Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw
© Zheng et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 2 July 2012
Published: 23 July 2012
Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost.
The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm.
This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.
KeywordsFilamentous fungi Mortierella isabellina Microbial lipid Biodiesel Lignocellulosic biomass Wheat straw
Detoxified liquid hydrolysate
Non-detoxified liquid hydrolysate
Single cell oil
Fatty acid methyl ester
Dry cell weight
Polyunsaturated fatty acid
Energy Independence and Security Act
The traditional feedstocks for biodiesel production are vegetable oils and animal fats resulting in competition with the food industry. Single cell oil (SCO) from microbes is considered as an alternative oil source due to the high productivity and low land requirement . Among different oleaginous microorganisms, increasing attention has been paid to filamentous fungi due to multiple advantages: (1) Accumulate up to 80% of lipid and produce some value-added fatty acids . Aggelis  cultured Cunninghamella echinulata to achieve 46.6% of cellular lipid with a γ-linolenic acid (GLA) content of 14.1%. Moreover, it was demonstrated that the arachidonic acid (AA) content in Mortierella alpine was more than 16% of dry cell weight and the total lipid also reached 36% . (2) Show good lipid profiles for making high quality biodiesel. Vicente et al.  suggested that not all lipids extracted from microbes were suitable for biodiesel production but only saponifiable lipids and free fatty acids could be produced to fatty acid methyl esters (FAMEs). Their results showed that 98.0% of the total lipids extracted from Mucor circinelloides were saponifiable lipids and free fatty acids, and the fungus-derived biodiesel met the specifications of the current existing standards very well; (3) Use a variety of carbon sources for lipid production, such as monosugar, glycerol, acetic acid, cereal, corncob, sweet sorghum, wheat straw, orange peel, apple pomace and oil [3, 6–13]; (4) Produce oils through solid state fermentation with low capital cost and low energy expenditure ; (5) Tend to form pellets that not only reduce the viscosity of the fermentation broth to improve the mixing and mass transfer performance, but also are much easier to be harvested from cell broth by using simple filtration, compared with traditional high cost centrifugation methods .
Although SCO from filamentous fungi shows the promise for biodiesel production, the hurdle is the high production cost. It has been reported that up to 75% of the total costs came from the feedstocks or carbon sources required for producing microbial lipids . However, the cost will be reduced potentially if cheap feedstocks or waste materials can be used. Xue et al.  successfully grew the oleaginous yeast Rhodotorula glutinis with monosodium glutamate wastewater to produce 25 g L-1 biomass with 25% lipid content. André et al.  reported that the fungus Aspergillus niger could accumulate 41–57% of lipid on biodiesel derived waste glycerol. Moreover, food wastes have proven to be suitable substrates for production of lipid by yeast and microalgae [16, 17]. However, the availability of these sources is limited and not able to meet the increasing demand of alternative energy. It is very urgent, therefore, to investigate other renewable sources as feedstocks for microbial lipid production.
Lignocellulosic materials have attracted a lot of attention as feedstocks for biofuel production due to its abundance and relatively low cost. It was estimated that there would be potentially over 1.3 billion dry tons of lignocellulosic biomass produced in the US each year on a sustainable basis for biofuel production . The energy content of this amount of biomass is equivalent to 3.8 billion barrels of oil, which is approximately more than half of the US’s annual energy consumption . These inexpensive materials such as agricultural residues can result in a reasonable biofuel production cost . Some studies have been conducted to produce lipid from oleaginous yeast by feeding with lignocellulosic material. Huang et al.  obtained a cell density of 28.6 g L-1 with 40% lipid content by culturing the yeast Trichosporon fermentans with detoxified rice straw hydrolysate. Yu et al.  reported that the yeast Cryptococcus curvatus could grow with non-detoxified wheat straw hydrolysate and reach 17.2 g L-1 dry cell weight with 33.5% lipid content. However, cultivation of filamentous fungi for lipid production with lignocellulosic hydrolysate has not been well examined.
The purpose of this study is to investigate the feasibility of culturing the filamentous fungi with lignocellulosic materials and to screen the best lipid producing strain, especially using the non-detoxified hydrolysates. The very basic requirements for fungi to be used for this purpose are: (1) can use various sugars, especially xylose; (2) can adapt to the lignocellulosic biomass processing without extensively conditioning the sugar stream; (3) can accumulate high lipid contents while utilizing lignocellulosics as the carbon source; (4) can grow with proper morphology to facilitate downstream processing.To achieve these objectives, the lipid accumulation capability of eleven filamentous fungal strains was evaluated on glucose and xylose respectively. Then, the selected strains with high lipid contents were cultivated with hydrolysates from dilute sulfuric acid pretreated wheat straw. The biomass and lipid yields, fatty acid profiles, capability to tolerate inhibitors and pellet formation were studied. Finally, the fungal lipid based biodiesel yield and cost were estimated when lignocellulosic biomass was used as the feedstock.
