Feasibility of lipid production from waste paper by the oleaginous yeast Cryptococcus curvatus

Abstract

Waste paper was studied as a potential source for lipid production using the oleaginous yeast Cryptococcus curvatus for the first time. Three common types of waste paper, office paper, newspaper, and cardboard, were directly hydrolyzed by an enzyme cocktail to generate sugar-rich and nitrogen-limited hydrolysates. When these hydrolysates were used without any auxiliary nutrients by C. curvatus, the lipid content and lipid yield were higher than 50% and 200 mg/g, respectively. The nitrogen-rich enzyme cocktail exerted no negative effects on lipid production. Moreover, the integrated processes of enzymatic hydrolysis and lipid fermentation achieved comparable lipid yield to the separate hydrolysis and lipid production process. The resulting lipid samples had similar fatty acid compositional profiles to those of vegetable oils, which suggested their potential for biodiesel production. These findings strongly supported waste paper as appealing substrates for lipid production via oleaginous yeast, which provided cost-effective waste paper-to-lipids routes for sustainable biodiesel production.

Full Article

Feasibility of Lipid Production from Waste Paper by the Oleaginous Yeast Cryptococcus curvatus

Wenting Zhou,^a Zhiwei Gong,^a,* Linfang Zhang,^a Yi Liu,^a Jiabao Yan,^a and Mi Zhao ^b

Waste paper was studied as a potential source for lipid production using the oleaginous yeast Cryptococcus curvatus for the first time. Three common types of waste paper, office paper, newspaper, and cardboard, were directly hydrolyzed by an enzyme cocktail to generate sugar-rich and nitrogen-limited hydrolysates. When these hydrolysates were used without any auxiliary nutrients by C. curvatus, the lipid content and lipid yield were higher than 50% and 200 mg/g, respectively. The nitrogen-rich enzyme cocktail exerted no negative effects on lipid production. Moreover, the integrated processes of enzymatic hydrolysis and lipid fermentation achieved comparable lipid yield to the separate hydrolysis and lipid production process. The resulting lipid samples had similar fatty acid compositional profiles to those of vegetable oils, which suggested their potential for biodiesel production. These findings strongly supported waste paper as appealing substrates for lipid production via oleaginous yeast, which provided cost-effective waste paper-to-lipids routes for sustainable biodiesel production.

Keywords: Cryptococcus curvatus; Waste paper; Nitrogen limitation; Microbial lipid; Biodiesel

Contact information: a: School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, 947 Heping Road, Wuhan 430081, PR China; b: China Carbon Balance Energy and Tech LTD, 1 Jianguomenwai Avenue, Beijing 100004, PR China;

*Corresponding author: gongzhiwei@wust.edu.cn

INTRODUCTION

As all major economies race to search for regenerative energy sources, biodiesel has been recognized as a sustainable and renewable substitute for petroleum diesel. However, large-scale biodiesel production is severely hampered by the high cost and limited supply of raw materials such as vegetable oils, animal fats, waste cooking oil, etc. Microbial lipids prepared from oleaginous species have captured much attention in recent years as perfect candidates for biodiesel production and substitutes for high value-added exotic fats such as polyunsaturated fatty acids (PUFA) and cocoa butter equivalents (Raltedge 1993; Papanikolaou and Aggelis 2010). However, the costs for microbial lipid production remain too high to be competitive. Techno-economic evaluation has demonstrated that the substrates requirement and the fermentation process are the two principal elements hindering the commercialization of lipid production (Koutinas et al. 2014).

To reduce the cost, considerable efforts have been devoted to exploring low-cost substrates for lipid production (Huang et al. 2013). Meanwhile, attempts have also been made to achieve higher titer, yield, and productivity, which are pivotal for better techno-economics of microbial lipid technology (Li et al. 2007; Gong et al. 2014).

