Microbial lipids derived from oleaginous yeast could be a promising resource for biodiesel and other oleochemical materials. The objective of this study was to develop an efficient bioconversion process from lignocellulosic biomass to microbial lipids using three types of robust oleaginous yeast: T. oleaginosus, L. starkeyi, and C. albidus. Sorghum stalks and switchgrass were utilized as feed-stocks for lipid production. Among oleaginous yeast strains, T. oleaginous showed better performance for lipid production using sorghum stalk hydrolysates. Lipid titers of 13.1 g·L -1 were achieved by T. oleaginosus, using sorghum stalk hydrolysates with lipid content of 60% (wt·wt -1) and high lipid yield of 0.29 g·g -1, which was substantially higher than the value reported in literature. Assessment of overall lipid yield revealed a total of 14.3 g and 13.3 g lipids were produced by T. oleaginosus from 100 g of raw sorghum stalks and switchgrass, respectively. This study revealed that minimization of sugar loss during pretreatment and selection of appropriate yeast strains would be key factors to develop an efficient bioconversion process and improve the industrial feasibility in a lignocellulose-based biorefinery.
Microbial lipids are promising candidates for replacing traditional oil sources in the production of biodiesel, oleo-chemicals, and nutraceuticals, due to similar chemical composition and energy value [
Lignocellulosic biomass, such as agricultural residues and woody crops, is a strong alternative substrate for microbial lipid production due to their abundance, low-cost investment, and high content of polysaccharides (up to 75%) [
Several challenges remain for successful bioconversion of lignocellulosic biomass to microbial lipids. A broad array of monomer sugars is generated from lignocellulosic biomass including glucose, xylose, mannose, and arabinose. Typically, the ratio of hexoses to pentoses ranges from 1.5:1 to 3:1 [
Oleaginous yeast, which has an inherent ability to accumulate lipids from 20% to 70% as a percentage of cell dry weight, offers many advantages to overcome challenges associated with lignocellulose-based lipid production [
In this study, production of lignocellulose-based microbial lipids was investigated using three oleaginous yeast cultures: Trichosporon oleaginosus ATCC20509, Lipomyces starkeyi ATCC 56304, and Cryptococcus albidus ATCC10672. Sorghum stalks and switchgrass, which are typical bio-energy crops, were utilized as sugar suppliers for microbial lipid production. In addition, fermentation performance of T. oleaginosus, L. starkeyi, and C. albidus were evaluated using sorghum stalks and switchgrass hydrolysates. To our knowledge, C. albidus ATCC 10672 has not previously been evaluated for lipid production using lignocellulosic hydrolysates. Also, overall yield of microbial lipids from raw biomass was studied to evaluate the lipid production process.
Sorghum stalks were obtained from Texas A&M University, College Station, Texas, and ground by Mesa Associate Inc., Knoxville, Tennessee. Switchgrass was obtained from the Kansas State University agronomy farm, Manhattan, Kansas, and ground at a size of less than 1 mm, using a Tomas-Wiley laboratory mill (Model 4). Biomass composition was determined following the protocol of NREL/TP-510-42618 [
A schematic diagram of the process for lignocellulosic hydrolysate preparation was shown in
Pretreatment and hydrolysis conditions for each lignocellulosic biomass were optimized in our lab and it was followed in this study for lipid production [
Trichosporon oleaginosus ATCC 20509, Lipomyces starkeyi ATCC 56304, and Cryptococcus albidus ATCC 10672 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultivated in a yeast mold broth (YM broth, Difco, Detroit, MI, USA) at 25˚C and 200 rpm for 72 hr. All yeast cultures were preserved in YM agar plates at 4˚C, and transferred to fresh plates once a month.
Starter cultures of all yeast strains were begun by inoculating a single colony from a YM agar plate. T. oleaginosus, L. starkeyi, and C. albidus were grown in a YM broth at 25˚C and 200 rpm for 12 h, and cells were transferred into a 500 mL shake flask containing 100 mL of fermentation media. Sorghum stalks and switchgrass hydrolysates, containing a total of 50 g∙L−1 sugars, were utilized as carbon sources for lipid production. Nitrogen source of yeast extract (0.33 g∙L−1) and peptone (1 g∙L−1) was supplemented into the fermentation media. Fermentation was carried out at 25˚C and 200 rpm for 120 h in a shaking incubator (Innova 4300, New Brunswick Scientific, NJ).
Dry-cell weight (DCW) was used to determine cell concentrations. Cell pellets were washed with water two times, dried at 80˚C overnight, and measured for weight. Sugars and organic acid concentrations were analyzed via a high-per- formance liquid chromatography (HPLC; Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) equipped with a refractive index detector (RID) and a Rezex ROA organic acid column (150 × 7.8 mm, Phenomenex Inc., Torrance, CA, USA). Oven temperature was kept at 80˚C, and 0.005 N sulfuric acid was utilized as a mobile phase, with a pumping rate of 1.0 mL∙min−1.