Results and discussion
Screening oleaginous fungi with xylose assimilation capability
Fungal biomass and lipid production on glucose and xylose
As one of the most abundant carbohydrates in nature, xylose can be easily released from biomass by hydrolysis, which makes it a potential feedstock for biofuel production . However, compared with glucose (a more preferable substrate for most heterotrophic microbes), specific metabolic pathways are required for xylose utilization. Actually many microorganisms do not naturally use xylose as a substrate due to the lack of some key enzymes . For instance, the most commonly used yeast Saccharomyces cerevisiae for ethanol production cannot ferment xylose naturally, which limits its industrial application. Therefore, the capability to utilize xylose to accumulate lipids is a critical criterion for screening strains with industrial potential in biodiesel area. In this study, all the eleven fungi candidates showed satisfactory results on xylose assimilation and more than half of them exhibited comparable or even higher biomass production on xylose than on glucose. Particularly, A. terreus C. elegans M. isabellina M. vinacea R. oryzae and T. lanuginosus could accumulate more than 20% lipid on xylose (Table 1), which were selected for the following experiments utilizing wheat straw hydrolysate as the substrate.
Chemical compositions of hydrolysates
Culture oleaginous fungi with NDLH and DLH
Culture of the selected fungal strains with NDLH and DLH
Fatty acid compositions of selected lipid producing fungal strains grown on NDLH and DLH
Relative abundance of the total fatty acids (%, w/w)
PUFA (> = 4 double bonds)
Iodine valueb (g of I2/100 g)
Viscosity (mm2 s-1)b
Density (kg m-3)b
Higher heating value (MJ kg-1)b
Fungal pelletization when feeding with NDLH
It was important that the oleaginous fungi formed pellets when fed with NDLH for three reasons: (1) the pelletization could improve the mixing and mass transfer caused by viscosity, and was preferred in the microbial lipid production because of its easier harvesting compared with the traditional centrifugation ; (2) the gypsum produced during dilute acid pretreatment of lignocellulosics was considered to negatively affect the downstream ethanol production process, but it could be used as nuclei for the formation of fungal pellets to benefit the lipid production and reduce the cost for the addition of other nuclei or polymer ; (3) the fermentation process could be further simplified since the filtration of NDLH was not necessary.
The perspective of fungal lipid-based biodiesel production from lignocellulosic biomass
Estimated biodiesel yield from fungal lipids grown with wheat straw
Lipid yield (kg ton-1 glucose)
Lipid yield (kg ton-1 xylose)
Lipid yield (kg ton-1 wheat straw)b
Biodiesel yield (gal ton-1 glucose)
Biodiesel yield (gal ton-1 xylose)
Biodiesel yield (gal ton-1 wheat straw)c
Current biodiesel yield in US (billion gal y-1)d
Potential biodiesel yield in US (billion gal y-1)d
From the economical assessment aspect, carbon source attributes up to 75% of the total cost for producing biodiesel from SCO . By using commercial raw sugar as feedstock, with an average price at about $852 ton-1 (duty fee paid) based on the data of IntercontinentalExchange (ICE, http://www.theice.com) in 2011, the fungal lipid based biodiesel production cost is $20.8 gal-1 (based on biodiesel yield on glucose in this study, Table 4). However, 70% of this cost will be cut if lignocellulosic biomass is used as the feedstock. According to the latest estimations released from the National Renewable Energy Laboratory , the selling price for lignocellulosic biomass derived sugar is only $257 ton-1 (including the costs of feedstock, handling, pretreatment, enzymatic hydrolysis, waste treatment, fixed cost, capital depreciation and the associated tax), which will result in the biodiesel production cost of $6.3 gal-1. Based on the theoretical yield (on glucose) in Table 4, the biodiesel manufacturing cost can be potentially reduced to $3.8 gal-1 from lignocellulosic biomass, however, there is still a disparity with the US DOE’s target for renewable diesel of $2.8 per gallon by 2017 . Therefore, it is very necessary to make further technical improvements for economical application not only on the fermentation but also on other processes, such as harvesting, extraction, transesterification, high value co-product production, etc.
