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Fermentation of levoglucosan with oleaginous yeasts for lipid production
Bioresource Technology 133 (2013) 183–189
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Fermentation of levoglucosan with oleaginous yeasts for lipid production Jieni Lian, Manuel Garcia-Perez, Shulin Chen ⇑ Biological Systems Engineering, Washington State University, WA 99164, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
of levoglucosan to lipid was proposed. " Oleaginous yeasts Rhodotorula glutinis and Rhodosporidium toruloides showed best growth in LG. " Enzyme activities of LG kinase from R. glutinis and R. toruloides were determined. " LG aqueous phase by three detoxification methods was fermented by R. glutinis.
Lignocellulosic material
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 8 September 2012 Received in revised form 4 January 2013 Accepted 6 January 2013 Available online 22 January 2013 Keyword: Levoglucosan Oleaginous yeast Levoglucosan kinase Lipid fermentation Pyroligneous water
Pyrolysis Reactor (400-500 oC)
Condenser
" A new process of directly conversion
Yeast fermentation reactor
LGK
LG Mg2+ ADP ATP
Levoglucosan
Glucose-6phosphate
Lipids
Bio-char
Bio oil
Lipids
This paper reports the production of lipids from non-hydrolyzed levoglucosan (LG) by oleaginous yeasts Rhodosporidium toruloides and Rhodotorula glutinis. Enzyme activity tests of LG kinases from both yeasts indicated that the phosphorylation pathway of LG to glucose-6-phosphate existed. The highest enzyme activity obtained for R. glutinis was 0.22 U/mg of protein. The highest cell mass and lipid production by R. glutinis were 6.8 and 2.7 g/L, respectively from pure LG, and 3.3 and 0.78 g/L from a pyrolytic LG aqueous phase detoxified by ethyl acetate extraction, rotary evaporation and activated carbon. This corresponded to a lipid yield of 13.5 wt.% for pure LG and only 3.9 wt.% for LG in pyrolysis oil. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Bio-oil obtained from the pyrolysis of lignocellulosic materials is a promising feedstock for partially displacing petroleum crude usage (Zhang et al., 2007). Pyrolysis oil is a complex liquid formed by hundreds of compounds (Zhou et al., 2012; Garcia-Perez et al., 2007, 2008). The separation and utilization of different fractions is a major challenge for the refining of bio-oil (de Miguel Mercader et al., 2012). For example, hydrotreatment is a utilization strategy studied to produce transportation fuels from bio-oil (de Miguel Mercader et al., 2012; Zhang et al., 2007). The anhydrosugars pres⇑ Corresponding author. Address: Biological Systems Engineering Department, Washington State University, Pullman, WA 99164-6120, USA. E-mail address: [email protected] (S. Chen). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.031
ent in the oil are responsible for intensive coking, greater hydrogen consumption and catalyst deactivation in the hydrotreatment step (de Miguel Mercader et al., 2012), which prevents easy refining of bio-oil into useful products. Among the anhydrosugars in the biooil, levoglucosan (LG) is a major component produced from the pyrolysis of cellulose. Existing fast pyrolysis technologies typically convert around 20 mass% of the cellulose mass in lignocellulosic materials into LG. The rest is transformed into heavier molecules and charcoal with lower economic value. Conversion of cellulose to pyrolytic sugars can be improved, as proven by sugar yield as high as 59 mass% reported by other research groups (Shafizadeh et al., 1979; Scott, 1989). The separation and fermentation of LG is a promising approach to enhance the economic viability of bio-oil refineries (Garcia-Perez et al., 2010; Jarboe et al., 2011; Lian et al., 2010).
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The use of LG as a fermentative carbon and energy source in the fermentation industry has been studied for many years (Shafizadeh et al., 1979; Scott, 1989; Jarboe et al., 2011). The process to convert LG into ethanol or lipids (Lian et al., 2010) consists of a hydrolysis step to produce glucose from LG followed by the detoxification and fermentation of glucose into the products (Lian et al., 2010; Shafizadeh and Stevenson, 1982). LG hydrolysis could be carried out using acid catalysts in solution (Helle et al., 2007) or solid acid catalysts (Olson and Freel, 2007). After hydrolysis, the aqueous sugar rich phase needs to be neutralized prior to fermentation. Consequently, this step generates a waste stream. Ideally, if LG were directly utilized in fermentation, the process could improve reagent consumption efficiency and minimize waste production. Although many strains of bacteria, yeasts and fungi (Kitamura et al., 1991; Prosen et al., 1993) can use LG as a carbon source, the industrial utilization of these microorganisms is still in its infancy. Literature concerning LG fermentation is very scarce (Xie et al., 2006; Nakagawa et al., 1984; Zhuang et al., 2001). Only a few studies on direct LG conversion to valuable products in eukaryotic microorganisms have been reported. Yasui et al. (1991) found six colonies from soil, mostly coryneform bacteria, able to assimilate LG completely. Xie et al. (2006) also isolated some LG-assimilating fungi and yeasts (Alternaria alternata, Eupenicillium javanicum, Aspergillus niger, Penicillium herquei, Cryptococcus laurentii, Cryptococcus flavescens, Cryptococcus luteolus and R. aurantiaca) from soil. The diversity of LG-assimilating strains isolated offers a good platform to explore the use of these microorganisms to produce value-added products by fermentation. Itaconic acid was produced from pure LG by Aspergillus terreus K26 (Nakagawa et al., 1984) with the same yield and at the same rate as produced from glucose. Citric acid was produced from pure LG by A. niger CBX-2 with a very low conversion ratio (<10%) but an c-ray mutant of A. niger CBX-2 achieved a significantly higher conversion ratio (87%) (Zhuang et al., 2001). Some species of yeasts, such as Schwanniomyces castelli Labatt 1402, Saccharomyces diastaticus Labatt 1363, Candida utilis CMI 23311 (Prosen et al., 1993) produced ethanol from partly purified LG with a very low yield, about 10% as compared with 48% with glucose. The large number of microorganisms identified clearly shows that yeasts and fungi have large potential as LG fermentation candidates. However, very little is known about microorganisms with the capacity to directly utilize LG to valuable products for biofuel purposes. In yeasts and filamentous fungi, LG can be directly converted by phosphorylation to glucose-6-phosphate (G6P) (one-step) by a specific levoglucosan kinase (LGK) (Kitamura et al., 1991; Zhuang and Zhang, 2002). A novel cDNA of a LG kinase gene (lgk) from the yeast Lipomyces starkeyi YZ-215 was first identified by cloning and expression in Escherichia coli BL21 (Dai et al., 2009). In a recent study, an engineered ethanologenic E. coli KO11 with expression of the same lgk gene with modified codon bias was able to convert LG into ethanol (Layton et al., 2011). As many studies have demonstrated that L. starkeyi is a good candidate for lipid production (Gong et al., 2012), the study for an oleaginous yeast pathway to convert LG to lipid is quite feasible and should be explored. The 1,6-anhydro bond of LG is cleaved and simultaneously phosphorylated by LGK in the presence of Mg2+ and ATP. The G6P is then oxidized to produce acetyl-CoA, which is a precursor of fatty acid synthesis when the accessory acetyl-CoA accumulated. One-step direct bioconversion of LG in yeast is advantageous because it avoids intermediate product feedback inhibition in metabolic pathway. Microbial oil, a high energy density of long-chain fatty acid triglyceride mixture, has been investigated worldwide for the production of drop-in fuels and bio-diesel. Previous studies for the production of lipids (Chen et al., 2012; Huang et al., 2012; Yu et al., 2011) were conducted with lignocellulosic hydrolysates from