Lignocellulose, annually produced in enormous quantities, is mainly composed of 40% to 50% cellulose, 25% to 30% hemicelluloses, 15% to 20% lignin, and 3% to 10% proteins (Chundawat et al. 2011; Menon and Rao 2012). Glucose and xylose, the two major sugars derived from lignocellulose, can be co-consumed for lipid biogenesis (Hu et al. 2011; Tsigie et al. 2011; Ruan et al. 2012; Zikou et al. 2013; Gong et al. 2016a). Various lignocellulosic residues, such as corn stover (Ruan et al. 2013; Gong et al. 2014; Xue et al. 2015), rice straw (Huang et al. 2009), wheat straw (Yu et al. 2011), corncob (Huang et al. 2012), rice hulls (Economou et al. 2011), sugarcane bagasse (Tsigie et al. 2011), and sorghum bagasse (Liang et al. 2012), have been investigated for lipid production. Moreover, acetic acid when present together with lignocellulosic sugars can serve as a building block for lipid production (Ruan et al. 2015; Gong et al. 2016a). However, there are at least three factors hindering the cost competitiveness of the lignocellulose-to-lipids routes. First, high-cost pretreatment technologies are generally required to break the rigid structure of lignocellulose (Himmel et al. 2007). Second, various inhibitors, such as furan aldehydes, aliphatic acids, and phenolic compounds, are inevitably generated during the pretreatment process, which are toxic to the oleaginous yeasts, hindering cell growth and lipid accumulation (Yu et al. 2014). Third, lignocellulose contains roughly 3% to 10% of proteins and minor amounts of other nitrogenous components, which disfavor lipid production, because lipid accumulation is commonly induced by nitrogen limitation (Ratledge and Wynn 2002; Chundawat et al. 2011; Huang et al. 2011).

Waste paper, derived from cellulosic biomass, is roughly composed of 40% to 80% cellulose, 5% to 15% hemicellulose, and negligible amounts of lignin and proteins (Ioelovich 2014). More than 400 million tons of waste paper are generated annually around the world (Shi et al. 2009). Recently, waste paper has been reintegrated for various valuable bio-products production (Nishimura et al. 2016). However, it is scarcely studied for lipid production. There are two distinct advantages of using waste paper over virgin lignocellulose for lipid production. On the one hand, energy-intense or corrosive reagents mediated pretreatments are not required to alleviate the biomass recalcitrance of waste paper because pretreatment has already been conducted during the pulping process (Elliston et al. 2013). In contrast, most lignocellulose-derived inhibitors and nitrogenous components have been removed through washing during the upstream papermaking processes. Therefore, waste paper may be amenable to generate sugar-rich and nitrogen-limited hydrolysates through direct enzymatic hydrolysis, which can potentially serve as appealing substrates for cost-effective microbial lipid production.

Cryptococcus curvatus has several highly desirable features for lipid production, such as broad feedstock spectrum, high tolerance to lignocellulose derived inhibitors, and high lipid accumulation under a relatively low carbon-to-nitrogen (C/N) molar ratio (Yu et al. 2011; Gong et al. 2016b). Various low-cost lignocellulosic residues have been applied for lipid production by C. curvatus (Yu et al. 2011; Liang et al. 2012; Gong et al. 2016a,b). The goal of this study is to explore the possibility of waste paper as preferable substrates for lipid production. Three typical categories of waste paper; office paper, newspaper, and cardboard, were evaluated for lipid production by C. curvatus. The enzymatic hydrolysates, without auxiliary nutrient supplementation, were investigated for lipid production. Further simultaneous saccharification and lipid production (SSLP) on office paper was investigated. To the authors’ knowledge, this is the first report on the feasibility of waste paper-to-lipids routes by oleaginous species, which provides valuable information for cost-effective lipid production.

EXPERIMENTAL

Materials

Strain and medium

Cryptococcus curvatus ATCC 20509 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), stored at 4 °C and propagated twice a month on yeast peptone dextrose (YPD) agar slants (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L, agar 15 g/L, pH 6.0).

Preparation of waste paper enzymatic hydrolysates

Shredded office paper with an average size of 2 mm × 8 mm and moisture content of 5.0% (w/w) was supplied by the Dean’s office of Wuhan University of Science and Technology (Wuhan, China). Cardboard and newspaper, with moisture contents of 8.7% and 7.5%, respectively, were obtained from the local reclamation depot (Wuhan, China). The samples were shredded into pieces with comparable sizes to the office paper. Cellulase, β-glucosidase, and xylanase were used as described (Gong et al. 2016a). Waste paper was adjusted to a pH of 4.8 with sulphuric acid (H₂SO₄), loaded at 10% (w/v) solid loading and hydrolyzed at 50 °C for 72 h in the presence of 15 FPU/g cellulase, 30 CBU/g β-glucosidase, 5 mg/g xylanase, and 50 μg/mL of ampicillin. The enzymatic hydrolysates were boiled for 10 min before vacuum filtration to remove the residual solids and precipitated proteins. The liquid hydrolysates, without any auxiliary nutrient supplementation, were adjusted to a pH of 5.5 and sterilized by autoclaving at 121 °C for 20 min before used as culture media.