This is the first report to investigate the capabilities of filamentous fungi for lipid production with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. All of the selected oleaginous fungi could grow on the hydrolysates with or without detoxification. Wherein, three fungal strains, including A. terreus, M. isabellina and M. vinacea, showed the highest tolerance to the inhibitors existing in the hydrolysate. The highest lipid content of 39.4% was achieved by M. isabellina on NDLH. In addition, the filamentous fungi could form proper pellets to benefit the downstream harvesting process when cultured on NDLH. Overall, cultivation of filamentous oleaginous fungi with lignocellulosic biomass showed great promise for biodiesel production.
Dilute sulfuric acid pretreatment of wheat straw
Wheat straw was obtained from Pullman, WA. The milled wheat straw was mixed with 2% (v/v) dilute sulfuric acid at a solid loading of 10% (w/v) and pretreated in an autoclave at 121°C for 60 min. After cooling, the liquid hydrolysate was separated by centrifugation. Calcium hydroxide was used to adjust the pH to 5.5. After 10-min settling, the supernatant was prepared as NDLH. And then the NDLH was filtered with a 0.22 μm membrane (Millipore, MA) for use as a fermentation substrate.
Detoxification of the hydrolysate
The detoxification process was similar with that described by Yu et al. . Briefly, the original liquid hydrolysate (without pH adjustment) was heated to 42°C while stirring, and then calcium hydroxide was added to increase the pH to 10.0. The temperature would increase to 50–52°C by addition of calcium hydroxide, and thereafter the mixtures were kept stirring at 50°C for 30 min. After detoxification, the liquid was separated and re-acidified to pH 5.5 with sulfuric acid, followed by passing through a 0.22 μm membrane (Millipore, MA).
Strains and media
Eleven potential lipid producing fungi were investigated: A. niger (NRRL 364), A. terreus (NRRL 1960), C. globosum (NRRL 1870), C. elegans (NRRL 2310), M. isabellina (NRRL 1757), M. vinacea (ATCC 20034), M. circinelloides (NRRL 3628), N. fischeri (NRRL 181), R. oryzae (NRRL 1526), M. plumbeus (CBS 295.63), T. lanuginosus (ATCC 76323). All the strains were kept on potato dextrose agar (PDA) at 4°C. The compositions of the basic medium were (g L-1): (NH4)2SO4, 0.5; KH2PO4, 7.0; Na2HPO4, 2.0; MgSO4·7H2O, 1.5; CaCl2·2H2O, 0.1; FeCl3·6H2O, 0.008; ZnSO4·7H2O, 0.001; CuSO4·5H2O, 0.0001; Co(NO3)2·H2O, 0.0001; MnSO4·5H2O, 0.0001; yeast extract, 0.5 . Glucose (30 g L-1) and xylose (30 g L-1) were used as the carbon source respectively. Cultures were conducted in triplicate in 250 mL Erlenmeyer flasks containing 50 mL medium in an orbital shaker at a rotary rate of 200 rpm, and inoculated with 1 ml of spore suspension (1 × 107 spores). The temperature was maintained at 28°C, except T. lanuginosus (50°C).
To evaluate the capability of utilizing hydrolysates, the selected lipid producing fungi were cultured in 250 mL Erlenmeyer flasks containing 50 mL each of either NDLH or DLH, as well as 0.4 g L-1 MgSO4·7H2O, 2.0 g L-1 KH2PO4, 0.003 g L-1 MnSO4·H2O, 0.0001 g L-1 CuSO4·5H2O, and 1.5 g L-1 yeast extract. The culture conditions were the same as the description above.
The fungal biomass was harvested and washed three times by distilled water, and then freeze-dried to a constant weight. The analysis of fatty acids was performed by Hewlett Packard 5890 gas chromatograph with a Supelco SP-2560 capillary column (100 m × 0.25 mm × 0.20 μm). The conditions for GC were the same as the description by O'Fallon et al. . Tridecanoic acid (C13:0) was used as the internal standard.
Monosugars were analyzed using a Dionex ICS-3000 ion chromatography system equipped with a CarboPac TM PA 20 (4 × 50 mm) analytical column, and CarboPac TM PA 20 (3 × 30 mm) guard column (Dionex Corporation, CA) . Acetic acid, furfural and HMF were determined via High-performance liquid chromatography (HPLC) with a Biorad Aminex HPX-87 H column (Bio-Rad Laboratories, CA) and a refractive index detector as described by Sluiter et al. .
The experimental data were statistically analyzed with ANOVA using SAS 9.2 (SAS Institute Inc.). All values were presented as the average of three independent measurements with significance declared at P <0.05.