corncob and wheat straw. However, direct fermentation of LG (a product of thermochemical conversion) to lipid by oleaginous yeast is an unexplored but promising approach. This paper focuses on the identification of oleaginous yeasts that are able to directly ferment LG into lipids. The potential use of these strains for the conversion of anhydrosugars to lipid in a bio-oil refinery is explored. 2. Methods 2.1. Strain selection and medium Strains: Oleaginous yeasts were selected as promising candidates for LG utilization. Five well-known oleaginous yeasts with a high lipid yield were used for strain selection on LG mineral medium agar plates: L. starkeyi ATCC12659, Cryptococcus curvatus ATCC20509, Yarrowia lipolytica ATCC20460, Rhodosporidium toruloides ATCC10788 and Rhodotorula glutinis ATCC204091 (Ratledge and Cohen, 2008). R. toruloides and R. glutinis showed significantly better growth than the other three yeasts on agar plates using a LG mineral medium. Based on these results, R. toruloides and R. glutinis were selected for further LG fermentation studies for lipid production. Medium: The strains were mixed with 15 mass% glycerol and kept at 80 °C. YPD medium was used as the seed culture, and had a composition of 20 g/L glucose, 10 g/L yeast extract and 20 g/L peptone. LG mineral agar plates for selection contained 20 g/L LG (Carbosynth) and 10 g/L bacteria agar (without glucose or other carbon source) (Sigma–Aldrich) and additional mineral compositions of (NH4)2SO4 (1.5 g/L); KH2PO4 (2.4 g/L); NaH2PO412H2O (2.3 g/L); MgSO47H2O (1.5 g/L); FeSO47H2O (0.024 g/L) (Sigma–Aldrich). The agar plates for strain culture were stored at 30 °C for 5 days. 2.2. Separation and analysis of levoglucosan kinase (LGK) Yeasts were cultured in LG mineral medium for 96 h. Cells were collected from each yeast culture at 12, 24, 48, 72 and 96 h and washed with deionized water. The cells were resuspended in 2 mL bead beating tubes and lysed by glass bead-beating with mini bead beater (Cole-parmer). The crude extract containing LGK was separated by centrifugation at 4000 rpm. LGK activity for both yeasts was assessed by measuring NADPH (the reduced form of Nicotinamide adenine dinucleotide phosphate) formation with G6P dehydrogenase coupling system spectrophotometrically. The LGK present in the crude enzyme extract was measured by changes in NADPH concentration, determined by UV absorption at 340 nm. One unit of LGK activity was defined as the amount of enzyme which catalyzed the formation of 1 lmol of NADPH per minute at 30 °C. Enzyme assays were carried out with a reaction buffer of 50 mM Tris–HCl buffer (pH 9.0) and a reaction mixture containing 75 mM LG, 2 mM ATP, 0.2 mM NADP+, 10 mM MgCl2 and 1 U G6P dehydrogenase. The content of NADPH was quantified by a UV–Vis spectrophotometer UV-2550 (SHIMADZU Corporation). Total soluble protein concentration in the crude extract was analyzed by the Bradford method using bovine serum albumin (BSA) as standard. 2.3. LG fermentation with R. toruloides and R. glutinis The medium contained 20 g/L LG, 3 g/L yeast extract (about 10 mass% nitrogen) and the following mineral composition: KH2PO4 (2.4 g/L); NaH2PO412H2O (2.3 g/L); MgSO47H2O (1.5 g/ L); FeSO47H2O (0.024 g/L) (Sigma–Aldrich). The C:N ratio used for pure LG to lipid fermentation was 27 as nitrogen limited
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control. As a control, a medium containing glucose instead of LG was used with the same mineral composition. 10% v/v seed culture medium was pre-cultured and inoculated into the LG medium. The culture was standardized for 5 days by flask fermentation at 30 °C and 150 rpm. The cell mass and lipid production were analyzed at different fermentation times. Dry cell mass and lipid analysis: Samples of yeast cells collected at different times were washed twice with deionized water and oven-dried in a pre-weighed aluminum dish at 105 °C until a stable weight was achieved. To analyze the fatty acid profile, samples of the harvested yeast cells were freeze-dried overnight until constant weight. The lipids were transesterified to fatty acid methyl ester (FAME), then analyzed and quantified by gas chromatography (GC) detection (O’fallon et al., 2007). Briefly, the fatty acids in the yeast cell were released by alkaline cell lysis by addition of 10 N KOH. Under basic conditions, the lipids were transesterified by adding MeOH. Following transesterification, the FAME solution was neutralized using H2SO4. Hexane and water were added to the neutralized solution for FAME extraction. The hexane phase, rich in FAME, was injected in a GC with an FID (Hewlett Packard 3396 Series II integrator and 7673 controller with flame ionization detector) and a SP-2560 column (Supelco, Bellefonte) with split injection. The calibration curves for all the fatty acids analyzed (myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), gamma-linolenic (C18:3), linolenic (C18:3), arachidic (C20:0), behenic (C22:0) and lignoceric (C24:0)) were linear in the range of concentrations detected. 2.4. Bio-oil production and characterization Bio-oil production from Douglas fir: A continuous pyrolysis reactor operating at a throughput capacity between 1 and 2 kg/h of dry biomass (described elsewhere (Liaw et al., 2011)) was used to produce the oil studied in this paper. Briefly, the biomass was fed by a volumetric single screw stirring feeder, DSR28 from Brabender Technology. The reactor consisted of a 100 mm diameter stainless-steel tube placed in a Lindberg/Blue M (model HTF55322A) furnace with an auger driven by a 1 hp motor (maximum speed: 1725 rpm, 10.9 A). The pyrolysis vapors were evacuated from the reactor using 1 L/min of nitrogen as a carrier gas. A water-cooled jacket was installed between the hopper and the furnace to prevent heating the biomass in the hopper. The residence time of the biomass inside the reactor could be controlled by varying the speed of the auger via a manual controller. The charcoal was collected in a stainless-steel container located downstream. A vertical condenser followed by a series of ice-cooled traps was used to condense the pyrolysis vapors. The pressure inside the reactor was maintained a few millimeters of water below atmospheric pressure using a vacuum pump. The flow of nitrogen to the reactor was measured and controlled with two rotameters (one measuring the flow of nitrogen to the hopper and the other to the auger reactor). Although the reactor could be operated continuously, the capacity of the current charcoal pot limited the operational duration of the system to only one hour. A total of 300 g of charcoal could be produced per run. Bio-oil characterization: The volatile compounds of the bio-oil were identified and quantified using an Agilent 6890N GC coupled with an Agilent Technologies Inert XL Mass Spectrometry Detector (Agilent HP-5 MS) with a capillary column (HP19091S-433). Model compounds (hydroxyacetaldehyde, acetic acid, acetol, propionic acid, toluene, cyclopentanone, 2-furaldehyde, furfuryl alcohol, oxylene, furanone, phenol, o-cresol, eugenol, vanillin, syringaldehyde (chemicals above purchased from Sigma–Aldrich) and LG,) were used as standards. Calibration curves were obtained and used to calculate bio-oil chemical compositions following the procedure described elsewhere (Liaw et al., 2011).
Sugar analysis by IEC: The hydrophobic compounds of bio-oil were precipitated in cold water and the resulting aqueous phase was hydrolyzed under acid conditions. The sugar contents of the bio-oil were analyzed and quantified by ion exchange chromatography (IEC). The bio-oil was diluted 100 times by adding cold deionized water (4 °C) which precipitated the lignin-derived oligomers and produced an aqueous phase rich in sugars. (Garcia-Perez et al., 2008). The aqueous phase rich in sugars was hydrolyzed with 500 mM sulfuric acid as catalyst at 130 °C for 1 h. LG was completely converted to glucose during the hydrolysis process. The solution was neutralized using NaOH and the content of sugars quantified by IEC (a Dionex ICS-3000 system equipped with an AS50 auto-Sampler, GP50 gradient pump and ED50 electrochemical detector). Model sugars (LG, fucose, arabinose, galactose, glucose, mannose, xylose, fructose and ribose) were used for standard curve calibration. The separation of sugars was performed with a Carbopac PA20 column. The mobile phase was an aqueous NaOH solution at a flow rate of 0.50 mL/min. Deionized water was used to prepare the mobile phase. 10 mM sodium hydroxide storage solution was added to the postcolumn to maintain pH 10.4 in the detector. The injection volume of the sample was 10 lL and the column temperature was a constant (35 °C). The calibration curves for the sugars analyzed were linear in the range of concentrations studied. 2.5. Lipid fermentation Fig. 1 shows the scheme used for the conversion of LG contained in the bio-oil to lipids. A strategy for separation and detoxification of LG phase was tested first. The production of cell mass and lipids by R. toruloides and R. glutinis fermentation was investigated using three LG aqueous phases obtained from bio-oil by three different detoxification treatments.
Douglas fir Pyrolysis Reactor
Bio-oil Condenser
Gases
Hot water extraction
Precipitation in 4 C
NaOH
Lignin oligmers
Neutralization Ethyl acetate Extraction
Rotary Evaporation Activated carbon
Phenolics
Volatile inhibitors (Acetol)
Detoxification
Filtration
Activated carbon
Fermentation
Lipid Fig. 1. Scheme of the process used to ferment the LG present in bio-oil to produce lipids.