Lipid production on waste paper enzymatic hydrolysates

Pre-cultures were grown in 50 mL YPD liquid medium (yeast extract 10 g/L, peptone 10 g/L, glucose 20 g/L, pH 6.0) at 30 °C for 24 h unless otherwise specified. Lipid production cultures were initiated by adding 5 mL of pre-culture to 45 mL of the waste paper enzymatic hydrolysates in 250-mL unbaffled conical flasks, and incubated at a rotary rate of 200 rpm at 30 °C. The cultivation pH was adjusted to 5.5 in 12 h time intervals. Experiments were conducted in triplicates and data were presented as mean value ± standard deviation.

SSLP on waste paper

Experiments were conducted in 250-mL conical flasks with 5.0 g of office paper at 10% (w/v) solid loading. The culture pH was adjusted to 5.2 with H₂SO₄ and sterilized by autoclaving at 121 °C for 20 min. Next, an enzyme cocktail containing 15 FPU/g cellulase, 30 CBU/g β-glucosidase, and 5 mg/g xylanase was then supplemented. Cultures were initiated by adding 5.0 mL of the pre-culture, and held at 30 °C, 200 rpm for 120 h. The cultivation pH was adjusted to 5.2 in 12 h time intervals.

Methods

Glucose was determined using a glucose analyzer (SBA-40E, Shandong Academy of Sciences, Jinan, China). Xylose was measured by a K-XYLOSE assay kit from Megazyme (Wicklow, Ireland). The total reducing sugars (TRS) were quantified according to the dinitrosalicylate (DNS) method with glucose as the standard (Miller 1959).

Nitrogen was determined according to the Kjeldahl method (Morgan et al. 1957). Total nitrogen concentration and C/N ratio were calculated according to the published equations (Gong et al. 2016b). The crude proteins content was roughly calculated by multiplying Kjeldahl nitrogen by a factor of 6.25.

Structural carbohydrates, lignin, and ash contents of waste paper were analyzed according to the procedures of the National Renewable Energy Laboratory (NREL) (Sluiter et al. 2008a, 2008b).

Cells from 30 mL of the culture broth were harvested by centrifugation and washed twice with distilled water. The cell mass was determined gravimetrically after drying the wet cells at 105 °C overnight.

Lipid extraction was performed according to a previously published procedure (Gong et al. 2012). Specifically, dried cells were digested with 4 M HCl at 78 °C for 1 h and extracted twice with chloroform/methanol (1:1, v/v). The extracts were washed with 0.1% NaCl, dried over anhydrous sodium sulphate (Na₂SO₄) and then evaporated in a vacuum. The residue was dried at 105 °C overnight to a constant weight. For the SSLP process, lipids were obtained via extraction according to the above method followed by a petroleum ether partition step (Gong et al. 2014). Lipid content was expressed as gram lipid per gram lipid-containing dry cell weight. Lipid yield was calculated as gram lipid per gram sugars consumed.

The fatty acid compositional profiles of lipid samples were analyzed using a Shimadzu 2010 plus gas chromatography (GC) instrument (Shimadzu, Kyoto, Japan) after transmethylation according to a published procedure (Gong et al. 2016a).

Statistical analysis

An analysis of variance (ANOVA) and multiple comparisons were performed to identify the significant differences. Tukey’s post hoc test was conducted to analyze significant differences among the experimental data of various substrates. A P value of less than 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS software (SPSS Inc., Version 17.0, Chicago, USA).

