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
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.
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
Authors acknowledge Jim O’Fallon for his assistance with the fatty acid analysis.
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