P.; Zhou, X. Z.; Accepted September 1, 2010Liu, S. M. Determination of nicotine-related alkaloids in tobacco andcigarette smoke by GC-FID. Chromatographia 2005, 62, 63–68. IE100329Z