RESULTS AND DISCUSSION

Compositional Analysis and Enzymatic Hydrolysis of Waste Paper

The chemical composition of the three types of waste paper was measured according to the procedures of the NREL (Sluiter et al. 2008a, 2008b). As shown in Table 1, the office paper consisted of 60.3% glucan, 11.7% xylan, 1.4% lignin, 0.4% crude proteins, and 23.4% ash. It contained high amounts of sugar polymers (72%) and minimal lignin, as alkaline delignification during papermaking was conducted to concentrate cellulose and remove lignin (Elliston et al. 2013). The nitrogen content was negligible, which indicated nitrogen-limited hydrolysates could have been generated. The relative high ash content was mainly due to kaolin and calcite used as mineral fillers and CaCO₃ used as white pigment during papermaking (Ioelovich 2014). Similarly, newspaper and cardboard also contained large amounts of sugar polymers and negligible amounts of proteins (Table 1), which could also favor the generation of hydrolysates with high C/N ratios. However, lignin exceeding 10% was observed in both newspaper and cardboard, which were noticeably higher than that in office paper.

Table 1. Chemical Composition of Three Common Types of Waste Paper

Office paper, newspaper, and cardboard were directly hydrolyzed using an enzyme cocktail containing 15 FPU/g cellulase, 30 CBU/g β-glucosidase, and 5 mg/g xylanase, respectively. The hydrolysis was conducted for 72 h at 10% (w/v) solid loading. The calcium carbonate (CaCO₃) exerted a strong inhibitory effect on cellulase, while calcium sulfate (CaSO₄) caused no negative effects (Wang et al. 2011). Thus, H₂SO₄ was used to adjust the pH of the waste paper slurries. The results are shown in Fig. 1. A one-way ANOVA followed by Tukey’s post hoc test demonstrated that TRS yield was decreased significantly (P < 0.05) among different substrates following the order of office paper, newspaper, and cardboard (Fig. 1). For office paper, glucose and xylose was 37.3 g/L and 7.3 g/L, which corresponded to 55.7% and 54.9%, respectively, of the theoretical yields. The formation of 49.2 g/L TRS suggested that roughly 61% of the polymeric carbohydrates were hydrolyzed to soluble reducing sugars. Thus, office paper could be remarkably hydrolyzed by an enzyme cocktail, as pretreatment had already been conducted during the pulping process. Glucose and xylose in newspaper was 29.0 g/L and 6.5 g/L, respectively, which was inferior to those in office paper. The hydrolytic processes of cardboard generated 27.7 g/L glucose, 4.9 g/L xylose, and 35.8 g/L TRS. This data was 25.7%, 32.9%, and 27.2% lower than those in office paper. It was indicated that cardboard was the most recalcitrant substrate for enzymes accessibility, probably because of the lower levels of delignification. Overall, the sugar polymers that ranged from 50% to 60% were deconstructed into monomeric sugars.

Lipid Production on Waste Paper Enzymatic Hydrolysates by C. curvatus

Office paper, newspaper, and cardboard enzymatic hydrolysates were then investigated for their feasibility as sole nutrient sources for lipid production. The hydrolysate was boiled for 10 min and filtered to remove precipitated enzymes before use. The results are summarized in Fig. 2 and Table 2. The liquid hydrolysates of office paper contained 33.3 g/L glucose and 6.6 g/L xylose. The C/N ratio was calculated as 45.9, which was significantly lower than expected. This was because some nitrogenous components were introduced into the hydrolysates along with the enzyme cocktail. When the hydrolysates, without any auxiliary nutrients, were used to culture C. curvatus, glucose and xylose were both below the limit of detection within 48 h (Fig. 2A). The overall sugar consumption rate reached 0.83 g/L/h, which was even significantly (P < 0.05) higher than that on 40 g/L of glucose or xylose (Gong et al. 2016a). The cell mass, lipid titre, lipid content, and lipid productivity was 17.3 g/L, 9.1 g/L, 52.5%, and 4.4 g/L/d, respectively (Table 2). The lipid yield reached 201.4 mg/g, which indicated that C. curvatus favored lipid biosynthesis rather than cell growth. As shown in Fig. 3, the lipid yield was equal to 98.2 g lipid per kg raw office paper supplied, which was identical to that from corn stover through simultaneous saccharification and an enhanced lipid production process (Gong et al. 2014). Furthermore, significantly better lipid production results, i.e., lipid content, lipid yield, and lipid productivity, were achieved on the office paper hydrolysates than those on other low-cost substrates rich in glucose and xylose such as rice straw hydrolysates (Huang et al. 2009), wheat straw hydrolysates (Yu et al. 2011), corn stover hydrolysates (Hu et al. 2011; Huang et al. 2011; Ruan et al. 2013), corncob hydrolysates (Huang et al. 2012), and corncob residues hydrolysates (Gao et al. 2014). These superior results might have been attributed mainly to the nitrogen limited condition and the absence of lignocellulose-derived inhibitors.

