Appl Biochem Biotechnol (2015) 176:1801–1814 DOI 10.1007/s12010-015-1679-y
Lipid Production from Hemicellulose and Holocellulose Hydrolysate of Palm Empty Fruit Bunches by Newly Isolated Oleaginous Yeasts Srikanya Tampitak 1 & Yasmi Louhasakul 1 & Benjamas Cheirsilp 1 & Poonsuk Prasertsan 1
Received: 4 September 2014 / Accepted: 25 May 2015 / Published online: 31 May 2015 # Springer Science+Business Media New York 2015
Abstract Palm empty fruit bunches (EFBs) are abundant lignocellulosic wastes from palm oil mills. They are potential sources of sugars which can be converted to microbial lipids by oleaginous yeasts. To produce sugars from EFB, two-step and one-step hydrolysis reactions were performed. In the first step, the use of diluted sulfuric acid (0.5 % w/v) hydrolyzed hemicelluloses and released mainly pentoses, and in the second step of hydrolysis of residual pulp using 2.5 % (w/v), sulfuric acid released more hexoses. The use of 2.5 % (w/v) sulfuric acid in one-step hydrolysis of holocelluloses released the highest amount of sugars (38.3 g/L), but it also produced high concentration of potential inhibitors (>1 g/L). Three oleaginous yeasts, Rhodotorula mucilaginosa, Kluyveromyces marxianus, and Candida tropicalis, were isolated and screened for their ability to convert EFB hydrolysates into lipids. These yeasts grew well and produced lipids from EFB hemicellulose and holocellulose hydrolysate after potential inhibitors were removed. This study shows that EFB can be used for lipid production. Keywords Acid hydrolysis . Holocellulose . Hemicellulose . Oleaginous yeast . Palm empty fruit bunches
Introduction Biodiesel is an attractive replacement for petroleum diesel because it is domestically available, biodegradable, and compatible with existing diesel engines. At present, the high production cost of biodiesel, especially its raw material cost, is a major barrier to its commercialization. In addition, the use of pure vegetable oil as a raw material for biodiesel production could compete
* Benjamas Cheirsilp [email protected] 1
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
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with its use as an edible oil. Microbial lipids have been considered to be new sources of biodiesel feedstocks due to their plant-like oil composition. Microorganisms that can accumulate lipid up to more than 20 % of their biomass are defined as oleaginous species. Oleaginous yeasts have many advantages due to their fast growth rate, high lipid content, and the resemblance of their triacylglycerol fraction to plant oil. So far, many yeast species such as Cryptococcus albidus, Cryptococcus curvatus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans, and Yarrowia lipolytica have been intensively used for lipid production. Some of these species can accumulate intracellular lipid as high as 60 % of their dry weight [1, 2]. Bioconversion of lignocellulosic wastes into valuable products is of considerable interest because of their low cost, renewable nature, and abundance. The carbon sources for oleaginous microbes need to be extended to include lignocellulosic wastes, which allow for the production of large volumes of cheap microbial lipids. Currently, the palm oil industry has considerably expanded its production in Thailand, and this expansion has produced large volumes of lignocellulosic waste by-products including 13.5 % (w/w) palm pressed fiber (PPF), 22 % (w/w) palm empty fruit bunches (EFB), and 5.5 % (w/w) palm kernel cake (PK) [3]. Among these byproducts, EFBs are the most interesting biomass due to their high content of holocelluloses which are composed of celluloses (41.3–46.5 %, w/w) and hemicelluloses (25.3–33.8 %, w/w) [4]. They are considered to be potential sugar sources that can be converted to microbial lipid by oleaginous yeasts. EFB can be hydrolyzed into sugars by acid hydrolysis. However, the acid treatment generates various by-products resulting from the degradation of sugars and lignin, which are potential inhibitors of the yeasts [5]. One method to reduce the toxic by-products from the lignin is alkali pretreatment of lignocellulosic materials. This process yields holocellulose pulp that can be further hydrolyzed by acid. The pentoses in the hemicellosic fraction can be easily released by dilute acid hydrolysis due to the amorphous structure of the hemicelluloses [6]. In contrast, high concentrations of acid are required to release glucose from the highly crystallized cellulose fraction [7]. However, the use of such a high concentration of acid would result in further degradation of pentoses to furfural and glucose to 5-hydroxymethylfurfural (HMF) [8]. To keep the concentration of these by-products at a minimum, it is necessary to use diluted acid in the first step to hydrolyze hemicelluloses and then high concentrations of acid in the second step to hydrolyze the crystalline celluloses that remain as residual solid from the first step. Although this two-step hydrolysis can keep the concentration of by-products at a minimum, it is quite time-consuming. Because some microorganisms are potentially tolerant to the by-products or may even be able to consume them during fermentation [9], the hydrolysis of holocelluloses in a one-step process would be highly advantageous for such microorganisms. Recently, the use of hemicellulose hydrolysates as carbon sources for lipid production by oleaginous yeasts has been reported [5, 10–12], but only a few attempts have been made to use either cellulose hydrolysates [9] or holocellulose hydrolysates [13]. The aim of this study was to exploit EFB as a cheap and renewable raw material for the production of microbial lipids by newly isolated oleaginous yeasts. Although there have been many studies on the production of sugars from EFB [7, 14, 15], to our knowledge, there have been no attempts to use these sugars for lipid production. In this study, both two-step and one-step hydrolysis procedures were performed to convert EFBs into fermentable sugars. The oleaginous yeasts that can consume these sugars for growth and lipid production were screened and identified. The effect of detoxification methods on the growth and lipid production by the selected yeasts was also investigated.
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Materials and Methods Materials EFB samples from the Thai Tallow and Oil company (Suratthani, Thailand) were crushed using a high-speed rotary cutting mill (Patipong agriculture machinery, P5208, Thailand) to give particle sizes in the range of 0.1–0.5 cm. Alkaline pretreatment was employed to delignify the EFBs. Sodium hydroxide at 10 % (w/v) was used to pretreat EFBs at a ratio of 10:1 [16]. The mixture was treated at a temperature of 100 °C for 30 min. The pretreated samples were then washed and dried overnight at 60 °C.
Acid Hydrolysis of Palm EFBs The delignified EFB was hydrolyzed through either a two-step or one-step process using sulfuric acid solutions. In the two-step process, diluted sulfuric acid (0.5 % w/v) was used in the first step to hydrolyze the hemicellulose fraction of the delignified EFB in the first step to avoid degradation of sugar monomers into toxic by-products. The liquid fraction from this step was termed the “hemicellulose hydrolysate.” In the second step, sulfuric acid at a concentration of 2.5 % (w/v) was used to hydrolyze the residual pulp from the dilute acid hydrolysis step. The hydrolysate from the residual pulp was termed the Bresidual pulp hydrolysate.” The delignified EFB (holocellulose) was also hydrolyzed using 2.5 % (w/v) sulfuric acid in a one-step process. The hydrolysate from this step was termed the Bholocellulose hydrolysate.^ This reaction was carried out at 120 °C for 60 min in an autoclave. After the reaction was complete, the solids were separated from the aqueous solution by filtration. The filtrate was analyzed for xylose, arabinose, glucose, galactose, furfural, and HMF.
Screening and Identification of Oleaginous Yeasts The waste samples containing potential isolates were collected from soil and wastes from palm oil mills in the southern region of Thailand. Ten percent samples were enriched in yeast extract peptone dextrose (YPD) medium composed of 10 g/L yeast extract (HiMedia, India), 10 g/L peptone (HiMedia, India), and 20 g/L of either glucose or xylose as carbon sources with a pH of 4.0. Then, 0.1 mL diluted culture was inoculated on YPD agar medium using the spread plate technique and grown for 72 h at room temperature. The isolated yeasts were stained using the Sudan black B technique and observed under a phase contrast microscope for the presence of blue or grayish lipid globules within the cells. The yeast strains containing large lipid globules were selected for further quantitative analysis of their lipid content. The selected yeasts were then inoculated in EFB hemicellulose hydrolysate-based medium (20 g/L sugars from dilute acid hydrolysate, 2 g/L (NH4)2SO4, 0.2 g/L MgSO4·7H20, 0.5 g/L KH2PO4, 0.1 g/ L CaCl2·2H2O, pH 6.0) and incubated for 72 h at room temperature with shaking at 140 rpm. Selected oleaginous yeasts were identified based on their 26S ribosomal DNA (rDNA) sequence. The 26S rDNA was PCR-amplified using universal primer sets of F63-Forward (5′GCA TAT CAA GCG GAG GAA AAG-3′) and LR-Reverse (5′-GGT CCG TGT TTC AAG ACG G-3′) and sequenced using automated sequencer. The resulting sequences were BLAST searched against the National Center for Biotechnology Information (NCBI) database. The sequences that shared over 99 % similarity with currently known sequences were considered to be the same species and used to construct a phylogenetic tree.
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Biomass and Lipid Production by Oleaginous Yeasts Shake flask cultures in 250-mL Erlenmeyer flasks contained 90 mL of sugar- or hydrolysatebased medium. The pH was adjusted to 6.0. The medium was sterilized by autoclaving before use. The cultures were initiated with 10 % of a 24-h-old seed culture (approximately 107 cells/ mL) and incubated at room temperature (30±2 °C) on a rotary shaker at 140 rpm for 72 h. The samples were taken to measure biomass and lipid production.