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LG separation from bio-oil was conducted in two steps: a hot water extraction step and a cold water separation step. LG in the bio-oil was first released by dilution with deionized water at a 1:1 volume ratio and stirred at 60 °C for an hour in a hot water bath. At this temperature the hydrogen bonds between lignin structures present in the bio-oil break and bound LG is released into the aqueous phase (Garcia-Perez et al., 2008). After mixing, the solution was stored at 4 °C overnight to precipitate non-polar lignin oligmeric material in the suspension. The aqueous phase was neutralized to pH 7 with sodium hydroxide. However, the color of the aqueous phase obtained remained red-brown, indicating the presence of phenolic and furanic inhibitors. GC–MS analysis conducted for the samples from this phase showed the presence of these inhibitors (data not shown in this paper). Thus an additional detoxification step was needed to remove the inhibitors and improve yeast growth and lipid accumulation. Several detoxification strategies were studied. These included ethyl acetate extraction (EE) for phenolic inhibitors, rotary evaporation (RE) for volatile inhibitors and activated carbon absorption (AA) for inhibitor residues. The strategies tested include three combinations as followed (a) AA, (b) RE/AA and (c) EE/RE/AA. The resulting slurries were stored overnight at 4 °C and the suspensions rich in sugar were achieved by filtration. Three detoxified aqueous phase were diluted to a final concentration of 20 g/L LG based on IEC measurement and mixed with yeast extract (3 g/L) and the same mineral composition as that of the medium of pure LG to lipid fermentation. Cell mass production was measured as a function of fermentation time. The lipid accumulation of the yeast was also analyzed after a culture at 5 days. 3. Results and discussion Both R. toruloides and R. glutinis showed much better growth by the third day than other three yeasts. C. curvatus, L. starkeyi and Y. lipolytica showed limited growth on the plate after a 10-days culture. Based on the growth rate observed, R. toruloides and R. glutinis were selected as candidates for levoglucosan fermentation study. 3.1. Analysis of levoglucosan kinase (LGK) activity The enzyme activities of LGK of R. glutinis and R. toruloides at different culture times are shown in Fig. 2. The LGK enzyme activity suggests that LG can be phosphorylated to G6P in both yeasts by LGK in the presence of Mg2+ and ATP. The G6P could be further oxidized to acetyl CoA as the precursor for lipid synthesis. Although the G6P pathway seems to be viable, other pathways cannot be ruled out. Eukaryotic microorganisms have a different LG metabolic pathway than prokaryotic microorganisms. In Arthrobacter bacteria, LG was transformed into glucose through three enzymatic reactions (Nakahara et al., 1994). But in yeasts and filamentous fungi, LG was reported to prefer directly conversion to G6P (one-step) by LGK (Kitamura et al., 1991). During fermentation in a 20 g/L LG medium, the LGK enzyme activity of both yeasts similarly increased during the first 24 h, followed by a slight decrease. The highest enzyme activity of R. glutinis (0.22 U/mg of protein) was obtained at 24 h. It was statistically (T-test) higher than that found for R. toruloides (0.17 U/mg of protein) at the same time. The LGK enzyme activity obtained was similar to that of other yeasts reported: R. aurantiaca (0.26 U/mg of protein), C. laurentii (0.32 U/mg of protein), C. flavescens (0.30 U/ mg of protein), C. luteolus (0.25 U/mg of protein) (Xie et al., 2006) and Cryptococcu albidus (0.31 U/mg of protein) (Kitamura et al., 1991). The LGK reported is similar to glucokinase based on the high Km (low substrate affinity), high substrate specificity (not able to catalyze glucose, galactose, mannose, even mannosan or
Levoglucosan kinase activity of R. glutinis and R. toruloides 0.3 Enzyme activity (U/mg protein)
186
0.25
R. glutinis R. toruloides
0.2 0.15 0.1 0.05 0 0
20
40
60 Time (hour)
80
100
120
Fig. 2. Enzyme analysis of levoglucosan kinase of oleaginous yeast R. glutinis and R. toruloides.
galactosan (as same intramolecular linkage as LG)) and negligible G6P product feedback inhibition at high concentrations (10 mM), which is in contrast to hexokinases I, II, and III. These similarities of LGK to glucoskinase indicate the huge potential of LG utilization as carbon and energy source. 3.2. Levoglucosan fermentation Fig. 3 shows the cell mass and lipid production measured for R. glutinis and R. toruloides when using pure LG (20 g/L) and glucose (20 g/L) as carbon sources. Yeast cell mass and lipid accumulation increased with the culture time. The highest cell mass achieved with R. glutinis was 6.8 g/L with LG culture and 8.1 g/L with glucose culture, while the highest lipid accumulation was 2.7 g/L with LG and 2.9 g/L with glucose. The highest cell mass achieved with R.
Glucose and LG fermentation with R. glutinis 10 9 8 7 6 5 4 3 2 1 0
Dry cell mass in LG medium (g/L) Lipid in LG medium (g/L) Dry cell mass in glucose medium (g/L) Lipid in glucose medium (g/L)
0
24
48
72 Time (hour)
96
120
144
Glucose and LG fermentation with R. toruloides
10
Dry cell mass in LG medium (g/L) Lipid in LG medium (g/L) Dry cell mass in glucose medium (g/L) Lipid in glucose medium (g/L)
9 8 7 6 5 4 3 2 1 0 0
24
48
72 Time (hour)
96
120
144
Fig. 3. Levoglucosan and glucose fermentation with oleaginous yeast R. glutinis and R. toruloides.
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toruloides was 5.8 g/L with LG culture and 6.5 g/L with glucose culture. The highest lipid accumulation achieved when using R. toruloides was 2.0 g/L with LG and 2.4 g/L with glucose. The dry cell mass and lipid accumulation with glucose was higher than with levoglucosan. However, levoglucosan could be directly used as a carbon source by the oleaginous yeasts resulting in a reasonable growth rate and level of lipid accumulation. Comparison of the cell mass and lipid accumulation during fermentation of both sugars indicated that LG is a comparable carbon source to glucose for both yeasts. The lipids could be further refined to produce biodiesel through a simple transesterification process. . The lipid yield from R. glutinis (13.5 mass%) was relatively low compared with the theoretical yield (33 mass%), it was higher than the lipid yield (about 10 mass%) from R. toruloides Y4 obtained by Li et al. (2007) with glucose in a flask batch culture. Given the previous study results of 106.5 g/L dry cell mass and 51.1 g/L lipid achieved with glucose fermentation (Li et al., 2007), the fed-batch culture model could be considered a promising method to improve the cell mass and lipid production. The biomass and lipid accumulation during LG and glucose fermentation indicates that LG is a comparable carbon source to glucose for both yeasts studied. However, the LG transport limitation is unknown and may lower LG utilization. Direct utilization of LG as a carbon source by the oleaginous yeasts is a novel technique that can be applied for the production of value added products and to develop the refinery concept for pyrolysate. Comparing the cell mass and lipid accumulation of both yeasts, R. glutinis was a better LG consuming strain than R. toruloides. The fatty acid profiles of R. glutinis and R. toruloides obtained on a dry cell basis are presented in Table 1. The total fatty acid content of R. glutinis was 42.2 mass% for LG culture and 44.8 mass% when cultured in glucose. The total fatty acid content for R. toruloides cultured in LG was 35.6 and 38.1 mass% when grown in glucose. These results demonstrate that LG could be used to support lipid accumulation in yeast cell nearly as well as glucose. Although there were similarities in the fatty acid profiles produced using LG and glucose for both yeasts studied, the content of total saturated fatty acids (palmitic and stearic acids) decreased and the content of total unsaturated fatty acids (oleic, linoleic, and linolenic) increased when LG was used as the carbon source instead of glucose. The triglycerides produced by R. glutinis and R. toruloides are composed