Yeast cells were harvested via centrifugation (Sorvall Super T21, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 8500 rpm for 20 min. Cells were washed two times with water and concentrated to 109 cells mL−1. The concentrated cells were preserved at −80˚C for one day prior to lipid extraction. Thawed cell pellets (0.5 mL) were transferred into a 2.5 mL polypropylene microvial, followed by adding 0.5 mL of methanol, 0.5 mL of chloroform, and 1 mL of 0.5 mm cubic zirconia beads. Bead beating was performed using a bead-beater homogenizer (Mini-Beadbeater-24, BioSpec Products, Inc., Bartlesville, OK, USA) in 45 sec intervals, with a cooling of 10 min on ice repeated six times. Lipid extraction was conducted by following a modified Bligh and Dyer method [
Fatty acids in the lipids were converted to fatty acid methyl esters (FAMEs) via a transesterification for compositional analysis. Lipid samples were transferred into a 7 mL Kimax tube with 25 nmol of internal standard (pentadecanoic acids) and the chloroform was evaporated under nitrogen gas at 40˚C. For transesterification, 1 mL of methanolic hydrochloric acid (3 M) was added into each tube and incubated at 78˚C for 30 min in the heating block. After cooling down the samples, 2 ml of water were added, followed by 1.6 mL of chloroform and 0.4 mL of hexane. The layers were then separated via centrifugation at 4000 rpm for 5 min. The lower layer was transferred into a clean Kimax tube and the organic phase was dried down under nitrogen gas. One hundred µL of hexane were added to solubilize FAMEs, and then transferred into a glass vial. FAMEs were analyzed by injecting 1 µL of the sample into a gas chromatograph (GC-2014, Shimadzu Scientific Instruments, Columbia, MD, USA) equipped with a flame- ionization detector (FID) and an aqueous-stable polyethylene glycol capillary column (Zebron ZB-Waxplus 30 m × 0.25 mm × 0.25 μm, Phenomenex, Torrance, CA, USA). The initial oven temperature of 160˚C was gradually increased to 200 ˚C at a rate of 5 ˚C min−1, and detector temperature was 250˚C. The FAME mixture (Supelco, 37 component FAME mix) was utilized as an external standard to identify fatty acid composition in the lipids.
SAS software (SAS v9.4, SAS institute, Cary, NC, USA) was used to analyze all data by performing PROC GLM for the least-significant difference (LSD) test at a 95% confidence level (P < 0.05).
The composition of ground sorghum stalks and switchgrass is shown in
After saccharification of each pretreated biomass, sugar recoveries from lignocellulosic biomass were investigated in
Sorghum stalks and switchgrass hydrolysates were utilized as feedstocks for lipid production using T. oleaginosus, L. starkeyi, and C.albidus. Both lignocellulosic
hydrolysates contained acetic acid and citric acid as byproducts of enzymatic hydrolysis process. Acetic acid is normally released from acetylated hemicellulose [
Sugar consumption rate of T. oleaginosus was the fastest, compared with the other two strains. T. oleaginosus consumed all sugars in sorghum stalks and switchgrass hydrolysates at 72 h. L. starkeyi consumed all glucose in the biomass hydrolysates at 72 h, and started using xylose. C. albidus slowly consumed only glucose for 120 h. Citrate utilization was only observed by T. oleaginosus. T. oleaginosus consumed a total of 6 g∙L−1 citrate in both biomass hydrolysates after all glucose was utilized at 48 h. L. starkeyi and C. albidus did not use citrate as nutrients. Instead of utilization, citrate accumulation was observed by C. albidus during lipid production. A total of 3 g∙L−1 of citric acid was produced as a
secondary metabolite. It is known that citric acid is an important substrate of ATP citrate lyase (ACL) to improve lipid accumulation in oleaginous yeast; ACL enzymes have responsibility to increase cytosolic Acetyl-CoA pool which is major substrate for lipid synthesis [
A high lipid yield of 0.29 g∙g−1 was obtained by T. oleaginosus using sorghum stalk hydrolysates. This product yield was a close value to the economically feasible lipid yield suggested by Lennen and F.Pfleger; 0.3 - 0.4 g∙g−1 would be theoretical limit to replace current petrochemical technologies [
aLipid content (%) | Lipid concentration (g∙L−1) | bLipid yield (g∙g−1) | Dry-cell weight (g∙L−1) | Sugar consumption (g∙L−1) | |
---|---|---|---|---|---|
Sorghum stalk hydrolysates | |||||
T. oleaginosus | 60 ± 2.5A | 13.1 ± 0.7A | 0.29 ± 0.0A | 21.7 ± 0.3A | 45 ± 0.7B |
L. starkeyi | 44 ± 2.0B | 7.9 ± 0.3C | 0.16 ± 0.0C | 18.1 ± 0.1B | 48 ± 0.7A |
C. albidus | 42 ± 2.0B,C | 4.6 ± 0.2E | 0.17 ± 0.0D | 11.1 ± 0.1D | 27 ± 0.6E |
Switchgrass hydrolysates | |||||
T. oleaginosus | 58 ± 2.6A | 12.3 ± 0.2B | 0.27 ± 0.0B | 21.1 ± 0.6A | 46 ± 1.1B |
L. starkeyi | 39 ± 0.1C | 6.5 ± 0.3D | 0.17 ± 0.0D | 16.6 ± 0.4C | 38 ± 0.9C |
C. albidus | 44 ± 0.0B | 4.7 ± 0.1E | 0.16 ± 0.0C | 10.7 ± 0.3D | 29 ± 1.4D |
The data represent average value of triplicate experiments ± sample standard deviation. Values with the same letters, in superscripts, within the same column, are not significantly different at a 95% confidence level. aLipid content was defined as weight of extractable lipid relative to weight of dry cell mass. bLipid yield was calculated by dividing amount of lipids by amount of sugar consumed.