Analytical Methods The cell pellet harvested following centrifugation was washed twice with distilled water and dried at 60 °C for 72 h. The final biomass was measured and expressed as dry cell weight (DCW). The extraction of lipid from the biomass was performed according to the modified procedure of Folch et al. [17]. One gram of dried biomass was ground into fine powder and blended with 20 mL of chloroform/methanol (2:1, v/v). The mixture was sonicated (Elma, E30H, Switzerland) for 30 min, and the solvent phase was recovered using centrifugation. The extraction process was repeated twice more. All collected solvent was evaporated using a rotary evaporator (EYELA, SB-651, Rikakikai Co. Ltd. Tokyo, Japan) and dried at 60 °C to constant weight (at least 48 h). The lipid content is expressed as a percentage of gram lipid per gram dried biomass. The lipid production was expressed as the gram lipid per liter of medium (g/L). The sugar composition was analyzed with high-performance liquid chromatography (HPLC) using an Agilent Technology 1100 series RID with an Aminex-87P column operated at 45 °C and a mobile phase containing 0.01 N of sulfuric acid pumped at a rate of 0.6 mL/min [18]. The furans in the samples were determined with a DU800 spectrophotometer. The maximum absorbance spectra of furfural and HMF were 276 and 282 nm, respectively. The concentrations of furfural and HMF in the samples were calculated using standards of known concentration [19]. All experiments were performed with at least three replicates. Analysis of variance was performed to identify any significant differences in the treatment mean values, and the least significant difference (p≤0.05), calculated using SPSS software, was used to separate means.
Results and Discussion Acid Hydrolysis of Palm EFBs Palm EFBs are considered to be potential sources of hemicelluloses and celluloses that could be converted to useful products. The hemicellulose, cellulose, holocellulose, and lignin contents of EFBs used in this study were 23.1±1.2 % (w/w), 40.8±1.2 % (w/w), 63.8± 1.9 % (w/w), and 16.9±1.2 % (w/w), respectively. The cellulose content in the EFBs was higher than that in other lignocellulosic biomasses such as rice straw (36.6 %, w/w), poplar (33.1 %, w/w), and switchgrass (29.7 %, w/w) [20]. The high cellulose content is one important advantage in using EFBs as potential feedstock for glucose production. To reduce lignin and increase the content of holocelluloses, an alkali pretreatment was applied to the EFBs. Alkaline pretreatment of lignocelluloses with sodium hydroxide was employed because of its effectiveness not only in reducing the lignin content but also in swelling and disrupting the crystalline structure of the cellulose [16, 21]. In this study, after alkaline pretreatment, lignin content was
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reduced to 11.6±0.3 % (w/w), and holocellulose content was increased up to at most 80.2± 0.9 % (w/w). The delignified EFB was sequentially hydrolyzed to avoid degradation of pentoses to byproducts at high acid concentrations. The first step in this process was the hydrolysis of hemicelluloses using diluted acid at 0.5 % (w/v), which resulted in a liquid fraction of hemicellulose hydrolysate. The residual pulp was further hydrolyzed using 2.5 % (w/v) acid, concentration that produced the highest total sugar (data not shown). It should be noted that at acid concentrations >2.5 % (w/v), the total sugar concentration decreased. This decrease was due to the degradation of pentoses to furfural and hexoses to HMF. The liquid fraction from this second step was the residual pulp hydrolysate. The delignified EFBs were also hydrolyzed using 2.5 % (w/v) sulfuric acid in a one-step process. This single-step process hydrolyzed both the hemicellulose and cellulose fractions in the delignified EFBs. The resulting hydrolysate was the holocellulose hydrolysate. Figure 1 shows the sugar composition in the hydrolysate from the two-step and one-step acid hydrolysis procedures. Dilute acid hydrolysis of the delignified EFBs mainly produced xylose from xylans and arabinose from arabinans in the hemicellulose fraction (Fig. 1a). These products formed because the hemicellulose fraction is more susceptible to hydrolysis with mild acid treatment than is the cellulose fraction, which needs more severe treatment because of its crystalline nature. Although xylose and arabinose were the main sugars found in the hemicellulose hydrolysate, other by-products such as glucose, furfural, and HMF were also produced in low amounts. It has been reported that the amount and type of sugars released during hydrolysis depend on the operating conditions [8]. When the residual pulp from the dilute acid hydrolysis was further hydrolyzed using 2.5 % (w/v) sulfuric acid, more glucose was released together with xylose. The concentrations of furfural and HMF in this residual pulp hydrolysate were higher than those found in the hemicellulose hydrolysate. The one-step hydrolysis of the holocellulose fraction using 2.5 % (w/v) sulfuric acid produced the highest amount of total sugars (38.3 g/L) together with high concentrations of furfural and HMF (0.57 and 0.44 g/L, respectively). Each hydrolysate was then diluted to adjust the sugar concentration to 20 g/L before being used as a carbon source for cultivation of the yeasts. It should be noted that after
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Fig. 1 Composition of sugars and inhibitors in the hydrolysate following two-step and one-step acid hydrolysis
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adjustment of the sugar concentration, the concentrations of furfural and HMF in the holocellulose hydrolysate were lower than those detected in the residual pulp hydrolysate (Fig. 1b).
Screening and Identification of Oleaginous Yeasts One hundred and thirty-eight strains of yeast were isolated from soils and wastes of the palm oil mill in the southern region of Thailand. These yeast strains were placed in YPD medium (pH 4.0) using glucose or xylose as a carbon source. The isolates were then stained with Sudan black B and observed under a phase contrast microscope to identify the presence of blue or grey lipid globules within the cells. Among the 138 isolates, 16 isolates and 11 isolates that showed large lipid globules when using glucose and xylose as a carbon source, respectively, were selected as potential lipid producers. These selected isolates were pre-cultured in an inoculum medium for 24 h and then inoculated into medium containing EFB hemicellulose hydrolysate as a carbon source. The biomass and lipid production of all yeasts was compared after 72 h (data not shown). When using hemicellulose hydrolysate as a carbon source in the screening medium, only three isolates of G43, X32, and X37 produced lipid concentrations of higher than 1.08 g/L. These three isolates were then identified based on their 26S rDNA sequences as Rhodotorula mucilaginosa (accession no. AB976563), Kluyveromyces marxianus (accession no. AB976564), and Candida tropicalis (accession no. AB976565) with 99 % similarity. Table 1 shows the fatty acid composition of the lipids from these three isolates. Over 70 % of the fatty acids in the yeast lipids were palmitic, stearic, and oleic acids, and these were similar to those of the plant oil, indicating their potential use as an alternative feedstock for biodiesel production. A phylogenetic tree was constructed as described in the methods. A search for similarities between D1/D2 26S rDNA of the isolates and that of those in the NCBI database showed that many phylogenetically related yeast species are similar to the isolates obtained in this study (Fig. 2). Although Rodotorula mucilaginosa and C. tropicalis are known as oleaginous yeasts [2, 22], this is the first time that K. marxianus has been shown to accumulate a high lipid content.
Biomass and Lipid Production of Yeasts Using Glucose and Xylose The three newly isolated oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), and C. tropicalis X37 (CX37), together with the reference
Table 1 Fatty acid composition of lipid from selected oleaginous yeasts Strain
Fatty acid composition (% w/w)a C12:0
C14:0
C16:0
Rodotorula mucilaginosa G43
0.22
1.49
25.1
K. marxianus X32
0.44
2.57
36.4
C. tropicalis X37
0.81
2.66
26.2
a
C16:1
C18:0
C18:1
C18:2
1.07
6.89
46.2
13.9
11.7 4.77
8.63 9.47
0.00 24.7
27.4 18.5
C18:3
C20:0
3.58
0.39
11.2 9.30
0.00 0.00
Percentage of fatty acids including lauric (C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), and arachidic acid (C20:0)
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Fig. 2 Phylogenetic tree of the newly isolated oleaginous yeast strains and previously reported oleaginous yeast strains. Strain number and sequence accession numbers are given. All strains shown are type strains. The three new isolates are depicted in bold
oleaginous yeast Rhodotorula glutinis TISTR 5159 (R5159) [23], were cultivated using glucose and xylose as carbon sources. Their biomass, lipid production, and lipid content are shown in Fig. 3. All yeasts grew well with xylose and yielded a higher biomass than those grown with glucose. Although the biomass values of all yeasts grown with xylose were not
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Fig. 3 Biomass and lipid production by the three newly isolated oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37), and reference oleaginous yeast Rhodotorula glutinis TISTR 5159 (R5159) cultivated with a glucose and b xylose. The lipid content was expressed as a percentage of the gram lipid per gram dried biomass
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much different, Rodotorula mucilaginosa G43 had the highest lipid production, which reached 1.04 g/L. The lipid contents of all yeasts grown with glucose were higher than those grown with xylose. Because all the tested yeasts can assimilate xylose better than glucose, it is feasible to use these yeasts to convert lignocellulosic sugars into lipid. It has been reported that the regulation of sugar transport across cell membranes plays a key role in determining how effectively microorganisms can assimilate xylose [24, 25]. According to those studies, xylose uptake in yeast occurs by both facilitated diffusion and active transport processes. Because xylose molecules are small enough to easily penetrate the yeast cell membrane, the yeast grow faster with xylose than with glucose. Several yeasts use xylose for growth and production of lipids such as Rhodotorula graminis [9], Rhodotorula mucilaginosa TJY15a [2], and Y. lipolytica Po1g [13]. However, the lipid content generated when using xylose is lower than that generated when using glucose.