primarily of long-chain fatty acids with 16 and 18 carbon atoms. The major fatty acids in the profile of both yeasts studied were palmitic (C16:0), stearic (C18:0) and oleic (C18:1). The content of saturated fatty acids was 39.1 mass%, the content of unsaturated fatty acids was 60.6 mass%. The fatty acid profile was similar to vegetable oils (10–45 mass% saturated fatty acids and 55–90 mass% unsaturated fatty acids) for bio-diesel production (Ramos et al., 2009). Although the unsaturated fatty acids (low cetane numbers and high iodine values) are known to be responsible for the poor oxidation stability of bio-diesel, the unsaturated methyl and ethyl esters could improve the cold flow properties of biodiesel (Ramos et al., 2009) 3.3. Bio-oil characterization Table 2 shows the identification and the quantification of the compounds in the oil analyzed by GC/MS, Karl Fischer Titration and IEC. The compounds quantified were accounted for 57.8 mass% of oils. The remaining fraction was composed of very light compounds (chiefly hydroxyl acetaldehyde, acetol, acetic acid) or heavy oligomers (anhydrosugars and lignin derived oligomers), comparable to previous research results reported (Jarboe et al., 2011; Hoekstra et al., 2011; Zhang et al., 2007). The most important compounds quantified were water (26.7 mass%), glycoaldehyde (1.4 mass%), acetic acid (2.6 mass%), acetol (2.6 mass%), phenol, 2-methoxy- (4.0 mass%), phenol, 2-methoxy4-(1-propenyl)-, (E)- (1.3 mass%), and LG (7.7 mass%) (see results in Table 2). The content of all chemical compounds quantified in the bio-oil studied were similar compared to other fast pyrolysis oils reported in the literature (Zhang et al., 2007; Liaw et al., 2012; Hoekstra et al., 2011). The large amount inhibitors, including phenolics, glycoaldehyde and acetol (Lian et al., 2012), showed the importance and necessity of detoxification to remove the inhibition to yeast fermentation. The glucose detected was derived mainly from the hydrolysis of LG (the content of glucose in these oils (7.7 mass%) by IEC was very close to the content of LG quantified by GC/MS). Other sugars, derived from hemicelluloses (fucose, arabinose, galactose, mannose/xylose, fructose, ribose), accounted for only 2 mass% of this oil. Therefore, LG was considered to be the main anhydrosugar carbon source in the oil to be used for fermentation. The concentration of LG (7.7 mass% in GC/MS) was
Table 1 Fatty acid profiles of R. glutinis and R. toruloides obtained at with levoglucosan (LG) and glucose medium. R. glutinis
R. toruloides
LG
Glucose
LG
Glucose
Mass% FA 0.4 24.3 0.2 0.2 10.1 53.2 6.8 1 0.1 0.3 0.7 2.4
Mass% FA 0.5 18.1 1.2 0.1 24.4 44.6 5.6 0.7 0.9 1.6 0.6 1.7
Mass% FA 0.2 25.1 0.8 0.1 9.2 50.8 7.2 1.1 0.7 0.4 0.7 3.5
Mass% FA 0.6 19.7 0.9 0.4 17.3 43.5 9.8 0.6 0.2 1.2 0.8 4.7
Saturated MUFA PUFA Omega-3 Omega-6 Identified Unknown
39.1 53.4 7.2 0.3 6.9 99.7 0.3
46.1 45.8 8.1 1.6 6.5 100 0
39.9 51.6 8.3 0.4 7.9 99.8 0.2
44.1 44.4 11.2 1.2 10 99.7 0.3
Total fat
Mass% in dry cell 42.2
Mass% in dry cell 44.8
Mass% in dry cell 35.6
Mass% in dry cell 38.1
Fatty acid Myristic Palmitic Palmitoleic Heptadecanoic Stearic Oleic Linoleic Gamma-Linolenic Linolenic Arachidic Behenic Lignoceric
Structure C14:0 C16:0 C16:1n7 C17:0 C18:0 C18:1n9 C18:2n6 C18:3n6 C18:3n3 C20:0 C22:0 C24:0
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Table 2 Characterization of bio-oil from Douglas fir by GC–MS analysis and the content of hydrolysable sugars by IEC.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Water Glycoaldehyde Acetic acid Acetol Propionic acid Toluene Cyclopentanone 2-Furaldehyde Furfuryl alcohol 2(5H)-Furanone 2-Furanethanol, b-methoxy-(S)Phenol o-Cresol Phenol, 4-ethylPyrocatechol Benzene, 1-methyl-2-(1-methylethyl)Phenol, 2-methoxyPhenol, 2-methoxy-4-methylPhenol, 4-ethyl-2-methoxyEugenol Vanilin 1,2-Benzenediol, 4-methylPhenol, 2-methoxy-4-propylPhenol, 2-methoxy-4-(1-propenyl)-, (E)1,6-Anhydro-b-D-glucose (levoglucosan) (by GC–MS) Fucose (after hydrolysis) Arabinose (after hydrolysis) Galactose (after hydrolysis) Mannose/xylose (after hydrolysis) Fructose (after hydrolysis) Ribose Total
Concentration (mass%) 26.7 1.4 4.8 2.6 0.3 0.6 0.5 0.4 0.5 0.6 0.5 0.3 0.1 0.1 0.6 0.1 4.0 0.6 0.2 0.2 0.9 0.1 0.7 1.3 7.6 0.1 0.2 0.2 1.1 0.3 0.1 57.8
sufficient for fermentation after some dilution under the experiment design.
3.4. Detoxification of levoglucosan rich phase and fermentation After detoxifying the aqueous phase with only activated carbon, R. glutinis did not show growth. The evolution of the dry R. glutinis cell mass by fermenting LG from detoxified aqueous phases is shown in Fig. 4. The yeast dry cell mass results showed significant difference that clearly indicated the effect of inhibitors on microbial growth and lipid accumulation. Ethyl acetate extraction was used first to separate phenolics in the aqueous phase. The ethyl acetate remaining in the aqueous phase, as well as the C1–C4 small volatile molecules (chiefly, acetol, hydroxyacetaldehyde) were removed with the aid of a rotary evaporator operated with a water bath at 80 °C. Under these conditions most of the toxic organic compounds such as acetol (boiling point at 145 °C), hydroxyacetaldehyde (boiling point at 132 °C) and furfural (boiling point at 161.7 °C) were removed from the aqueous phase. Activated carbon was used to absorb the furanics, mono-phenols and lignin oligomeric compounds left in the aqueous phase. When using a single detoxification step with activated carbon the maximum cell mass achieved was very low, at 0.74 g/L. The combination with rotary evaporation increased the cell mass to 1.6 g/L. The addition of an ethyl acetate extraction step further increased cell mass to 3.3 g/L. Compared with the highest cell mass produced of 6.8 g/L and the highest lipid of 2.7 g/L in R. glutinis with pure LG medium, it was clear that yeast growth was affected by inhibitors present in bio-oil. The combination of detoxification methods resulted in higher yeast lipid production (0.1 g/L only using activated carbon, 0.24 g/L with rotary evaporation-activated
a
b
c
3.5 Dry cell mass (g/L)
Compound
4
3 2.5 2 1.5 1 0.5 0 0
24
48
72 Time (hour)
96
120
144
Fig. 4. The cell mass analysis with culture time in medium after 5 days culture of oleaginous yeast R. glutinis in the levoglucosan fermentation of three bio-oil aqueous phases detoxified with three different methods: (a) detoxification with activated carbon, (b) detoxification with rotary evaporation and activated carbon, (c) detoxification with ethyl acetate extraction, rotary evaporation and activated carbon.
carbon and 0.78 g/L with ethyl acetate extraction- rotary evaporation-activated carbon). The inhibitors also affected lipid accumulation and production in the yeast cells. The main inhibitors in bio-oils are likely the phenolic compounds, acetol and hydroxyacetaldehyde (Lian et al., 2012). Phenolic compounds, such as syringaldehyde and vanillin, also showed inhibition on growth and lipid accumulation of oleaginous yeast Trichosporon fermentans (Hu et al., 2009; Huang et al., 2011). The inhibition observed is similar to inhibition of citric acid production by the c-ray mutant strain A. niger CBX-209 when using pyrolytic LG after activated carbon detoxification, yielding less than 10% compared with a yield of 87.5% using pure LG (Zhuang et al., 2001). The results suggest the possibility exists for the conversion of LG to renewable hydrocarbons via the production of lipids as an intermediate. To the best of our knowledge this was the first study on the use of LG by oleaginous yeasts for the production of lipids, which showed LG as a possible carbon source for biofuel production and improved the concept of biological conversion of pyrolytic anhydrosugars. The energy consumption and chemical waste generation of traditional anhydrosugar hydrolysis processes could also be avoided if the oleaginous yeasts studied would be used. Our results suggested that LG separation, detoxification and fermentation were the important steps during this process. Detoxification methods for inhibitors in pyrolytic oil and metabolic evolution for robust strains require further investigation. 4. Conclusion This study demonstrated that LG can be used directly as a carbon source for lipids production by oleaginous yeasts R. glutinis and R. toruloides. It also showed that LGK in these microorganisms functions in LG phosphorylation to G6P. Oleaginous yeasts could be good candidates for pyrolytic sugar utilization with high conversion yield of LG to lipid production (similar to that of glucose). The utilization of anhydrosugars is critical to ensure the economic viability of biofuel production from the pyrolysis of lignocellulosic materials because of the significant concentration of LG in the pyrolysis oil. Acknowledgements This project was financially supported by the Sun-Grant Initiative (Interagency Agreement: T0013G-A and C04432GB) and to the Washington State Agricultural Research Center. The authors are very thankful for their support.