obtained in the sorghum stalk hydrolysates. It was because of that fewer amounts of sugars were consumed in the switchgrass hydrolysates. Results of statistical analysis showed that lipid accumulation of C. albidus was similar with L. starkeyi in the sorghum stalks hydrolysates, but C. albidus produced the lowest concentration of lipids in both biomass hydrolysates. This was because lower amounts of DCW were obtained using both biomass hydrolysates. These results demonstrated that both lipid content and DCW were important factors to achieve high titers of lipids by oleaginous yeast, because lipids are intracellular products.
which is preferable in the oleochemical industry [
Our bioconversion process of lignocellulose-based microbial lipid production was evaluated by calculating the overall yield of lipid from raw sorghum stalks and switchgrass (
The highest lipid yield was achieved by T. oleaginosus from both lignocellulosic biomasses, since T. oleaginosus showed the best fermentation performance among other yeast strains during lipid production. T. oleaginosus produced 8% higher amounts of lipids from sorghum stalks containing a 14% lower con-
tent of polysaccharides compared with switchgrass. This might be due to a significant hemicellulose loss during pretreatment of switchgrass. It showed another key factor to attaining high lipid yields from biomass was to maximize sugar recoveries during pretreatment and enzymatic hydrolysis for sugar production.
Lipid yields obtained by C. albidus and L. starkeyi were not substantially different because they showed similar fermentation performance during lipid production. C. albidus produced higher amounts of lipids from sorghum stalks compared with switchgrass, although low-lipid concentrations were obtained during fermentation. It was due to that higher product yield was achieved during fermentation using sorghum stalk hydrolysates. Whereas, lower amounts of lipids were obtained by L. starkeyi using sorghum stalks compared with switchgrass, even though higher lipid concentrations and contents were attained during fermentation of L. starkeyi using sorghum stalk hydrolysates. This was because lower sugar consumptions and product yields were observed by L. starkeyi in sorghum stalk hydrolysates. To sum up these results, maximization of sugar recoveries during sugar production, and selection of proper microbial strains for lipid production, were key factors to achieve high yields of microbial lipids from lignocellulosic biomass.
Microbial lipid production from lignocellulosic biomass such as sorghum stalks and switchgrass was investigated using three oleaginous yeast strains; T. oleaginosus, L. starkeyi and C. albidus. High-sugar recoveries (89% of TY) from sorghum stalks were obtained via an alkaline pretreatment whereas total sugar recoveries from switchgrass were 67% of the TY. T. oleaginosus showed the best fermentation performance using both biomass hydrolysates among oleaginous yeast cultures. Lipid titers of 13.2 g∙L−1 and lipid yield of 0.29 g∙g−1 were achieved by T. oleaginosus using sorghum stalk hydrolysates. Results of overall lipid yield assessment revealed that a key matrix to improve industrial feasibility of bioconversion for lignocelluose-based microbial lipid production is maximal recovery of fermentable sugars from raw biomass and strain development to attain better fermentation performance.
This work was funded by the Development Initiative Competitive Grants Program (BRDI; grant number: 2012-10008-20263). The authors are also grateful to Novozymes Inc. for the donation of enzyme samples, and Dr. Yadhu N. Guragain for his valuable suggestions on pretreatment process. Author PVV thanks the Lortscher Endowment for their support.
Lee, J.-E., Vadlani, P.V. and Min, D. (2017) Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures. Journal of Sustainable Bioenergy Systems, 7, 36-50. https://doi.org/10.4236/jsbs.2017.71004