Authors acknowledge Jim O’Fallon for his assistance with the fatty acid analysis.
- Chi Z, Zheng Y, Ma J, Chen S: Oleaginous yeast Cryptococcus curvatus culture with dark fermentation hydrogen production effluent as feedstock for microbial lipid production. Int J Hydrogen Energy 2011, 36:9542–9550.View Article
- Subramaniam R, Dufreche S, Zappi M, Bajpai R: Microbial lipids from renewable resources: production and characterization. J Ind Microbiol Biotechnol 2010, 37:1271–1287.View Article
- Gema H, Kavadia A, Dimou D, Tsagou V, Komaitis M, Aggelis G: Production of γ-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel. Appl Microbiol Biotechnol 2002, 58:303–307.View Article
- Eroshin VK, Satroutdinov AD, Dedyukhina EG, Chistyakova TI: Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis. Process Biochem 2000, 35:1171–1175.View Article
- Vicente G, Bautista LF, Gutiérrez FJ, Rodríguez R, Martínez V, Rodríguez-Frómeta RA, Ruiz-Vázquez RM, Torres-Martínez S, Garre V: Direct transformation of fungal biomass from submerged cultures into biodiesel. Energy Fuel 2010, 24:3173–3178.View Article
- Andre A, Diamantopoulou P, Philippoussis A, Sarris D, Komaitis M, Papanikolaou S: Biotechnological conversions of bio-diesel derived waste glycerol into added-value compounds by higher fungi: production of biomass, single cell oil and oxalic acid. Ind Crops Prod 2010, 31:407–416.View Article
- Du Preez JC, Immelman M, Kock JLF, Kilian SG: Production of gamma-linolenic acid by Mucor circinelloides and Mucor rouxii with acetic acid as carbon substrate. Biotechnol Lett 1995, 17:933–938.View Article
- Conti E, Stredansky M, Stredanska S, Zanetti F: γ-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals. Bioresour Technol 2001, 76:283–286.View Article
- Venkata Subhash G, Venkata Mohan S: Biodiesel production from isolated oleaginous fungi Aspergillus sp. using corncob waste liquor as a substrate. Bioresour Technol 2011, 102:9286–9290.View Article
- Economou CN, Makri A, Aggelis G, Pavlou S, Vayenas DV: Semi-solid state fermentation of sweet sorghum for the biotechnological production of single cell oil. Bioresour Technol 2010, 101:1385–1388.View Article
- Peng X, Chen H: Single cell oil production in solid-state fermentation by Microsphaeropsis sp. from steam-exploded wheat straw mixed with wheat bran. Bioresour Technol 2008, 99:3885–3889.View Article
- Stredansky M, Conti E, Salaris A: Production of polyunsaturated fatty acids by Pythium ultimum in solid-state cultivation. Enzyme Microb Technol 2000, 26:304–307.View Article
- Weber RWS, Tribe HT: Oil as a substrate for Mortierella species. Mycologist 2003, 17:134–139.View Article
- Xia C, Zhang J, Zhang W, Hu B: A new cultivation method for microbial oil production: cell pelletization and lipid accumulation by Mucor circinelloides. Biotechnol Biofuels 2011, 4:15.View Article
- Xue F, Miao J, Zhang X, Luo H, Tan T: Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium. Bioresour Technol 2008, 99:5923–5927.View Article
- Chi Z, Zheng Y, Jiang A, Chen S: Lipid production by culturing oleaginous yeast and algae with food waste and municipal wastewater in an integrated process. Appl Biochem Biotechnol 2011, 165:442–453.View Article
- Zheng Y, Chi Z, Lucker B, Chen S: Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production. Bioresour Technol 2012, 103:484–488.View Article
- Perlack RD, Wright LL, Turhollow AF, Graham RL, Stokes BJ, Erbach DC: Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply. Oak Ridge National Laboratory Technical Report, ORNL/TM-2005/66, Oak Ridge, TN; 2005.View Article
- Kumar A, Jones D, Hanna M: Thermochemical biomass gasification: a review of the current status of the technology. Energies 2009, 2:556–581.View Article
- Sassner P, Galbe M, Zacchi G: Techno-economic evaluation of bioethanol production from three different lignocellulosic materials. Biomass Bioenergy 2008, 32:422–430.View Article
- Huang C, Zong M-h WH, Liu Q-: Microbial oil production from rice straw hydrolysate by Trichosporon fermentans. Bioresour Technol 2009, 100:4535–4538.View Article
- Yu X, Zheng Y, Dorgan KM, Chen S: Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour Technol 2011, 102:6134–6140.View Article