Biomass and Lipid Production of Yeasts Using EFB Hydrolysate The biomass and lipid production of oleaginous yeasts using hemicellulose hydrolysate, residual pulp hydrolysate, and holocellulose hydrolysate of delignified EFB is shown in Fig. 4a–c, respectively. However, the biomass and lipid production of yeasts using all types of EFB hydrolysate, especially residual pulp hydrolysate, yielded poor results compared to that using pure glucose and xylose (Fig. 3). It should be noted that although the sugar composition in the residual pulp hydrolysate was similar to that in the holocellulose hydrolysate, no growth or lipid production was observed in the residual pulp hydrolysate. Higher concentrations of inhibitors in the residual pulp hydrolysate might contribute to this phenomenon (Fig. 1b). Hu et al. [26] reported that >1 mM furfural (>0.096 g/L) and >14.7 mM HMF (>1.85 g/L) inhibited cell growth and lipid production of the oleaginous yeast Rhodosporidium toruloides Y4. They also showed that these inhibitors most likely repressed cell growth more severely than did lipid biosynthesis. Depending on the source of the biomass and the type of hydrolysis employed, the concentration of these inhibitors in the lignocellulosic hydrolysates can range from 0.5 to 11 g/L [27]. These inhibitors are thought to damage cell walls and membranes and inhibit RNA synthesis in microorganisms [28, 29]. The acid hydrolysis of sugarcane bagasse produced furfural and HMF concentrations as high as 0.83 and 0.15 g/L, respectively [13].
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Fig. 4 Biomass and lipid production by the oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37), and Rhodotorula glutinis TISTR 5159 (R5159) using a hemicellulose hydrolysate, b residual pulp hydrolysate, and c holocellulose hydrolysate of delignified EFBs. The lipid content is expressed as a percentage of the gram lipid per gram dried biomass
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In this study, the concentrations of HMF in all types of hydrolysate ranged from 0.1 to 0.4 g/L which were lower than the previously reported inhibitory levels (1.8–3.0 g/L) [11, 12, 26]. In contrast, the concentrations of furfural were higher than the previously reported inhibitory levels (0.09–0.2 g/L) [11, 12, 26]. The furfural concentrations in hemicellulose, residual pulp, and holocellulose hydrolysates were 0.22, 0.41, and 0.30 g/L, respectively. Therefore, the inhibitory effect on yeast cell growth was most likely due to the presence of furfural. In this study, growth and lipid accumulation was better using hemicellulose hydrolysate than using holocellulose hydrolysate. This result can be explained by the lower amount of furfural present in the hemicellulose hydrolysate. Thus, the detoxification of hydrolysate required to remove these inhibitors is thought to be essential in facilitating its efficient use as an alternative carbon source for lipid production by these oleaginous yeasts. In addition, the comparison of these three types of hydrolysates indicated that dilute acid hydrolysis is the most suitable method to hydrolyze the lignocellulosic materials because it yielded the lowest amount of inhibitory by-products, but the amount of sugars obtained was also lowest. Therefore, if the amount of total sugar and simple process was the most concern factors, the one-step hydrolysis would be more appropriate.
Detoxification of EFB Hydrolysate Normally, hydrolysate is neutralized with alkali prior to use. Other diverse methods have been proposed to accomplish this process including the following: adsorption by activated carbon, overliming (neutralization with lime), and filtration; partial removal of furfural and soluble lignin by molecular sieves; and vapor stripping for the removal of volatile inhibitors [30]. Because activated carbon is often used to remove compounds from the liquid phase, it was used to remove inhibitors from EFB hydrolysates in this study. To simultaneously neutralize acidic compounds and remove inhibitors, the EFB hydrolysate was detoxified using the overliming method. In this process, dried calcium hydroxide was added to the acidic hydrolysates for conversion to gypsum, which has commercial value as plaster of Paris. This method has also been used to remove volatile inhibitory compounds such as furfural and HMF [6]. The three types of EFB hydrolysates were then detoxified using activated carbon adsorption and overliming. Table 2 shows the effect of the detoxification method on the removal of inhibitors. Each potent inhibitor was reduced using either method. Furfural was reduced by Table 2 Effect of detoxification method on the concentrations of inhibitors present in the medium Hydrolysate
Detoxification method
Furfural (g/L)
HMF (g/L)
Hemicellulose
Non-detoxified
0.22±0.08
0.09±0.04
hydrolysate
Absorption
0.07±0.00 (68 %)
0.05±0.01 (44 %)
Overliming
0.10±0.01 (55 %)
0.07±0.01 (22 %)
Residual pulp hydrolysate
Non-detoxified Absorption
0.41±0.08 0.09±0.01 (78 %)
0.31±0.12 0.09±0.01 (71 %)
Overliming
0.12±0.00 (71 %)
0.13±0.01 (58 %)
Holocellulose
Non-detoxified
0.30±0.12
0.23±0.08
Absorption
0.08±0.01 (73 %)
0.08±0.01 (65 %)
Overliming
0.11±0.01 (63 %)
0.17±0.02 (26 %)
hydrolysate
Data in parentheses are percent removal of inhibitors
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68–78 % to a yield less than 0.10 g/L using the adsorption method. The overliming method also effectively removed furfural with a 55–71 % reduction. HMF was reduced by 44–71 and 22–58 % through adsorption and overliming, respectively. Overall, the adsorption method was better at removing the inhibitors than the overliming method. The percent removal also depended on the initial concentration of the inhibitors. Tsigie et al. [13] reported that the growth of Y. lipolytica Po1g was limited in non-detoxified sugarcane bagasse hydrolysate due to the presence of furfural and HMF at concentrations of 0.12 and 0.61 g/L, respectively. Overliming reduced the concentrations of furfural and HMF by 24.8 and 21.3 %, respectively. The yeast grew faster and better in the detoxified sugarcane bagasse hydrolysate. According to Huang et al. [10], overliming completely removed furfural while adsorption was more effective for the removal of HMF. Their results also showed that the oleaginous yeast Trichosporon fermentans was able to grow and efficiently accumulate lipids on rice straw hydrolysate after detoxification. Huang et al. [12] reported that T. fermentans grew and accumulated lipid better on sugarcane bagasse hydrolysate after detoxification.
Biomass and Lipid Production of Yeasts Using Detoxified EFB Hydrolysates Figures 5 and 6 show the biomass and lipid production of oleaginous yeasts on EFB hydrolysates detoxified by adsorption and overliming, respectively. All yeasts grew better and yielded a higher lipid content in the detoxified EFB hydrolysates than in the nondetoxified hydrolysates. This finding was most likely due to the lower concentration of inhibitors in the detoxified hydrolysates. All yeasts except Rhodotorula glutinis TISTR 5159 performed better in the detoxified hydrolysates than in medium containing pure glucose and xylose as carbon sources (Fig. 3). It is possible that the hydrolysate might contain some additional nutrients that could be used by the yeasts. Furthermore, the results of this study were consistent to those of Tsigie et al. [13], who also found that detoxified sugarcane bagasse hydrolysates yielded a higher biomass of Y. lipolytica Po1g than did pure glucose and xylose. In the detoxified hydrolysates, C. tropicalis X37 performed the best, followed by Rodotorula mucilaginosa G43 and K. marxianus X32, while Rhodotorula glutinis TISTR 5159 yielded the lowest biomass and lipid content. This yeast might be very sensitive to the inhibitors even at very low concentrations.