Fig. 2B & 2C. Profiles of substrates consumption, cell growth, and lipid production on enzymatic hydroylsates of office paper (A), newspaper (B), and cardboard (C) by C. curvatus

Similarly, newspaper and cardboard hydrolysates contained few amounts of nitrogenous components, which was beneficial for lipid production. The C/N ratios reached 47.2 and 53.7, respectively. When the culture was initiated on the newspaper hydrolysates containing 26.3 g/L glucose and 5.9 g/L xylose, all glucose was consumed and residual xylose was only 0.7 g/L within 36 h (Fig. 2B).

Table 2. Results of Lipid Production on Various Waste Paper Enzymatic Hydrolysates by C. curvatus

^a: Office paper, newspaper, and cardboard enzymatic hydrolysates are abbreviated as OPEH, NPEH, and CBEH, respectively

Cell mass, lipid content, and lipid yield was 14.7 g/L, 51.4%, and 208.9 mg/g, respectively (Table 2, Entry 2). Similarly, when cardboard hydrolysate was used to culture C. curvatus, glucose and xylose was consumed within 36 h. Lipid content and lipid yield reached 56.4% and 224.4 mg/g, respectively, which was the highest of the three (Table 2, Entry 3). However, the lipid yield corresponded to 75.7 kg per ton raw cardboard, which was significantly (P<0.05) lower than that on office paper, due to lower sugar yield during enzymatic hydrolysis (Fig. 3). It was crucial to further improve the sugar yields during the waste paper hydrolysis process, which should provide hydrolysates with more building blocks and higher C/N ratios to advance lipid production. Higher sugar yield is expected through the demineralization of the waste paper (Ioelovich 2014).

As shown in Fig. 2, C. curvatus assimilated glucose quickly from 0 h to 24 h, whereas xylose almost kept constant, regardless of the variations in substrates. Xylose consumption started until the glucose concentration dropped below 10.0 g/L, suggesting that the yeast strongly prefers glucose over xylose (Kim et al. 2010). This phenomenon was in line with the results of C. curvatus and Mortierella isabellina on lignocellulosic hydrolysates (Ruan et al. 2015; Gong et al. 2016b). However, this finding was inconsistent with results reported for other oleaginous species such as Trichosporon cutaneum, Trichosporon fermentans, Thamnidium elegans, etc., probably due to the strain diversities (Hu et al. 2011; Zikou et al. 2013; Liu et al. 2015). The sequential manner of sugars assimilation was probably because glucose can repress the utilization of xylose via a catabolite repression mechanism or allosteric competition for sugar transporters (Kawaguchi et al. 2006).

The theoretical lipid yields on glucose and xylose by oleaginous species are 330 mg/g and 340 mg/g, respectively (Ratledge 1988). However, the experimental results were rarely higher than 220 mg/g (Papanikolaou and Aggelis 2011). The high lipid yields that ranged from 201.4 mg/g to 224.4 mg/g suggested that carbon sources in the hydrolysates were mainly channeled into lipid biosynthesis. These data were comparable to those obtained by Yarrowia lipolytica and Rhodosporidium toruloides on glucose (Qiao et al. 2015; Tchakouteu et al. 2017). Overall, the authors’ results indicated that waste paper hydrolysates could serve as appealing substrates for lipid production.