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Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Fermentation of levoglucosan with oleaginous yeasts for lipid production Jieni Lian, Manuel Garcia-Perez, Shulin Chen ⇑ Biological Systems Engineering, Washington State University, WA 99164, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
of levoglucosan to lipid was proposed. " Oleaginous yeasts Rhodotorula glutinis and Rhodosporidium toruloides showed best growth in LG. " Enzyme activities of LG kinase from R. glutinis and R. toruloides were determined. " LG aqueous phase by three detoxification methods was fermented by R. glutinis.
Lignocellulosic material
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 8 September 2012 Received in revised form 4 January 2013 Accepted 6 January 2013 Available online 22 January 2013 Keyword: Levoglucosan Oleaginous yeast Levoglucosan kinase Lipid fermentation Pyroligneous water
Pyrolysis Reactor (400-500 oC)
Condenser
" A new process of directly conversion
Yeast fermentation reactor
LGK
LG Mg2+ ADP ATP
Levoglucosan
Glucose-6phosphate
Lipids
Bio-char
Bio oil
Lipids
This paper reports the production of lipids from non-hydrolyzed levoglucosan (LG) by oleaginous yeasts Rhodosporidium toruloides and Rhodotorula glutinis. Enzyme activity tests of LG kinases from both yeasts indicated that the phosphorylation pathway of LG to glucose-6-phosphate existed. The highest enzyme activity obtained for R. glutinis was 0.22 U/mg of protein. The highest cell mass and lipid production by R. glutinis were 6.8 and 2.7 g/L, respectively from pure LG, and 3.3 and 0.78 g/L from a pyrolytic LG aqueous phase detoxified by ethyl acetate extraction, rotary evaporation and activated carbon. This corresponded to a lipid yield of 13.5 wt.% for pure LG and only 3.9 wt.% for LG in pyrolysis oil. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Bio-oil obtained from the pyrolysis of lignocellulosic materials is a promising feedstock for partially displacing petroleum crude usage (Zhang et al., 2007). Pyrolysis oil is a complex liquid formed by hundreds of compounds (Zhou et al., 2012; Garcia-Perez et al., 2007, 2008). The separation and utilization of different fractions is a major challenge for the refining of bio-oil (de Miguel Mercader et al., 2012). For example, hydrotreatment is a utilization strategy studied to produce transportation fuels from bio-oil (de Miguel Mercader et al., 2012; Zhang et al., 2007). The anhydrosugars pres⇑ Corresponding author. Address: Biological Systems Engineering Department, Washington State University, Pullman, WA 99164-6120, USA. E-mail address: [email protected] (S. Chen). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.031
ent in the oil are responsible for intensive coking, greater hydrogen consumption and catalyst deactivation in the hydrotreatment step (de Miguel Mercader et al., 2012), which prevents easy refining of bio-oil into useful products. Among the anhydrosugars in the biooil, levoglucosan (LG) is a major component produced from the pyrolysis of cellulose. Existing fast pyrolysis technologies typically convert around 20 mass% of the cellulose mass in lignocellulosic materials into LG. The rest is transformed into heavier molecules and charcoal with lower economic value. Conversion of cellulose to pyrolytic sugars can be improved, as proven by sugar yield as high as 59 mass% reported by other research groups (Shafizadeh et al., 1979; Scott, 1989). The separation and fermentation of LG is a promising approach to enhance the economic viability of bio-oil refineries (Garcia-Perez et al., 2010; Jarboe et al., 2011; Lian et al., 2010).
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The use of LG as a fermentative carbon and energy source in the fermentation industry has been studied for many years (Shafizadeh et al., 1979; Scott, 1989; Jarboe et al., 2011). The process to convert LG into ethanol or lipids (Lian et al., 2010) consists of a hydrolysis step to produce glucose from LG followed by the detoxification and fermentation of glucose into the products (Lian et al., 2010; Shafizadeh and Stevenson, 1982). LG hydrolysis could be carried out using acid catalysts in solution (Helle et al., 2007) or solid acid catalysts (Olson and Freel, 2007). After hydrolysis, the aqueous sugar rich phase needs to be neutralized prior to fermentation. Consequently, this step generates a waste stream. Ideally, if LG were directly utilized in fermentation, the process could improve reagent consumption efficiency and minimize waste production. Although many strains of bacteria, yeasts and fungi (Kitamura et al., 1991; Prosen et al., 1993) can use LG as a carbon source, the industrial utilization of these microorganisms is still in its infancy. Literature concerning LG fermentation is very scarce (Xie et al., 2006; Nakagawa et al., 1984; Zhuang et al., 2001). Only a few studies on direct LG conversion to valuable products in eukaryotic microorganisms have been reported. Yasui et al. (1991) found six colonies from soil, mostly coryneform bacteria, able to assimilate LG completely. Xie et al. (2006) also isolated some LG-assimilating fungi and yeasts (Alternaria alternata, Eupenicillium javanicum, Aspergillus niger, Penicillium herquei, Cryptococcus laurentii, Cryptococcus flavescens, Cryptococcus luteolus and R. aurantiaca) from soil. The diversity of LG-assimilating strains isolated offers a good platform to explore the use of these microorganisms to produce value-added products by fermentation. Itaconic acid was produced from pure LG by Aspergillus terreus K26 (Nakagawa et al., 1984) with the same yield and at the same rate as produced from glucose. Citric acid was produced from pure LG by A. niger CBX-2 with a very low conversion ratio (<10%) but an c-ray mutant of A. niger CBX-2 achieved a significantly higher conversion ratio (87%) (Zhuang et al., 2001). Some species of yeasts, such as Schwanniomyces castelli Labatt 1402, Saccharomyces diastaticus Labatt 1363, Candida utilis CMI 23311 (Prosen et al., 1993) produced ethanol from partly purified LG with a very low yield, about 10% as compared with 48% with glucose. The large number of microorganisms identified clearly shows that yeasts and fungi have large potential as LG fermentation candidates. However, very little is known about microorganisms with the capacity to directly utilize LG to valuable products for biofuel purposes. In yeasts and filamentous fungi, LG can be directly converted by phosphorylation to glucose-6-phosphate (G6P) (one-step) by a specific levoglucosan kinase (LGK) (Kitamura et al., 1991; Zhuang and Zhang, 2002). A novel cDNA of a LG kinase gene (lgk) from the yeast Lipomyces starkeyi YZ-215 was first identified by cloning and expression in Escherichia coli BL21 (Dai et al., 2009). In a recent study, an engineered ethanologenic E. coli KO11 with expression of the same lgk gene with modified codon bias was able to convert LG into ethanol (Layton et al., 2011). As many studies have demonstrated that L. starkeyi is a good candidate for lipid production (Gong et al., 2012), the study for an oleaginous yeast pathway to convert LG to lipid is quite feasible and should be explored. The 1,6-anhydro bond of LG is cleaved and simultaneously phosphorylated by LGK in the presence of Mg2+ and ATP. The G6P is then oxidized to produce acetyl-CoA, which is a precursor of fatty acid synthesis when the accessory acetyl-CoA accumulated. One-step direct bioconversion of LG in yeast is advantageous because it avoids intermediate product feedback inhibition in metabolic pathway. Microbial oil, a high energy density of long-chain fatty acid triglyceride mixture, has been investigated worldwide for the production of drop-in fuels and bio-diesel. Previous studies for the production of lipids (Chen et al., 2012; Huang et al., 2012; Yu et al., 2011) were conducted with lignocellulosic hydrolysates from