- Ratledge C: Microbial lipids. In Biotechnology. 2nd edition. Edited by: Kleinkauf H, Dohren H. Wiley-VCH, Weinheim; 1997:133–197.View Article
- Freer SN, Skory CD, Bothast RJ: D-Xylose metabolism in Rhodosporidium toruloides. Biotechnol Lett 1997, 19:1119–1122.View Article
- Jeffries TW: Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 2006, 17:320–326.View Article
- Narendranath NV, Thomas KC, Ingledew WM: Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium. J Ind Microbiol Biotechnol 2001, 26:171–177.View Article
- Ranatunga T, Jervis J, Helm R, McMillan J, Hatzis C: Identification of inhibitory components toxic toward Zymomonas mobilis CP4(pZB5) xylose fermentation. Appl Biochem Biotechnol 1997, 67:185–198.View Article
- Chen X, Li Z, Zhang X, Hu F, Ryu D, Bao J: Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds. Appl Biochem Biotechnol 2009, 159:591–604.View Article
- Hu C, Zhao X, Zhao J, Wu S, Zhao ZK: Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides. Bioresour Technol 2009, 100:4843–4847.View Article
- Ramírez-Verduzco LF, Rodríguez-Rodríguez JE, Jaramillo-Jacob AR: Predicting cetane number, kinematic viscosity, density and higher heating value of biodiesel from its fatty acid methyl ester composition. Fuel 2012, 91:102–111.View Article
- Vicente G, Bautista LF, Rodríguez R, Gutiérrez FJ, Sádaba I, Ruiz-Vázquez RM, Torres-Martínez S, Garre V: Biodiesel production from biomass of an oleaginous fungus. Biochem Eng J 2009, 48:22–27.View Article
- Gutiérrez T, Buszko M, Ingram L, Preston J: Reduction of furfural to furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose. Appl Biochem Biotechnol 2002, 98–100:327–340.View Article
- Karimi K, Emtiazi G, Taherzadeh MJ: Ethanol production from dilute-acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb Technol 2006, 40:138–144.View Article
- Junker B, Hesse M, Burgess B, Masurekar P, Connors N, Seeley A: Early phase process scale-up challenges for fungal and filamentous bacterial cultures. Appl Biochem Biotechnol 2004, 119:241–277.View Article
- Kootstra AMJ, Beeftink HH, Scott EL, Sanders JPM: Comparison of dilute mineral and organic acid pretreatment for enzymatic hydrolysis of wheat straw. Biochem Eng J 2009, 46:126–131.View Article
- Linde M, Jakobsson E-L, Galbe M, Zacchi G: Steam pretreatment of dilute H2SO4-impregnated wheat straw and SSF with low yeast and enzyme loadings for bioethanol production. Biomass Bioenergy 2008, 32:326–332.View Article
- Liu B, Zhao Z: Biodiesel production by direct methanolysis of oleaginous microbial biomass. J Chem Technol Biotechnol 2007, 82:775–780.View Article
- Papanikolaou S, Aggelis G: Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur J Lipid Sci Technol 2011, 113:1031–1051.View Article
- Sissine F: Energy Independence and Security Act of 2007: a summary of major provisions. Congressional Research Service Report for Congress, Washington, DC; 2007.
- Humbird D, Davis R, Tao L, Kinchin C, Hsu D, Aden A, Schoen P, Lukas J, Olthof B, Worley M, et al.: Process design and economics for biochemical conversion of lignocellulosic biomass to ethanol: dilute-acid pretreatment and enzymatic hydrolysis of corn stover. National Renewable Energy Laboratory Technical Report, NREL/TP-5100–47764, Golden, CO; 2011.View Article
- U.S. DOE: Biomass Program Multi-Year Program Plan. Office of Biomass Program, Energy Efficiency and Renewable Energy, U.S. Department of Energy, ; 2011. Available at: http://www1.eere.energy.gov/biomass
- Kavadia A, Komaitis M, Chevalot I, Blanchard F, Marc I, Aggelis G: Lipid and γ-linolenic acid accumulation in strains of zygomycetes growing on glucose. J Am Oil Chem Soc 2001, 78:341–346.View Article
- O'Fallon JV, Busboom JR, Nelson ML, Gaskins CT: A direct method for fatty acid methyl ester synthesis: application to wet meat tissues, oils, and feedstuffs. J Anim Sci 2007, 85:1511–1521.View Article
- Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D: Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Laboratory Analytical Procedure, National Renewable Energy Laboratory; 2006.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.