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Lipid content (%)
Biomass (g/L), Lipid (g/L)
Biomass Lipid Lipid content
Biomass (g/L), Lipid (g/L)
(a) Hemicellulose hydrolysate 8
0 RG43 KX32 CX37 R5159 Yeast strain
Fig. 5 Biomass and lipid production by the oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37), and Rhodotorula glutinis TISTR 5159 (R5159) using the EFB hydrolysates detoxified by the adsorption: a hemicellulose hydrolysate, b residual pulp hydrolysate, and c holocellulose hydrolysate. The lipid content is expressed as a percentage of the gram lipid per gram dried biomass
Appl Biochem Biotechnol (2015) 176:1801–1814
(b) Residual pulp hydrolysate
40
2
20 0 RG43 KX32 CX37 R5159 Yeast strain
Biomass (g/L), Lipid (g/L)
4
(c) Holocellulose hydrolysate
6
60
4
40
2
20
0
0 RG43 KX32 CX37 R5159 Yeast strain
8
80
Biomass Lipid Lipid content
80
6
60
4
40
2
20
0
Lipid content (%)
60
Biomass Lipid Lipid content
Lipid content (%)
6
0
8
80
Lipid content (%)
Biomass (g/L), Lipid (g/L)
Biomass Lipid Lipid content
Biomass (g/L), Lipid (g/L)
(a) Hemicellulose hydrolysate 8
1811
0 RG43 KX32 CX37 R5159 Yeast strain
Fig. 6 Biomass and lipid production by the oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37) and Rhodotorula glutinis TISTR 5159 (R5159) using EFB hydrolysates detoxified by overliming: a hemicellulose hydrolysate, b residual pulp hydrolysate, and c holocellulose hydrolysate. The lipid content is expressed as a percentage of the gram lipid per gram dried biomass
It should be noted that both detoxification methods gave similar results, but due to economic reasons, the overliming method was chosen. Moreover, this process also produced gypsum, a commercially valuable by-product after overliming. Among the yeasts tested, C. tropicalis X37 gave the highest lipid production of 2.73 g/L using residual pulp hydrolysates, suggesting that it is the most suitable strain for being cultivated in EFB hydrolysates. The higher lipid content in C. tropicalis X37 is likely due to the higher glucose content in the residual pulp hydrolysates since glucose has been shown to be more suitable for lipid accumulation (Fig. 3a). Table 3 compares the lipid production of oleaginous yeasts using various sources of lignocellulosic biomass. Lignocellulosic biomass is a potential low-cost feedstock due to its abundance and sustainability. Several attempts with lignocellulosic biomass have shown that it is possible to use hydrolysates from agricultural wastes for lipid production. However, a single crop residue/biomass would not be sufficient to meet the huge demand for biofuel. With this view, there is a need to explore new and abundant agricultural substrates for lipid production. Moreover, lipid production depends on many factors such as yeast species, sugar concentration, nitrogen sources and concentration, and the culture conditions. In most studies, costly nitrogen sources such as yeast extract, peptone, or a combination were used. In the study by Huang et al. [10, 12], T. fermentans yielded a very high concentration of lipid due to the high concentration of sugar used in their study. However, its lipid yield was lower than that of Y. lipolytica [13], C. curvatus [11], and C. tropicalis. These results indicate that palm EFBs are another lignocellulosic biomass source that could be used as feedstock for lipid production. Further optimization and development will improve the efficiency of the yeast in converting EFB hydrolysates into lipids.
Conclusions This study has shown that palm EFBs are potential low-cost feedstock for sugar production and microbial lipid production. The sugar and composition in the hydrolysates of palm EFBs were varied using one-step and sequential hydrolysis. One-step hydrolysis could release higher
Hemicellulose (116.9 g/L)
Hemicellulose (123.5 g/L)
Holocellulose (20 g/L)
Hemicellulose (20 g/L) Residual pulp (20 g/L) Holocellulose (20 g/L)
Rice straw
Sugarcane bagasse
Sugarcane bagasse
Palm empty fruit bunch
c
b
15.8
–a
–a
Y. lipolytica C. tropicalis
(NH4)2SO4 (2 g/L)
T. fermentans
1.61 2.73 1.31
6.81 6.18
6.68
3.5
6.44
11.4
11.5
14.7
L. starkeyi T. fermentans
4.6
13.8
Rhodotorula glutinis
5.8
17.2
C. curvatus
0.4
Lipid (g/L)
7.8
Biomass (g/L)
Y. lipolytica
Strain
Peptone (5 g/L)
Yeast extract (0.5 g/L) + peptone (2.2 g/L)b
Yeast extract (0.5 g/L) + peptone (1.8 g/L)
Yeast extract (1.5 g/L)
Nitrogen source (concentration)
Lipid yield was calculated by gram lipid per gram substrate
Concentration was calculated from C/N ratio of 165
Not available
Hemicellulose (40 g/L)
Wheat straw
a
Hydrolysate (sugar concentration)
Feedstock
Table 3 Lipid production from lignocellulosic biomass using oleaginous yeasts
0.07
0.14
0.08
0.33
0.13
0.09
0.12
0.09
0.15
0.01
Lipidc yield (g/g)
This work
Tsigie et al. [13]
Huang et al. [10] Huang et al. [12]
Yu et al. [11]
Ref.
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amount of sugars, but it also produced high concentration of potential inhibitors. In sequential hydrolysis, the use of diluted acid in the first step could effectively hydrolyze hemicelluloses and the use of higher concentration of acid in the second step could further hydrolyze celluloses in the residual pulp with low concentration of inhibitors. The newly isolated oleaginous yeasts Rhodotorula mucilaginosa G43, K. marxianus X32, and C. tropicalis X4 could grow and accumulate lipid in palm EFB hydrolysates after detoxification. Among them, C. tropicalis X4 is the most robust strain for lipid production. Since the use of palm EFBs will not conflict with food usage, it has great potential as a feedstock for industrialized lipid production. Acknowledgements This research was financially supported by the Graduate School of Prince of Songkla University and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission under Grant No. AGR540567c. The third and fourth authors are supported by Thailand Research Fund under Grant No. RTA5780002. Thanks are also given to Dr. Brian Hodgson for his assistance with the English language.
References 1. Li, Y. H., Zhao, Z. B., & Bai, F. W. (2007). High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme and Microbial Technology, 41, 312–317. 2. Li, M., Liu, G. L., Chi, Z., & Chi, Z. M. (2010). Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15. Biomass and Bioenergy, 34, 101–107. 3. Pua, F. L., Zakaria, S., Chia, C. H., Fan, S. P., Rosenau, T., Potthast, A., & Liebner, F. (2013). Solvolytic liquefaction of oil palm empty fruit bunches (EFB) fibres: analysis of product fractions using FTIR and pyrolysis-GCMS. Sains Malaysiana, 42, 793–799. 4. Kim, S., Park, J. M., Seo, J. W., & Kim, C. H. (2012). Sequential acid-/alkali-pretreatment of empty palm fruit bunches fiber. Bioresource Technology, 109, 229–233. 5. Chen, X., Li, Z., Zhang, X., Hu, F., Ryu, D. D. Y., & Bao, J. (2009). Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds. Applied Biochemistry and Biotechnology, 159, 591–604. 6. Chandel, A. K., Kapoor, R. K., Singh, A., & Kuhad, R. C. (2007). Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501. Bioresource Technology, 98, 1947–1950. 7. Rahman, S. H. A., Choudhury, J. P., & Ahmad, A. L. (2006). Production of xylose from oil palm empty fruit bunches fiber using sulfuric acid. Biochemical Engineering Journal, 30, 97–103. 8. Neureiter, M., Danner, H., Thomasser, C., Saidi, B., & Braun, R. (2002). Dilute-acid hydrolysis of sugarcane bagasse at varying conditions. Applied Biochemistry and Biotechnology, 98–100, 49–58. 9. Galafassi, S., Cucchetti, D., Pizza, F., Franzosi, G., Bianchi, D., & Compagno, C. (2012). Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis. Bioresource Technology, 111, 398–403. 10. Huang, C., Zong, M. H., Wu, H., & Liu, Q. P. (2009). Microbial oil production from rice straw hydrolysate by Trichosporon fermentans. Bioresource Technology, 100, 4535–4538. 11. Yu, X., Zheng, Y., Dorgan, K. M., & Chen, S. (2011). Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresource Technology, 102, 6134– 6140. 12. Huang, C., Wu, H., Li, R. F., & Zong, M. H. (2012). Improving lipid production from bagasse hydrolysate with Trichosporon fermentans by response surface methology. New Biotechnology, 29, 372–378. 13. Tsigie, Y. A., Wang, C. Y., Truong, C. T., & Ju, Y. H. (2011). Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate. Bioresource Technology, 102, 9216–9222. 14. Rahman, S. H. A., Choudhury, J. P., Ahmad, A. L., & Kamaruddin, A. H. (2007). Optimization studies on acid hydrolysis of oil palm empty fruit bunches fiber for production of xylose. Bioresource Technology, 98, 554–559. 15. Zhang, D., Ong, Y. L., Li, Z., & Wu, J. C. (2012). Optimization of dilute acid-catalyzed hydrolysis of oil palm empty fruit bunches for high yield production of xylose. Chemical Engineering Journal, 181–182, 636–642.