corncob and wheat straw. However, direct fermentation of LG (a product of thermochemical conversion) to lipid by oleaginous yeast is an unexplored but promising approach. This paper focuses on the identification of oleaginous yeasts that are able to directly ferment LG into lipids. The potential use of these strains for the conversion of anhydrosugars to lipid in a bio-oil refinery is explored. 2. Methods 2.1. Strain selection and medium Strains: Oleaginous yeasts were selected as promising candidates for LG utilization. Five well-known oleaginous yeasts with a high lipid yield were used for strain selection on LG mineral medium agar plates: L. starkeyi ATCC12659, Cryptococcus curvatus ATCC20509, Yarrowia lipolytica ATCC20460, Rhodosporidium toruloides ATCC10788 and Rhodotorula glutinis ATCC204091 (Ratledge and Cohen, 2008). R. toruloides and R. glutinis showed significantly better growth than the other three yeasts on agar plates using a LG mineral medium. Based on these results, R. toruloides and R. glutinis were selected for further LG fermentation studies for lipid production. Medium: The strains were mixed with 15 mass% glycerol and kept at 80 °C. YPD medium was used as the seed culture, and had a composition of 20 g/L glucose, 10 g/L yeast extract and 20 g/L peptone. LG mineral agar plates for selection contained 20 g/L LG (Carbosynth) and 10 g/L bacteria agar (without glucose or other carbon source) (Sigma–Aldrich) and additional mineral compositions of (NH4)2SO4 (1.5 g/L); KH2PO4 (2.4 g/L); NaH2PO412H2O (2.3 g/L); MgSO47H2O (1.5 g/L); FeSO47H2O (0.024 g/L) (Sigma–Aldrich). The agar plates for strain culture were stored at 30 °C for 5 days. 2.2. Separation and analysis of levoglucosan kinase (LGK) Yeasts were cultured in LG mineral medium for 96 h. Cells were collected from each yeast culture at 12, 24, 48, 72 and 96 h and washed with deionized water. The cells were resuspended in 2 mL bead beating tubes and lysed by glass bead-beating with mini bead beater (Cole-parmer). The crude extract containing LGK was separated by centrifugation at 4000 rpm. LGK activity for both yeasts was assessed by measuring NADPH (the reduced form of Nicotinamide adenine dinucleotide phosphate) formation with G6P dehydrogenase coupling system spectrophotometrically. The LGK present in the crude enzyme extract was measured by changes in NADPH concentration, determined by UV absorption at 340 nm. One unit of LGK activity was defined as the amount of enzyme which catalyzed the formation of 1 lmol of NADPH per minute at 30 °C. Enzyme assays were carried out with a reaction buffer of 50 mM Tris–HCl buffer (pH 9.0) and a reaction mixture containing 75 mM LG, 2 mM ATP, 0.2 mM NADP+, 10 mM MgCl2 and 1 U G6P dehydrogenase. The content of NADPH was quantified by a UV–Vis spectrophotometer UV-2550 (SHIMADZU Corporation). Total soluble protein concentration in the crude extract was analyzed by the Bradford method using bovine serum albumin (BSA) as standard. 2.3. LG fermentation with R. toruloides and R. glutinis The medium contained 20 g/L LG, 3 g/L yeast extract (about 10 mass% nitrogen) and the following mineral composition: KH2PO4 (2.4 g/L); NaH2PO412H2O (2.3 g/L); MgSO47H2O (1.5 g/ L); FeSO47H2O (0.024 g/L) (Sigma–Aldrich). The C:N ratio used for pure LG to lipid fermentation was 27 as nitrogen limited
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control. As a control, a medium containing glucose instead of LG was used with the same mineral composition. 10% v/v seed culture medium was pre-cultured and inoculated into the LG medium. The culture was standardized for 5 days by flask fermentation at 30 °C and 150 rpm. The cell mass and lipid production were analyzed at different fermentation times. Dry cell mass and lipid analysis: Samples of yeast cells collected at different times were washed twice with deionized water and oven-dried in a pre-weighed aluminum dish at 105 °C until a stable weight was achieved. To analyze the fatty acid profile, samples of the harvested yeast cells were freeze-dried overnight until constant weight. The lipids were transesterified to fatty acid methyl ester (FAME), then analyzed and quantified by gas chromatography (GC) detection (O’fallon et al., 2007). Briefly, the fatty acids in the yeast cell were released by alkaline cell lysis by addition of 10 N KOH. Under basic conditions, the lipids were transesterified by adding MeOH. Following transesterification, the FAME solution was neutralized using H2SO4. Hexane and water were added to the neutralized solution for FAME extraction. The hexane phase, rich in FAME, was injected in a GC with an FID (Hewlett Packard 3396 Series II integrator and 7673 controller with flame ionization detector) and a SP-2560 column (Supelco, Bellefonte) with split injection. The calibration curves for all the fatty acids analyzed (myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), gamma-linolenic (C18:3), linolenic (C18:3), arachidic (C20:0), behenic (C22:0) and lignoceric (C24:0)) were linear in the range of concentrations detected. 2.4. Bio-oil production and characterization Bio-oil production from Douglas fir: A continuous pyrolysis reactor operating at a throughput capacity between 1 and 2 kg/h of dry biomass (described elsewhere (Liaw et al., 2011)) was used to produce the oil studied in this paper. Briefly, the biomass was fed by a volumetric single screw stirring feeder, DSR28 from Brabender Technology. The reactor consisted of a 100 mm diameter stainless-steel tube placed in a Lindberg/Blue M (model HTF55322A) furnace with an auger driven by a 1 hp motor (maximum speed: 1725 rpm, 10.9 A). The pyrolysis vapors were evacuated from the reactor using 1 L/min of nitrogen as a carrier gas. A water-cooled jacket was installed between the hopper and the furnace to prevent heating the biomass in the hopper. The residence time of the biomass inside the reactor could be controlled by varying the speed of the auger via a manual controller. The charcoal was collected in a stainless-steel container located downstream. A vertical condenser followed by a series of ice-cooled traps was used to condense the pyrolysis vapors. The pressure inside the reactor was maintained a few millimeters of water below atmospheric pressure using a vacuum pump. The flow of nitrogen to the reactor was measured and controlled with two rotameters (one measuring the flow of nitrogen to the hopper and the other to the auger reactor). Although the reactor could be operated continuously, the capacity of the current charcoal pot limited the operational duration of the system to only one hour. A total of 300 g of charcoal could be produced per run. Bio-oil characterization: The volatile compounds of the bio-oil were identified and quantified using an Agilent 6890N GC coupled with an Agilent Technologies Inert XL Mass Spectrometry Detector (Agilent HP-5 MS) with a capillary column (HP19091S-433). Model compounds (hydroxyacetaldehyde, acetic acid, acetol, propionic acid, toluene, cyclopentanone, 2-furaldehyde, furfuryl alcohol, oxylene, furanone, phenol, o-cresol, eugenol, vanillin, syringaldehyde (chemicals above purchased from Sigma–Aldrich) and LG,) were used as standards. Calibration curves were obtained and used to calculate bio-oil chemical compositions following the procedure described elsewhere (Liaw et al., 2011).
Sugar analysis by IEC: The hydrophobic compounds of bio-oil were precipitated in cold water and the resulting aqueous phase was hydrolyzed under acid conditions. The sugar contents of the bio-oil were analyzed and quantified by ion exchange chromatography (IEC). The bio-oil was diluted 100 times by adding cold deionized water (4 °C) which precipitated the lignin-derived oligomers and produced an aqueous phase rich in sugars. (Garcia-Perez et al., 2008). The aqueous phase rich in sugars was hydrolyzed with 500 mM sulfuric acid as catalyst at 130 °C for 1 h. LG was completely converted to glucose during the hydrolysis process. The solution was neutralized using NaOH and the content of sugars quantified by IEC (a Dionex ICS-3000 system equipped with an AS50 auto-Sampler, GP50 gradient pump and ED50 electrochemical detector). Model sugars (LG, fucose, arabinose, galactose, glucose, mannose, xylose, fructose and ribose) were used for standard curve calibration. The separation of sugars was performed with a Carbopac PA20 column. The mobile phase was an aqueous NaOH solution at a flow rate of 0.50 mL/min. Deionized water was used to prepare the mobile phase. 10 mM sodium hydroxide storage solution was added to the postcolumn to maintain pH 10.4 in the detector. The injection volume of the sample was 10 lL and the column temperature was a constant (35 °C). The calibration curves for the sugars analyzed were linear in the range of concentrations studied. 2.5. Lipid fermentation Fig. 1 shows the scheme used for the conversion of LG contained in the bio-oil to lipids. A strategy for separation and detoxification of LG phase was tested first. The production of cell mass and lipids by R. toruloides and R. glutinis fermentation was investigated using three LG aqueous phases obtained from bio-oil by three different detoxification treatments.
Douglas fir Pyrolysis Reactor
Bio-oil Condenser
Gases
Hot water extraction
Precipitation in 4 C
NaOH
Lignin oligmers
Neutralization Ethyl acetate Extraction
Rotary Evaporation Activated carbon
Phenolics
Volatile inhibitors (Acetol)
Detoxification
Filtration
Activated carbon
Fermentation
Lipid Fig. 1. Scheme of the process used to ferment the LG present in bio-oil to produce lipids.