Enzymes were retained in the liquid hydrolysates when the enzymatic hydrolysates were directly filtered to remove residual solids without boiling. The cell mass, lipid titre, lipid content, and lipid yield reached 17.6 g/L, 9.2 g/L, 52.2%, and 204.1 mg/g, respectively, when hydrolytic enzymes were present in the office paper hydrolysates (Fig. 4). These data were comparable to those on the hydrolysates with enzymes removed, which demonstrated that proteins exerted no negative effect on lipid accumulation. Lipid accumulation by C. curvatus generally occurs under nutrients limitation, preferably nitrogen starvation (Ratledge and Wynn 2002). It indicated that proteins were invalid nitrogen sources, probably because C. curvatus was unable to secrete proteases, which might be the major reason that lipid accumulation by C. curvatus could be triggered under relatively lower C/N molar ratios than other oleaginous yeasts (Li et al. 2007; Angerbauer et al. 2008; Gong et al. 2016b). Overall, there was no need to remove the hydrolytic enzymes of the hydrolysates, which could simplify the processes.

SSLP on Waste Paper by C. curvatus

Integrating the processes of enzymatic hydrolysis and lipid production seemed to have great potential to avoid product inhibition, reduce enzyme usage, and simplify the processes for the conversion of cellulosic biomass into lipids. In fact, SSLP has been investigated for lipid production from lignocellulose by oleaginous species, such as C. curvatus and T. cutaneum (Liu et al. 2012; Gong et al. 2014). In this study, waste office paper was directly used according to the SSLP process by C. curvatus without auxiliary nutrients. As shown in Fig. 5, glucose and TRS rapidly accumulated up to 6.7 g/L and 11.4 g/L, respectively, within 6 h, which indicated that the substrates consumption was rate-limiting. The values then both rapidly dropped to 0.6 g/L and 3.1 g/L at 24 h. Xylose increased continuously and reached a plateau concentration of 1.5 g/L at 24 h, probably because of the glucose repression. Glucose and xylose were both below the limit of detection after 48 h, which suggested that the sugars became limiting for yeast assimilation. Lipid production increased over time. Lipid titre and lipid yield were 9.6 g/L and 96.2 mg/g, respectively, when the culture was terminated at 120 h. These values were three times higher than that of corn stover by T. cutaneum using the SSLP process (Liu et al. 2012), probably because less nitrogenous components existed in waste paper than in corn stover. Moreover, the lipid yield was comparable to that obtained from the separate hydrolysis and lipid production (SHLP) process. Thus, SSLP could simplify the processes while achieving comparable lipid production results. However, lipid productivity was only 1.9 g/L/d, which was significantly lower than that by the SHLP process, probably because of the limited supply of sugars during culture. The coordination of waste paper enzymatic hydrolysis and sugars assimilation is essential for further improving the efficiency of the SSLP process.

Fatty Acid Compositional Profiles of Lipid Samples

Lipid samples originated from the three waste paper types were transmethylated, and the resulting fatty acid methyl esters were analyzed by GC. The results are shown in Table 3. It was clear that those samples consisted mainly of long-chain fatty acids with 16 and 18 carbon atoms. The four major fatty acids were palmitic acid, stearic acid, oleic acid, and linoleic acid, regardless of the variations in substrates. Specifically, linoleic acid accounted for around 50% of the total fatty acids. It was demonstrated that these lipids could neither serve as substitutes for PUFA nor cocoa butter equivalents (Raltedge 1993). However, the fatty acid compositional profiles were comparable with those of vegetable oils, indicating that microbial lipids produced from waste paper could be excellent precursors for biodiesel production (Liu and Zhao 2007).

Table 3. Fatty Acid Profiles of Lipid Samples of C. curvatus on Various Types of Waste Paper

^a: Office paper, newspaper, and cardboard enzymatic hydrolysates are abbreviated as OPEH, NPEH, and CBEH, respectively; ^b: Lipid samples were derived from the SSLP process

CONCLUSIONS

1. Three common types of waste paper hydrolysates were found to be appealing substrates for lipid production by C. curvatus.
2. A nitrogen-rich enzyme cocktail exerted no negative effects on lipid production.
3. The SSLP process could simplify the processes and achieve comparable lipid yield to the SHLP process.
4. Microbial lipids from waste paper could serve as excellent precursors for biodiesel production.

ACKNOWLEDGMENTS

The authors are grateful for the support of the National Natural Science Foundation of China (51608400), the Science and Technology Research Project of Education Department of Hubei Province (Q20161114), the Young Scientists Foundation of Wuhan University of Science and Technology (2016XG002), and the innovative team of bioaugmentation and advanced treatment on metallurgical industry wastewater.