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16. Boonsawang, P., Subkaree, Y., & Srinorakutara, T. (2012). Ethanol production from palm pressed fiber by prehydrolysis prior to simultaneous saccharification and fermentation (SSF). Biomass and Bioenergy, 40, 127–132. 17. Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry, 226, 497–509. 18. Rodrigues, R. C. L. B., Sene, L., Matos, G. S., Roberto, I. C., Pessoa, A. J. R., & Felipe, M. G. A. (2006). Enhanced xylitol production by pre-cultivation of Candida guilliermondii cells in sugarcane bagasse hemicellulosic hydrolysate. Current Microbiology, 53, 53–59. 19. Zhang, Y., Han, B., & Ezeji, T. C. (2011). Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation. New Biotechnology, 29, 345–351. 20. Gnansounou, E., & Dauriat, A. (2010). Techno-economic analysis of lignocellulosic ethanol: a review. Bioresource Technology, 101, 4980–4991. 21. Hendriks, A. T. W. M., & Zeeman, G. (2009). Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource Technology, 100, 10–18. 22. Dey, P., & Maiti, M. K. (2013). Molecular characterization of a novel isolate of Candida tropicalis for enhanced lipid production. Journal of Applied Microbiology, 114, 1357–1368. 23. Saenge, C., Cheirsilp, B., Suksaroge, T., & Bourtoom, T. (2011). Potential use of oleaginous red yeast Rhodotorula glutinis for the bioconversion of crude glycerol from biodiesel plant to lipids and carotenoids. Process Biochemistry, 46, 210–128. 24. Alexander, M. A., Chapman, T. W., & Jeffries, T. W. (1988). Xylose metabolism by Candida shehatae in continuous culture. Applied Microbiology and Biotechnology, 28, 478–486. 25. Ligthelm, M. E., Prior, B. A., Preez, J. C., & Brandt, V. (1988). An investigation of D-{1-13C} xylose metabolism in Pichia stipitis under aerobic and anaerobic conditions. Applied Microbiology and Biotechnology, 28, 293–296. 26. Hu, C., Zhao, X., Zhao, J., Wu, S., & Zhao, Z. K. (2009). Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloide. Bioresource Technology, 100, 4843–4847. 27. Almeida, J. R. M., Bertilsson, M., Gorwa-Grauslund, M. F., Gorsich, S., & Lidén, G. (2009). Metabolic effect of furaldehyde and impact on biotechnological process. Applied Microbiology and Biotechnology, 82, 625–638. 28. Liu, Z.L., Blaschek, H.P. (2010). Biomass inhibitors and in situ detoxification. In Biomass to biofuels: strategies for global industries. Wiley pp. 233-259. 29. Heer, D., Heine, D., & Sauer, U. (2009). Resistance of Saccharomyces serevisiae to high concentrations of furfural is based on NADPH-dependent reduction by at least two oxidoreductases. Applied and Environmental Microbiology, 75, 7631–7638. 30. Olsson, L., & Hahn-Hägerdal, B. (1996). Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme and Microbial Technology, 18, 312–331.
Lipid Production from Hemicellulose and Holocellulose Hydrolysate of Palm Empty Fruit Bunches by Newly Isolated Oleaginous Yeasts Srikanya Tampitak 1 & Yasmi Louhasakul 1 & Benjamas Cheirsilp 1 & Poonsuk Prasertsan 1
Received: 4 September 2014 / Accepted: 25 May 2015 / Published online: 31 May 2015 # Springer Science+Business Media New York 2015
Abstract Palm empty fruit bunches (EFBs) are abundant lignocellulosic wastes from palm oil mills. They are potential sources of sugars which can be converted to microbial lipids by oleaginous yeasts. To produce sugars from EFB, two-step and one-step hydrolysis reactions were performed. In the first step, the use of diluted sulfuric acid (0.5 % w/v) hydrolyzed hemicelluloses and released mainly pentoses, and in the second step of hydrolysis of residual pulp using 2.5 % (w/v), sulfuric acid released more hexoses. The use of 2.5 % (w/v) sulfuric acid in one-step hydrolysis of holocelluloses released the highest amount of sugars (38.3 g/L), but it also produced high concentration of potential inhibitors (>1 g/L). Three oleaginous yeasts, Rhodotorula mucilaginosa, Kluyveromyces marxianus, and Candida tropicalis, were isolated and screened for their ability to convert EFB hydrolysates into lipids. These yeasts grew well and produced lipids from EFB hemicellulose and holocellulose hydrolysate after potential inhibitors were removed. This study shows that EFB can be used for lipid production. Keywords Acid hydrolysis . Holocellulose . Hemicellulose . Oleaginous yeast . Palm empty fruit bunches
Introduction Biodiesel is an attractive replacement for petroleum diesel because it is domestically available, biodegradable, and compatible with existing diesel engines. At present, the high production cost of biodiesel, especially its raw material cost, is a major barrier to its commercialization. In addition, the use of pure vegetable oil as a raw material for biodiesel production could compete
* Benjamas Cheirsilp [email protected] 1
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
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with its use as an edible oil. Microbial lipids have been considered to be new sources of biodiesel feedstocks due to their plant-like oil composition. Microorganisms that can accumulate lipid up to more than 20 % of their biomass are defined as oleaginous species. Oleaginous yeasts have many advantages due to their fast growth rate, high lipid content, and the resemblance of their triacylglycerol fraction to plant oil. So far, many yeast species such as Cryptococcus albidus, Cryptococcus curvatus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans, and Yarrowia lipolytica have been intensively used for lipid production. Some of these species can accumulate intracellular lipid as high as 60 % of their dry weight [1, 2]. Bioconversion of lignocellulosic wastes into valuable products is of considerable interest because of their low cost, renewable nature, and abundance. The carbon sources for oleaginous microbes need to be extended to include lignocellulosic wastes, which allow for the production of large volumes of cheap microbial lipids. Currently, the palm oil industry has considerably expanded its production in Thailand, and this expansion has produced large volumes of lignocellulosic waste by-products including 13.5 % (w/w) palm pressed fiber (PPF), 22 % (w/w) palm empty fruit bunches (EFB), and 5.5 % (w/w) palm kernel cake (PK) [3]. Among these byproducts, EFBs are the most interesting biomass due to their high content of holocelluloses which are composed of celluloses (41.3–46.5 %, w/w) and hemicelluloses (25.3–33.8 %, w/w) [4]. They are considered to be potential sugar sources that can be converted to microbial lipid by oleaginous yeasts. EFB can be hydrolyzed into sugars by acid hydrolysis. However, the acid treatment generates various by-products resulting from the degradation of sugars and lignin, which are potential inhibitors of the yeasts [5]. One method to reduce the toxic by-products from the lignin is alkali pretreatment of lignocellulosic materials. This process yields holocellulose pulp that can be further hydrolyzed by acid. The pentoses in the hemicellosic fraction can be easily released by dilute acid hydrolysis due to the amorphous structure of the hemicelluloses [6]. In contrast, high concentrations of acid are required to release glucose from the highly crystallized cellulose fraction [7]. However, the use of such a high concentration of acid would result in further degradation of pentoses to furfural and glucose to 5-hydroxymethylfurfural (HMF) [8]. To keep the concentration of these by-products at a minimum, it is necessary to use diluted acid in the first step to hydrolyze hemicelluloses and then high concentrations of acid in the second step to hydrolyze the crystalline celluloses that remain as residual solid from the first step. Although this two-step hydrolysis can keep the concentration of by-products at a minimum, it is quite time-consuming. Because some microorganisms are potentially tolerant to the by-products or may even be able to consume them during fermentation [9], the hydrolysis of holocelluloses in a one-step process would be highly advantageous for such microorganisms. Recently, the use of hemicellulose hydrolysates as carbon sources for lipid production by oleaginous yeasts has been reported [5, 10–12], but only a few attempts have been made to use either cellulose hydrolysates [9] or holocellulose hydrolysates [13]. The aim of this study was to exploit EFB as a cheap and renewable raw material for the production of microbial lipids by newly isolated oleaginous yeasts. Although there have been many studies on the production of sugars from EFB [7, 14, 15], to our knowledge, there have been no attempts to use these sugars for lipid production. In this study, both two-step and one-step hydrolysis procedures were performed to convert EFBs into fermentable sugars. The oleaginous yeasts that can consume these sugars for growth and lipid production were screened and identified. The effect of detoxification methods on the growth and lipid production by the selected yeasts was also investigated.
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Materials and Methods Materials EFB samples from the Thai Tallow and Oil company (Suratthani, Thailand) were crushed using a high-speed rotary cutting mill (Patipong agriculture machinery, P5208, Thailand) to give particle sizes in the range of 0.1–0.5 cm. Alkaline pretreatment was employed to delignify the EFBs. Sodium hydroxide at 10 % (w/v) was used to pretreat EFBs at a ratio of 10:1 [16]. The mixture was treated at a temperature of 100 °C for 30 min. The pretreated samples were then washed and dried overnight at 60 °C.
Acid Hydrolysis of Palm EFBs The delignified EFB was hydrolyzed through either a two-step or one-step process using sulfuric acid solutions. In the two-step process, diluted sulfuric acid (0.5 % w/v) was used in the first step to hydrolyze the hemicellulose fraction of the delignified EFB in the first step to avoid degradation of sugar monomers into toxic by-products. The liquid fraction from this step was termed the “hemicellulose hydrolysate.” In the second step, sulfuric acid at a concentration of 2.5 % (w/v) was used to hydrolyze the residual pulp from the dilute acid hydrolysis step. The hydrolysate from the residual pulp was termed the Bresidual pulp hydrolysate.” The delignified EFB (holocellulose) was also hydrolyzed using 2.5 % (w/v) sulfuric acid in a one-step process. The hydrolysate from this step was termed the Bholocellulose hydrolysate.^ This reaction was carried out at 120 °C for 60 min in an autoclave. After the reaction was complete, the solids were separated from the aqueous solution by filtration. The filtrate was analyzed for xylose, arabinose, glucose, galactose, furfural, and HMF.