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LG separation from bio-oil was conducted in two steps: a hot water extraction step and a cold water separation step. LG in the bio-oil was first released by dilution with deionized water at a 1:1 volume ratio and stirred at 60 °C for an hour in a hot water bath. At this temperature the hydrogen bonds between lignin structures present in the bio-oil break and bound LG is released into the aqueous phase (Garcia-Perez et al., 2008). After mixing, the solution was stored at 4 °C overnight to precipitate non-polar lignin oligmeric material in the suspension. The aqueous phase was neutralized to pH 7 with sodium hydroxide. However, the color of the aqueous phase obtained remained red-brown, indicating the presence of phenolic and furanic inhibitors. GC–MS analysis conducted for the samples from this phase showed the presence of these inhibitors (data not shown in this paper). Thus an additional detoxification step was needed to remove the inhibitors and improve yeast growth and lipid accumulation. Several detoxification strategies were studied. These included ethyl acetate extraction (EE) for phenolic inhibitors, rotary evaporation (RE) for volatile inhibitors and activated carbon absorption (AA) for inhibitor residues. The strategies tested include three combinations as followed (a) AA, (b) RE/AA and (c) EE/RE/AA. The resulting slurries were stored overnight at 4 °C and the suspensions rich in sugar were achieved by filtration. Three detoxified aqueous phase were diluted to a final concentration of 20 g/L LG based on IEC measurement and mixed with yeast extract (3 g/L) and the same mineral composition as that of the medium of pure LG to lipid fermentation. Cell mass production was measured as a function of fermentation time. The lipid accumulation of the yeast was also analyzed after a culture at 5 days. 3. Results and discussion Both R. toruloides and R. glutinis showed much better growth by the third day than other three yeasts. C. curvatus, L. starkeyi and Y. lipolytica showed limited growth on the plate after a 10-days culture. Based on the growth rate observed, R. toruloides and R. glutinis were selected as candidates for levoglucosan fermentation study. 3.1. Analysis of levoglucosan kinase (LGK) activity The enzyme activities of LGK of R. glutinis and R. toruloides at different culture times are shown in Fig. 2. The LGK enzyme activity suggests that LG can be phosphorylated to G6P in both yeasts by LGK in the presence of Mg2+ and ATP. The G6P could be further oxidized to acetyl CoA as the precursor for lipid synthesis. Although the G6P pathway seems to be viable, other pathways cannot be ruled out. Eukaryotic microorganisms have a different LG metabolic pathway than prokaryotic microorganisms. In Arthrobacter bacteria, LG was transformed into glucose through three enzymatic reactions (Nakahara et al., 1994). But in yeasts and filamentous fungi, LG was reported to prefer directly conversion to G6P (one-step) by LGK (Kitamura et al., 1991). During fermentation in a 20 g/L LG medium, the LGK enzyme activity of both yeasts similarly increased during the first 24 h, followed by a slight decrease. The highest enzyme activity of R. glutinis (0.22 U/mg of protein) was obtained at 24 h. It was statistically (T-test) higher than that found for R. toruloides (0.17 U/mg of protein) at the same time. The LGK enzyme activity obtained was similar to that of other yeasts reported: R. aurantiaca (0.26 U/mg of protein), C. laurentii (0.32 U/mg of protein), C. flavescens (0.30 U/ mg of protein), C. luteolus (0.25 U/mg of protein) (Xie et al., 2006) and Cryptococcu albidus (0.31 U/mg of protein) (Kitamura et al., 1991). The LGK reported is similar to glucokinase based on the high Km (low substrate affinity), high substrate specificity (not able to catalyze glucose, galactose, mannose, even mannosan or
Levoglucosan kinase activity of R. glutinis and R. toruloides 0.3 Enzyme activity (U/mg protein)
186
0.25
R. glutinis R. toruloides
0.2 0.15 0.1 0.05 0 0
20
40
60 Time (hour)
80
100
120
Fig. 2. Enzyme analysis of levoglucosan kinase of oleaginous yeast R. glutinis and R. toruloides.
galactosan (as same intramolecular linkage as LG)) and negligible G6P product feedback inhibition at high concentrations (10 mM), which is in contrast to hexokinases I, II, and III. These similarities of LGK to glucoskinase indicate the huge potential of LG utilization as carbon and energy source. 3.2. Levoglucosan fermentation Fig. 3 shows the cell mass and lipid production measured for R. glutinis and R. toruloides when using pure LG (20 g/L) and glucose (20 g/L) as carbon sources. Yeast cell mass and lipid accumulation increased with the culture time. The highest cell mass achieved with R. glutinis was 6.8 g/L with LG culture and 8.1 g/L with glucose culture, while the highest lipid accumulation was 2.7 g/L with LG and 2.9 g/L with glucose. The highest cell mass achieved with R.
Glucose and LG fermentation with R. glutinis 10 9 8 7 6 5 4 3 2 1 0
Dry cell mass in LG medium (g/L) Lipid in LG medium (g/L) Dry cell mass in glucose medium (g/L) Lipid in glucose medium (g/L)
0
24
48
72 Time (hour)
96
120
144
Glucose and LG fermentation with R. toruloides
10
Dry cell mass in LG medium (g/L) Lipid in LG medium (g/L) Dry cell mass in glucose medium (g/L) Lipid in glucose medium (g/L)
9 8 7 6 5 4 3 2 1 0 0
24
48
72 Time (hour)
96
120
144
Fig. 3. Levoglucosan and glucose fermentation with oleaginous yeast R. glutinis and R. toruloides.
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toruloides was 5.8 g/L with LG culture and 6.5 g/L with glucose culture. The highest lipid accumulation achieved when using R. toruloides was 2.0 g/L with LG and 2.4 g/L with glucose. The dry cell mass and lipid accumulation with glucose was higher than with levoglucosan. However, levoglucosan could be directly used as a carbon source by the oleaginous yeasts resulting in a reasonable growth rate and level of lipid accumulation. Comparison of the cell mass and lipid accumulation during fermentation of both sugars indicated that LG is a comparable carbon source to glucose for both yeasts. The lipids could be further refined to produce biodiesel through a simple transesterification process. . The lipid yield from R. glutinis (13.5 mass%) was relatively low compared with the theoretical yield (33 mass%), it was higher than the lipid yield (about 10 mass%) from R. toruloides Y4 obtained by Li et al. (2007) with glucose in a flask batch culture. Given the previous study results of 106.5 g/L dry cell mass and 51.1 g/L lipid achieved with glucose fermentation (Li et al., 2007), the fed-batch culture model could be considered a promising method to improve the cell mass and lipid production. The biomass and lipid accumulation during LG and glucose fermentation indicates that LG is a comparable carbon source to glucose for both yeasts studied. However, the LG transport limitation is unknown and may lower LG utilization. Direct utilization of LG as a carbon source by the oleaginous yeasts is a novel technique that can be applied for the production of value added products and to develop the refinery concept for pyrolysate. Comparing the cell mass and lipid accumulation of both yeasts, R. glutinis was a better LG consuming strain than R. toruloides. The fatty acid profiles of R. glutinis and R. toruloides obtained on a dry cell basis are presented in Table 1. The total fatty acid content of R. glutinis was 42.2 mass% for LG culture and 44.8 mass% when cultured in glucose. The total fatty acid content for R. toruloides cultured in LG was 35.6 and 38.1 mass% when grown in glucose. These results demonstrate that LG could be used to support lipid accumulation in yeast cell nearly as well as glucose. Although there were similarities in the fatty acid profiles produced using LG and glucose for both yeasts studied, the content of total saturated fatty acids (palmitic and stearic acids) decreased and the content of total unsaturated fatty acids (oleic, linoleic, and linolenic) increased when LG was used as the carbon source instead of glucose. The triglycerides produced by R. glutinis and R. toruloides are composed
primarily of long-chain fatty acids with 16 and 18 carbon atoms. The major fatty acids in the profile of both yeasts studied were palmitic (C16:0), stearic (C18:0) and oleic (C18:1). The content of saturated fatty acids was 39.1 mass%, the content of unsaturated fatty acids was 60.6 mass%. The fatty acid profile was similar to vegetable oils (10–45 mass% saturated fatty acids and 55–90 mass% unsaturated fatty acids) for bio-diesel production (Ramos et al., 2009). Although the unsaturated fatty acids (low cetane numbers and high iodine values) are known to be responsible for the poor oxidation stability of bio-diesel, the unsaturated methyl and ethyl esters could improve the cold flow properties of biodiesel (Ramos et al., 2009) 3.3. Bio-oil characterization Table 2 shows the identification and the quantification of the compounds in the oil analyzed by GC/MS, Karl Fischer Titration and IEC. The compounds quantified were accounted for 57.8 mass% of oils. The remaining fraction was composed of very light compounds (chiefly hydroxyl acetaldehyde, acetol, acetic acid) or heavy oligomers (anhydrosugars and lignin derived oligomers), comparable to previous research results reported (Jarboe et al., 2011; Hoekstra et al., 2011; Zhang et al., 2007). The most important compounds quantified were water (26.7 mass%), glycoaldehyde (1.4 mass%), acetic acid (2.6 mass%), acetol (2.6 mass%), phenol, 2-methoxy- (4.0 mass%), phenol, 2-methoxy4-(1-propenyl)-, (E)- (1.3 mass%), and LG (7.7 mass%) (see results in Table 2). The content of all chemical compounds quantified in the bio-oil studied were similar compared to other fast pyrolysis oils reported in the literature (Zhang et al., 2007; Liaw et al., 2012; Hoekstra et al., 2011). The large amount inhibitors, including phenolics, glycoaldehyde and acetol (Lian et al., 2012), showed the importance and necessity of detoxification to remove the inhibition to yeast fermentation. The glucose detected was derived mainly from the hydrolysis of LG (the content of glucose in these oils (7.7 mass%) by IEC was very close to the content of LG quantified by GC/MS). Other sugars, derived from hemicelluloses (fucose, arabinose, galactose, mannose/xylose, fructose, ribose), accounted for only 2 mass% of this oil. Therefore, LG was considered to be the main anhydrosugar carbon source in the oil to be used for fermentation. The concentration of LG (7.7 mass% in GC/MS) was
Table 1 Fatty acid profiles of R. glutinis and R. toruloides obtained at with levoglucosan (LG) and glucose medium. R. glutinis
R. toruloides
LG
Glucose
LG
Glucose
Mass% FA 0.4 24.3 0.2 0.2 10.1 53.2 6.8 1 0.1 0.3 0.7 2.4
Mass% FA 0.5 18.1 1.2 0.1 24.4 44.6 5.6 0.7 0.9 1.6 0.6 1.7
Mass% FA 0.2 25.1 0.8 0.1 9.2 50.8 7.2 1.1 0.7 0.4 0.7 3.5
Mass% FA 0.6 19.7 0.9 0.4 17.3 43.5 9.8 0.6 0.2 1.2 0.8 4.7
Saturated MUFA PUFA Omega-3 Omega-6 Identified Unknown
39.1 53.4 7.2 0.3 6.9 99.7 0.3
46.1 45.8 8.1 1.6 6.5 100 0
39.9 51.6 8.3 0.4 7.9 99.8 0.2
44.1 44.4 11.2 1.2 10 99.7 0.3
Total fat
Mass% in dry cell 42.2
Mass% in dry cell 44.8
Mass% in dry cell 35.6
Mass% in dry cell 38.1
Fatty acid Myristic Palmitic Palmitoleic Heptadecanoic Stearic Oleic Linoleic Gamma-Linolenic Linolenic Arachidic Behenic Lignoceric
Structure C14:0 C16:0 C16:1n7 C17:0 C18:0 C18:1n9 C18:2n6 C18:3n6 C18:3n3 C20:0 C22:0 C24:0
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Table 2 Characterization of bio-oil from Douglas fir by GC–MS analysis and the content of hydrolysable sugars by IEC.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Water Glycoaldehyde Acetic acid Acetol Propionic acid Toluene Cyclopentanone 2-Furaldehyde Furfuryl alcohol 2(5H)-Furanone 2-Furanethanol, b-methoxy-(S)Phenol o-Cresol Phenol, 4-ethylPyrocatechol Benzene, 1-methyl-2-(1-methylethyl)Phenol, 2-methoxyPhenol, 2-methoxy-4-methylPhenol, 4-ethyl-2-methoxyEugenol Vanilin 1,2-Benzenediol, 4-methylPhenol, 2-methoxy-4-propylPhenol, 2-methoxy-4-(1-propenyl)-, (E)1,6-Anhydro-b-D-glucose (levoglucosan) (by GC–MS) Fucose (after hydrolysis) Arabinose (after hydrolysis) Galactose (after hydrolysis) Mannose/xylose (after hydrolysis) Fructose (after hydrolysis) Ribose Total
Concentration (mass%) 26.7 1.4 4.8 2.6 0.3 0.6 0.5 0.4 0.5 0.6 0.5 0.3 0.1 0.1 0.6 0.1 4.0 0.6 0.2 0.2 0.9 0.1 0.7 1.3 7.6 0.1 0.2 0.2 1.1 0.3 0.1 57.8
sufficient for fermentation after some dilution under the experiment design.
3.4. Detoxification of levoglucosan rich phase and fermentation After detoxifying the aqueous phase with only activated carbon, R. glutinis did not show growth. The evolution of the dry R. glutinis cell mass by fermenting LG from detoxified aqueous phases is shown in Fig. 4. The yeast dry cell mass results showed significant difference that clearly indicated the effect of inhibitors on microbial growth and lipid accumulation. Ethyl acetate extraction was used first to separate phenolics in the aqueous phase. The ethyl acetate remaining in the aqueous phase, as well as the C1–C4 small volatile molecules (chiefly, acetol, hydroxyacetaldehyde) were removed with the aid of a rotary evaporator operated with a water bath at 80 °C. Under these conditions most of the toxic organic compounds such as acetol (boiling point at 145 °C), hydroxyacetaldehyde (boiling point at 132 °C) and furfural (boiling point at 161.7 °C) were removed from the aqueous phase. Activated carbon was used to absorb the furanics, mono-phenols and lignin oligomeric compounds left in the aqueous phase. When using a single detoxification step with activated carbon the maximum cell mass achieved was very low, at 0.74 g/L. The combination with rotary evaporation increased the cell mass to 1.6 g/L. The addition of an ethyl acetate extraction step further increased cell mass to 3.3 g/L. Compared with the highest cell mass produced of 6.8 g/L and the highest lipid of 2.7 g/L in R. glutinis with pure LG medium, it was clear that yeast growth was affected by inhibitors present in bio-oil. The combination of detoxification methods resulted in higher yeast lipid production (0.1 g/L only using activated carbon, 0.24 g/L with rotary evaporation-activated
a
b
c
3.5 Dry cell mass (g/L)
Compound
4
3 2.5 2 1.5 1 0.5 0 0
24
48
72 Time (hour)
96
120
144
Fig. 4. The cell mass analysis with culture time in medium after 5 days culture of oleaginous yeast R. glutinis in the levoglucosan fermentation of three bio-oil aqueous phases detoxified with three different methods: (a) detoxification with activated carbon, (b) detoxification with rotary evaporation and activated carbon, (c) detoxification with ethyl acetate extraction, rotary evaporation and activated carbon.
carbon and 0.78 g/L with ethyl acetate extraction- rotary evaporation-activated carbon). The inhibitors also affected lipid accumulation and production in the yeast cells. The main inhibitors in bio-oils are likely the phenolic compounds, acetol and hydroxyacetaldehyde (Lian et al., 2012). Phenolic compounds, such as syringaldehyde and vanillin, also showed inhibition on growth and lipid accumulation of oleaginous yeast Trichosporon fermentans (Hu et al., 2009; Huang et al., 2011). The inhibition observed is similar to inhibition of citric acid production by the c-ray mutant strain A. niger CBX-209 when using pyrolytic LG after activated carbon detoxification, yielding less than 10% compared with a yield of 87.5% using pure LG (Zhuang et al., 2001). The results suggest the possibility exists for the conversion of LG to renewable hydrocarbons via the production of lipids as an intermediate. To the best of our knowledge this was the first study on the use of LG by oleaginous yeasts for the production of lipids, which showed LG as a possible carbon source for biofuel production and improved the concept of biological conversion of pyrolytic anhydrosugars. The energy consumption and chemical waste generation of traditional anhydrosugar hydrolysis processes could also be avoided if the oleaginous yeasts studied would be used. Our results suggested that LG separation, detoxification and fermentation were the important steps during this process. Detoxification methods for inhibitors in pyrolytic oil and metabolic evolution for robust strains require further investigation. 4. Conclusion This study demonstrated that LG can be used directly as a carbon source for lipids production by oleaginous yeasts R. glutinis and R. toruloides. It also showed that LGK in these microorganisms functions in LG phosphorylation to G6P. Oleaginous yeasts could be good candidates for pyrolytic sugar utilization with high conversion yield of LG to lipid production (similar to that of glucose). The utilization of anhydrosugars is critical to ensure the economic viability of biofuel production from the pyrolysis of lignocellulosic materials because of the significant concentration of LG in the pyrolysis oil. Acknowledgements This project was financially supported by the Sun-Grant Initiative (Interagency Agreement: T0013G-A and C04432GB) and to the Washington State Agricultural Research Center. The authors are very thankful for their support.
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