Screening and Identification of Oleaginous Yeasts The waste samples containing potential isolates were collected from soil and wastes from palm oil mills in the southern region of Thailand. Ten percent samples were enriched in yeast extract peptone dextrose (YPD) medium composed of 10 g/L yeast extract (HiMedia, India), 10 g/L peptone (HiMedia, India), and 20 g/L of either glucose or xylose as carbon sources with a pH of 4.0. Then, 0.1 mL diluted culture was inoculated on YPD agar medium using the spread plate technique and grown for 72 h at room temperature. The isolated yeasts were stained using the Sudan black B technique and observed under a phase contrast microscope for the presence of blue or grayish lipid globules within the cells. The yeast strains containing large lipid globules were selected for further quantitative analysis of their lipid content. The selected yeasts were then inoculated in EFB hemicellulose hydrolysate-based medium (20 g/L sugars from dilute acid hydrolysate, 2 g/L (NH4)2SO4, 0.2 g/L MgSO4·7H20, 0.5 g/L KH2PO4, 0.1 g/ L CaCl2·2H2O, pH 6.0) and incubated for 72 h at room temperature with shaking at 140 rpm. Selected oleaginous yeasts were identified based on their 26S ribosomal DNA (rDNA) sequence. The 26S rDNA was PCR-amplified using universal primer sets of F63-Forward (5′GCA TAT CAA GCG GAG GAA AAG-3′) and LR-Reverse (5′-GGT CCG TGT TTC AAG ACG G-3′) and sequenced using automated sequencer. The resulting sequences were BLAST searched against the National Center for Biotechnology Information (NCBI) database. The sequences that shared over 99 % similarity with currently known sequences were considered to be the same species and used to construct a phylogenetic tree.
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Biomass and Lipid Production by Oleaginous Yeasts Shake flask cultures in 250-mL Erlenmeyer flasks contained 90 mL of sugar- or hydrolysatebased medium. The pH was adjusted to 6.0. The medium was sterilized by autoclaving before use. The cultures were initiated with 10 % of a 24-h-old seed culture (approximately 107 cells/ mL) and incubated at room temperature (30±2 °C) on a rotary shaker at 140 rpm for 72 h. The samples were taken to measure biomass and lipid production.
Analytical Methods The cell pellet harvested following centrifugation was washed twice with distilled water and dried at 60 °C for 72 h. The final biomass was measured and expressed as dry cell weight (DCW). The extraction of lipid from the biomass was performed according to the modified procedure of Folch et al. [17]. One gram of dried biomass was ground into fine powder and blended with 20 mL of chloroform/methanol (2:1, v/v). The mixture was sonicated (Elma, E30H, Switzerland) for 30 min, and the solvent phase was recovered using centrifugation. The extraction process was repeated twice more. All collected solvent was evaporated using a rotary evaporator (EYELA, SB-651, Rikakikai Co. Ltd. Tokyo, Japan) and dried at 60 °C to constant weight (at least 48 h). The lipid content is expressed as a percentage of gram lipid per gram dried biomass. The lipid production was expressed as the gram lipid per liter of medium (g/L). The sugar composition was analyzed with high-performance liquid chromatography (HPLC) using an Agilent Technology 1100 series RID with an Aminex-87P column operated at 45 °C and a mobile phase containing 0.01 N of sulfuric acid pumped at a rate of 0.6 mL/min [18]. The furans in the samples were determined with a DU800 spectrophotometer. The maximum absorbance spectra of furfural and HMF were 276 and 282 nm, respectively. The concentrations of furfural and HMF in the samples were calculated using standards of known concentration [19]. All experiments were performed with at least three replicates. Analysis of variance was performed to identify any significant differences in the treatment mean values, and the least significant difference (p≤0.05), calculated using SPSS software, was used to separate means.
Results and Discussion Acid Hydrolysis of Palm EFBs Palm EFBs are considered to be potential sources of hemicelluloses and celluloses that could be converted to useful products. The hemicellulose, cellulose, holocellulose, and lignin contents of EFBs used in this study were 23.1±1.2 % (w/w), 40.8±1.2 % (w/w), 63.8± 1.9 % (w/w), and 16.9±1.2 % (w/w), respectively. The cellulose content in the EFBs was higher than that in other lignocellulosic biomasses such as rice straw (36.6 %, w/w), poplar (33.1 %, w/w), and switchgrass (29.7 %, w/w) [20]. The high cellulose content is one important advantage in using EFBs as potential feedstock for glucose production. To reduce lignin and increase the content of holocelluloses, an alkali pretreatment was applied to the EFBs. Alkaline pretreatment of lignocelluloses with sodium hydroxide was employed because of its effectiveness not only in reducing the lignin content but also in swelling and disrupting the crystalline structure of the cellulose [16, 21]. In this study, after alkaline pretreatment, lignin content was
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reduced to 11.6±0.3 % (w/w), and holocellulose content was increased up to at most 80.2± 0.9 % (w/w). The delignified EFB was sequentially hydrolyzed to avoid degradation of pentoses to byproducts at high acid concentrations. The first step in this process was the hydrolysis of hemicelluloses using diluted acid at 0.5 % (w/v), which resulted in a liquid fraction of hemicellulose hydrolysate. The residual pulp was further hydrolyzed using 2.5 % (w/v) acid, concentration that produced the highest total sugar (data not shown). It should be noted that at acid concentrations >2.5 % (w/v), the total sugar concentration decreased. This decrease was due to the degradation of pentoses to furfural and hexoses to HMF. The liquid fraction from this second step was the residual pulp hydrolysate. The delignified EFBs were also hydrolyzed using 2.5 % (w/v) sulfuric acid in a one-step process. This single-step process hydrolyzed both the hemicellulose and cellulose fractions in the delignified EFBs. The resulting hydrolysate was the holocellulose hydrolysate. Figure 1 shows the sugar composition in the hydrolysate from the two-step and one-step acid hydrolysis procedures. Dilute acid hydrolysis of the delignified EFBs mainly produced xylose from xylans and arabinose from arabinans in the hemicellulose fraction (Fig. 1a). These products formed because the hemicellulose fraction is more susceptible to hydrolysis with mild acid treatment than is the cellulose fraction, which needs more severe treatment because of its crystalline nature. Although xylose and arabinose were the main sugars found in the hemicellulose hydrolysate, other by-products such as glucose, furfural, and HMF were also produced in low amounts. It has been reported that the amount and type of sugars released during hydrolysis depend on the operating conditions [8]. When the residual pulp from the dilute acid hydrolysis was further hydrolyzed using 2.5 % (w/v) sulfuric acid, more glucose was released together with xylose. The concentrations of furfural and HMF in this residual pulp hydrolysate were higher than those found in the hemicellulose hydrolysate. The one-step hydrolysis of the holocellulose fraction using 2.5 % (w/v) sulfuric acid produced the highest amount of total sugars (38.3 g/L) together with high concentrations of furfural and HMF (0.57 and 0.44 g/L, respectively). Each hydrolysate was then diluted to adjust the sugar concentration to 20 g/L before being used as a carbon source for cultivation of the yeasts. It should be noted that after
2.0
30
1.5
20
1.0
10
0.5
0
0.0 Hemicellulose Residual pulp Holocellulose hydrolysate hydrolysate hydrolysate Glucose Furfural
Arabinose HMF
Xylose
50
2.5
40
2.0
30
1.5
20
1.0
10
0.5
0
Furfural (g/L), HMF (g/L)
40
Sugar (g/L)
(b) After sugar concentration adjustment 2.5 Furfural (g/L), HMF (g/L)
Sugar (g/L)
(a) Before sugar concentration adjustment 50
0.0 Hemicellulose Residual pulp Holocellulose hydrolysate hydrolysate hydrolysate Glucose Furfural
Arabinose HMF
Xylose
Fig. 1 Composition of sugars and inhibitors in the hydrolysate following two-step and one-step acid hydrolysis
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adjustment of the sugar concentration, the concentrations of furfural and HMF in the holocellulose hydrolysate were lower than those detected in the residual pulp hydrolysate (Fig. 1b).
Screening and Identification of Oleaginous Yeasts One hundred and thirty-eight strains of yeast were isolated from soils and wastes of the palm oil mill in the southern region of Thailand. These yeast strains were placed in YPD medium (pH 4.0) using glucose or xylose as a carbon source. The isolates were then stained with Sudan black B and observed under a phase contrast microscope to identify the presence of blue or grey lipid globules within the cells. Among the 138 isolates, 16 isolates and 11 isolates that showed large lipid globules when using glucose and xylose as a carbon source, respectively, were selected as potential lipid producers. These selected isolates were pre-cultured in an inoculum medium for 24 h and then inoculated into medium containing EFB hemicellulose hydrolysate as a carbon source. The biomass and lipid production of all yeasts was compared after 72 h (data not shown). When using hemicellulose hydrolysate as a carbon source in the screening medium, only three isolates of G43, X32, and X37 produced lipid concentrations of higher than 1.08 g/L. These three isolates were then identified based on their 26S rDNA sequences as Rhodotorula mucilaginosa (accession no. AB976563), Kluyveromyces marxianus (accession no. AB976564), and Candida tropicalis (accession no. AB976565) with 99 % similarity. Table 1 shows the fatty acid composition of the lipids from these three isolates. Over 70 % of the fatty acids in the yeast lipids were palmitic, stearic, and oleic acids, and these were similar to those of the plant oil, indicating their potential use as an alternative feedstock for biodiesel production. A phylogenetic tree was constructed as described in the methods. A search for similarities between D1/D2 26S rDNA of the isolates and that of those in the NCBI database showed that many phylogenetically related yeast species are similar to the isolates obtained in this study (Fig. 2). Although Rodotorula mucilaginosa and C. tropicalis are known as oleaginous yeasts [2, 22], this is the first time that K. marxianus has been shown to accumulate a high lipid content.
Biomass and Lipid Production of Yeasts Using Glucose and Xylose The three newly isolated oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), and C. tropicalis X37 (CX37), together with the reference
Table 1 Fatty acid composition of lipid from selected oleaginous yeasts Strain
Fatty acid composition (% w/w)a C12:0
C14:0
C16:0
Rodotorula mucilaginosa G43
0.22
1.49
25.1
K. marxianus X32
0.44
2.57
36.4
C. tropicalis X37
0.81
2.66
26.2
a
C16:1
C18:0
C18:1
C18:2
1.07
6.89
46.2
13.9
11.7 4.77
8.63 9.47
0.00 24.7
27.4 18.5
C18:3
C20:0
3.58
0.39
11.2 9.30
0.00 0.00
Percentage of fatty acids including lauric (C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), and arachidic acid (C20:0)
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Fig. 2 Phylogenetic tree of the newly isolated oleaginous yeast strains and previously reported oleaginous yeast strains. Strain number and sequence accession numbers are given. All strains shown are type strains. The three new isolates are depicted in bold
oleaginous yeast Rhodotorula glutinis TISTR 5159 (R5159) [23], were cultivated using glucose and xylose as carbon sources. Their biomass, lipid production, and lipid content are shown in Fig. 3. All yeasts grew well with xylose and yielded a higher biomass than those grown with glucose. Although the biomass values of all yeasts grown with xylose were not
Biomass Lipid Lipid content
60
4
40
2
20 0 RG43 KX32 CX37 R5159 Yeast strain
(b) Xylose Biomass Lipid Lipid content
80
6
60
4
40
2
20
0
Lipid content (%)
6
0
8
80 Biomass (g/L), Lipid (g/L)
(a) Glucose
Lipid content (%)
Biomass (g/L), Lipid (g/L)
8
0 RG43 KX32 CX37 R5159 Yeast strain
Fig. 3 Biomass and lipid production by the three newly isolated oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37), and reference oleaginous yeast Rhodotorula glutinis TISTR 5159 (R5159) cultivated with a glucose and b xylose. The lipid content was expressed as a percentage of the gram lipid per gram dried biomass
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much different, Rodotorula mucilaginosa G43 had the highest lipid production, which reached 1.04 g/L. The lipid contents of all yeasts grown with glucose were higher than those grown with xylose. Because all the tested yeasts can assimilate xylose better than glucose, it is feasible to use these yeasts to convert lignocellulosic sugars into lipid. It has been reported that the regulation of sugar transport across cell membranes plays a key role in determining how effectively microorganisms can assimilate xylose [24, 25]. According to those studies, xylose uptake in yeast occurs by both facilitated diffusion and active transport processes. Because xylose molecules are small enough to easily penetrate the yeast cell membrane, the yeast grow faster with xylose than with glucose. Several yeasts use xylose for growth and production of lipids such as Rhodotorula graminis [9], Rhodotorula mucilaginosa TJY15a [2], and Y. lipolytica Po1g [13]. However, the lipid content generated when using xylose is lower than that generated when using glucose.
Biomass and Lipid Production of Yeasts Using EFB Hydrolysate The biomass and lipid production of oleaginous yeasts using hemicellulose hydrolysate, residual pulp hydrolysate, and holocellulose hydrolysate of delignified EFB is shown in Fig. 4a–c, respectively. However, the biomass and lipid production of yeasts using all types of EFB hydrolysate, especially residual pulp hydrolysate, yielded poor results compared to that using pure glucose and xylose (Fig. 3). It should be noted that although the sugar composition in the residual pulp hydrolysate was similar to that in the holocellulose hydrolysate, no growth or lipid production was observed in the residual pulp hydrolysate. Higher concentrations of inhibitors in the residual pulp hydrolysate might contribute to this phenomenon (Fig. 1b). Hu et al. [26] reported that >1 mM furfural (>0.096 g/L) and >14.7 mM HMF (>1.85 g/L) inhibited cell growth and lipid production of the oleaginous yeast Rhodosporidium toruloides Y4. They also showed that these inhibitors most likely repressed cell growth more severely than did lipid biosynthesis. Depending on the source of the biomass and the type of hydrolysis employed, the concentration of these inhibitors in the lignocellulosic hydrolysates can range from 0.5 to 11 g/L [27]. These inhibitors are thought to damage cell walls and membranes and inhibit RNA synthesis in microorganisms [28, 29]. The acid hydrolysis of sugarcane bagasse produced furfural and HMF concentrations as high as 0.83 and 0.15 g/L, respectively [13].
40
2
20
0
0 RG43 KX32 CX37 R5159 Yeast strain
Biomass (g/L), Lipid (g/L)
4
(c) Holocellulose hydrolysate
6
60
4
40
2
20
0
0 RG43 KX32 CX37 R5159 Yeast strain
8
80
6
Biomass Lipid Lipid content
80 60
4
40
2
20
0
Lipid content (%)
60
Biomass Lipid Lipid content
Lipid content (%)
Biomass (g/L), Lipid (g/L)
(b) Residual pulp hydrolysate 8
80
Lipid content (%)
6
Biomass Lipid Lipid content
Biomass (g/L), Lipid (g/L)
(a) Hemicellulose hydrolysate 8
0 RG43 KX32 CX37 R5159 Yeast strain
Fig. 4 Biomass and lipid production by the oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37), and Rhodotorula glutinis TISTR 5159 (R5159) using a hemicellulose hydrolysate, b residual pulp hydrolysate, and c holocellulose hydrolysate of delignified EFBs. The lipid content is expressed as a percentage of the gram lipid per gram dried biomass
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In this study, the concentrations of HMF in all types of hydrolysate ranged from 0.1 to 0.4 g/L which were lower than the previously reported inhibitory levels (1.8–3.0 g/L) [11, 12, 26]. In contrast, the concentrations of furfural were higher than the previously reported inhibitory levels (0.09–0.2 g/L) [11, 12, 26]. The furfural concentrations in hemicellulose, residual pulp, and holocellulose hydrolysates were 0.22, 0.41, and 0.30 g/L, respectively. Therefore, the inhibitory effect on yeast cell growth was most likely due to the presence of furfural. In this study, growth and lipid accumulation was better using hemicellulose hydrolysate than using holocellulose hydrolysate. This result can be explained by the lower amount of furfural present in the hemicellulose hydrolysate. Thus, the detoxification of hydrolysate required to remove these inhibitors is thought to be essential in facilitating its efficient use as an alternative carbon source for lipid production by these oleaginous yeasts. In addition, the comparison of these three types of hydrolysates indicated that dilute acid hydrolysis is the most suitable method to hydrolyze the lignocellulosic materials because it yielded the lowest amount of inhibitory by-products, but the amount of sugars obtained was also lowest. Therefore, if the amount of total sugar and simple process was the most concern factors, the one-step hydrolysis would be more appropriate.
Detoxification of EFB Hydrolysate Normally, hydrolysate is neutralized with alkali prior to use. Other diverse methods have been proposed to accomplish this process including the following: adsorption by activated carbon, overliming (neutralization with lime), and filtration; partial removal of furfural and soluble lignin by molecular sieves; and vapor stripping for the removal of volatile inhibitors [30]. Because activated carbon is often used to remove compounds from the liquid phase, it was used to remove inhibitors from EFB hydrolysates in this study. To simultaneously neutralize acidic compounds and remove inhibitors, the EFB hydrolysate was detoxified using the overliming method. In this process, dried calcium hydroxide was added to the acidic hydrolysates for conversion to gypsum, which has commercial value as plaster of Paris. This method has also been used to remove volatile inhibitory compounds such as furfural and HMF [6]. The three types of EFB hydrolysates were then detoxified using activated carbon adsorption and overliming. Table 2 shows the effect of the detoxification method on the removal of inhibitors. Each potent inhibitor was reduced using either method. Furfural was reduced by Table 2 Effect of detoxification method on the concentrations of inhibitors present in the medium Hydrolysate
Detoxification method
Furfural (g/L)
HMF (g/L)
Hemicellulose
Non-detoxified
0.22±0.08
0.09±0.04
hydrolysate
Absorption
0.07±0.00 (68 %)
0.05±0.01 (44 %)
Overliming
0.10±0.01 (55 %)
0.07±0.01 (22 %)
Residual pulp hydrolysate
Non-detoxified Absorption
0.41±0.08 0.09±0.01 (78 %)
0.31±0.12 0.09±0.01 (71 %)
Overliming
0.12±0.00 (71 %)
0.13±0.01 (58 %)
Holocellulose
Non-detoxified
0.30±0.12
0.23±0.08
Absorption
0.08±0.01 (73 %)
0.08±0.01 (65 %)
Overliming
0.11±0.01 (63 %)
0.17±0.02 (26 %)
hydrolysate
Data in parentheses are percent removal of inhibitors
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68–78 % to a yield less than 0.10 g/L using the adsorption method. The overliming method also effectively removed furfural with a 55–71 % reduction. HMF was reduced by 44–71 and 22–58 % through adsorption and overliming, respectively. Overall, the adsorption method was better at removing the inhibitors than the overliming method. The percent removal also depended on the initial concentration of the inhibitors. Tsigie et al. [13] reported that the growth of Y. lipolytica Po1g was limited in non-detoxified sugarcane bagasse hydrolysate due to the presence of furfural and HMF at concentrations of 0.12 and 0.61 g/L, respectively. Overliming reduced the concentrations of furfural and HMF by 24.8 and 21.3 %, respectively. The yeast grew faster and better in the detoxified sugarcane bagasse hydrolysate. According to Huang et al. [10], overliming completely removed furfural while adsorption was more effective for the removal of HMF. Their results also showed that the oleaginous yeast Trichosporon fermentans was able to grow and efficiently accumulate lipids on rice straw hydrolysate after detoxification. Huang et al. [12] reported that T. fermentans grew and accumulated lipid better on sugarcane bagasse hydrolysate after detoxification.
Biomass and Lipid Production of Yeasts Using Detoxified EFB Hydrolysates Figures 5 and 6 show the biomass and lipid production of oleaginous yeasts on EFB hydrolysates detoxified by adsorption and overliming, respectively. All yeasts grew better and yielded a higher lipid content in the detoxified EFB hydrolysates than in the nondetoxified hydrolysates. This finding was most likely due to the lower concentration of inhibitors in the detoxified hydrolysates. All yeasts except Rhodotorula glutinis TISTR 5159 performed better in the detoxified hydrolysates than in medium containing pure glucose and xylose as carbon sources (Fig. 3). It is possible that the hydrolysate might contain some additional nutrients that could be used by the yeasts. Furthermore, the results of this study were consistent to those of Tsigie et al. [13], who also found that detoxified sugarcane bagasse hydrolysates yielded a higher biomass of Y. lipolytica Po1g than did pure glucose and xylose. In the detoxified hydrolysates, C. tropicalis X37 performed the best, followed by Rodotorula mucilaginosa G43 and K. marxianus X32, while Rhodotorula glutinis TISTR 5159 yielded the lowest biomass and lipid content. This yeast might be very sensitive to the inhibitors even at very low concentrations.
(b) Residual pulp hydrolysate
4
40
2
20
0
0 RG43 KX32 CX37 R5159 Yeast strain
(c) Holocellulose hydrolysate 80
6
60
4
40
2
20
0
0 RG43 KX32 CX37 R5159 Yeast strain
8
Biomass Lipid Lipid content
80
6
60
4
40
2
20
0
Lipid content (%)
60
Biomass Lipid Lipid content
Lipid content (%)
6
8
Biomass (g/L), Lipid (g/L)
80
Lipid content (%)
Biomass (g/L), Lipid (g/L)
Biomass Lipid Lipid content
Biomass (g/L), Lipid (g/L)
(a) Hemicellulose hydrolysate 8
0 RG43 KX32 CX37 R5159 Yeast strain
Fig. 5 Biomass and lipid production by the oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37), and Rhodotorula glutinis TISTR 5159 (R5159) using the EFB hydrolysates detoxified by the adsorption: a hemicellulose hydrolysate, b residual pulp hydrolysate, and c holocellulose hydrolysate. The lipid content is expressed as a percentage of the gram lipid per gram dried biomass
Appl Biochem Biotechnol (2015) 176:1801–1814
(b) Residual pulp hydrolysate
40
2
20 0 RG43 KX32 CX37 R5159 Yeast strain
Biomass (g/L), Lipid (g/L)
4
(c) Holocellulose hydrolysate
6
60
4
40
2
20
0
0 RG43 KX32 CX37 R5159 Yeast strain
8
80
Biomass Lipid Lipid content
80
6
60
4
40
2
20
0
Lipid content (%)
60
Biomass Lipid Lipid content
Lipid content (%)
6
0
8
80
Lipid content (%)
Biomass (g/L), Lipid (g/L)
Biomass Lipid Lipid content
Biomass (g/L), Lipid (g/L)
(a) Hemicellulose hydrolysate 8
1811
0 RG43 KX32 CX37 R5159 Yeast strain
Fig. 6 Biomass and lipid production by the oleaginous yeasts Rodotorula mucilaginosa G43 (RG43), K. marxianus X32 (KX32), C. tropicalis X37 (CX37) and Rhodotorula glutinis TISTR 5159 (R5159) using EFB hydrolysates detoxified by overliming: a hemicellulose hydrolysate, b residual pulp hydrolysate, and c holocellulose hydrolysate. The lipid content is expressed as a percentage of the gram lipid per gram dried biomass
It should be noted that both detoxification methods gave similar results, but due to economic reasons, the overliming method was chosen. Moreover, this process also produced gypsum, a commercially valuable by-product after overliming. Among the yeasts tested, C. tropicalis X37 gave the highest lipid production of 2.73 g/L using residual pulp hydrolysates, suggesting that it is the most suitable strain for being cultivated in EFB hydrolysates. The higher lipid content in C. tropicalis X37 is likely due to the higher glucose content in the residual pulp hydrolysates since glucose has been shown to be more suitable for lipid accumulation (Fig. 3a). Table 3 compares the lipid production of oleaginous yeasts using various sources of lignocellulosic biomass. Lignocellulosic biomass is a potential low-cost feedstock due to its abundance and sustainability. Several attempts with lignocellulosic biomass have shown that it is possible to use hydrolysates from agricultural wastes for lipid production. However, a single crop residue/biomass would not be sufficient to meet the huge demand for biofuel. With this view, there is a need to explore new and abundant agricultural substrates for lipid production. Moreover, lipid production depends on many factors such as yeast species, sugar concentration, nitrogen sources and concentration, and the culture conditions. In most studies, costly nitrogen sources such as yeast extract, peptone, or a combination were used. In the study by Huang et al. [10, 12], T. fermentans yielded a very high concentration of lipid due to the high concentration of sugar used in their study. However, its lipid yield was lower than that of Y. lipolytica [13], C. curvatus [11], and C. tropicalis. These results indicate that palm EFBs are another lignocellulosic biomass source that could be used as feedstock for lipid production. Further optimization and development will improve the efficiency of the yeast in converting EFB hydrolysates into lipids.
Conclusions This study has shown that palm EFBs are potential low-cost feedstock for sugar production and microbial lipid production. The sugar and composition in the hydrolysates of palm EFBs were varied using one-step and sequential hydrolysis. One-step hydrolysis could release higher
Hemicellulose (116.9 g/L)
Hemicellulose (123.5 g/L)
Holocellulose (20 g/L)
Hemicellulose (20 g/L) Residual pulp (20 g/L) Holocellulose (20 g/L)
Rice straw
Sugarcane bagasse
Sugarcane bagasse
Palm empty fruit bunch
c
b
15.8
–a
–a
Y. lipolytica C. tropicalis
(NH4)2SO4 (2 g/L)
T. fermentans
1.61 2.73 1.31
6.81 6.18
6.68
3.5
6.44
11.4
11.5
14.7
L. starkeyi T. fermentans
4.6
13.8
Rhodotorula glutinis
5.8
17.2
C. curvatus
0.4
Lipid (g/L)
7.8
Biomass (g/L)
Y. lipolytica
Strain
Peptone (5 g/L)
Yeast extract (0.5 g/L) + peptone (2.2 g/L)b
Yeast extract (0.5 g/L) + peptone (1.8 g/L)
Yeast extract (1.5 g/L)
Nitrogen source (concentration)
Lipid yield was calculated by gram lipid per gram substrate
Concentration was calculated from C/N ratio of 165
Not available
Hemicellulose (40 g/L)
Wheat straw
a
Hydrolysate (sugar concentration)
Feedstock
Table 3 Lipid production from lignocellulosic biomass using oleaginous yeasts
0.07
0.14
0.08
0.33
0.13
0.09
0.12
0.09
0.15
0.01
Lipidc yield (g/g)
This work
Tsigie et al. [13]
Huang et al. [10] Huang et al. [12]
Yu et al. [11]
Ref.
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amount of sugars, but it also produced high concentration of potential inhibitors. In sequential hydrolysis, the use of diluted acid in the first step could effectively hydrolyze hemicelluloses and the use of higher concentration of acid in the second step could further hydrolyze celluloses in the residual pulp with low concentration of inhibitors. The newly isolated oleaginous yeasts Rhodotorula mucilaginosa G43, K. marxianus X32, and C. tropicalis X4 could grow and accumulate lipid in palm EFB hydrolysates after detoxification. Among them, C. tropicalis X4 is the most robust strain for lipid production. Since the use of palm EFBs will not conflict with food usage, it has great potential as a feedstock for industrialized lipid production. Acknowledgements This research was financially supported by the Graduate School of Prince of Songkla University and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission under Grant No. AGR540567c. The third and fourth authors are supported by Thailand Research Fund under Grant No. RTA5780002. Thanks are also given to Dr. Brian Hodgson for his assistance with the English language.
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