REFERENCES CITED

Angerbauer, C., Siebenhofer, M., Mittelbach, M., and Guebitz, G. M. (2008). “Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production,” Bioresource Technol. 99(8), 3051-3056. DOI: 10.1016/j.biortech.2007.06.045

Chundawat, S. P. S., Beckham, G. T., Himmel, M. E., and Dale, B. E. (2011). “Deconstruction of lignocellulosic biomass to fuels and chemicals,” Annu. Rev. Chem. Biomol. Eng. 2, 121-145. DOI: 10.1146/annurev-chembioeng-061010-114205

Economou, C. N., Aggelis, G., Pavlou, S., and Vayenas, D. V. (2011). “Single cell oil production from rice hulls hydrolysate,” Bioresource Technol. 102(20), 9737-9742. DOI: 10.1016/j.biortech.2011.08.025

Elliston, A., Collins, S. R. A., Wilson, D. R., Roberts, I. N., and Waldron, K. W. (2013). “High concentrations of cellulosic ethanol achieved by fed batch semi simultaneous saccharification and fermentation of waste-paper,” Bioresource Technol. 134, 117-126. DOI: 10.1016/j.biortech.2013.01.084

Gao, Q. Q., Cui, Z. Y., Zhang, J., and Bao, J. (2014). “Lipid fermentation of corncob residues hydrolysate by oleaginous yeast Trichosporon cutaneum,” Bioresource Technol. 152, 552-556. DOI: 10.1016/j.biortech.2013.11.044

Gong, Z. W., Wang, Q., Shen, H. W., Hu, C. M., Jin, G. J., and Zhao, Z. K. (2012). “Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production,” Bioresource Technol. 117, 20-24. DOI: 10.1016/j.biortech.2012.04.063

Gong, Z. W., Shen, H. W., Yang, X. B., Wang, Q., Xie, H. B., and Zhao, Z. K. (2014). “Lipid production from corn stover by the oleaginous yeast Cryptococcus curvatus,” Biotechnol. Biofuel. 7, 158. DOI: 10.1186/s13068-014-0158-y

Gong, Z. W., Zhou, W. T., Shen, H. W., Yang, Z. H., Wang, G. H., Zuo, Z. Y., Hou, Y. L., and Zhao, Z. K. (2016a). “Co-fermentation of acetate and sugars facilitating microbial lipid production on acetate-rich biomass hydrolysates,” Bioresource Technol. 207, 102-108. DOI: 10.1016/j.biortech.2016.01.122

Gong, Z. W., Zhou, W. T., Shen, H. W., Zhao, Z. K., Yang, Z. H., Yan, J. B., and Zhao, M. (2016b). “Co-utilization of corn stover hydrolysates and biodiesel-derived glycerol by Cryptococcus curvatus for lipid production,” Bioresource Technol. 219, 552-558. DOI: 10.1016/j.biortech.2016.08.021

Himmel, M. E., Ding, S. Y., Johnson, D. K., Adney, W. S., Nimlos, M. R., Brady, J. W., and Foust, T. D. (2007). “Biomass recalcitrance: Engineering plants and enzymes for biofuels production,” Science 315(5813), 804-807. DOI: 10.1126/science.1137016

Huang, C., Zong, M. H., Wu, H., and Liu, Q. P. (2009). “Microbial oil production from rice straw hydrolysate by Trichosporon fermentans,” Bioresource Technol. 100(19), 4535-4538. DOI: 10.1016/j.biortech.2009.04.022

Huang, X., Wang, Y. M., Liu, W., and Bao, J. (2011). “Biological removal of inhibitors leads to the improved lipid production in the lipid fermentation of corn stover hydrolysate by Trichosporon cutaneum,” Bioresource Technol. 102(20), 9705-9709. DOI: 10.1016/j.biortech.2011.08.024

Huang, C., Chen, X. F., Xiong, L., Chen, X. D., and Ma, L. L. (2012). “Oil production by the yeast Trichosporon dermatis cultured in enzymatic hydrolysates of corncobs,” Bioresource Technol. 110, 711-714. DOI: 10.1016/j.biortech.2012.01.077

Huang, C., Chen, X. F., Xiong, L., Chen, X. D., Ma, L. L., and Chen, Y. (2013). “Single cell oil production from low-cost substrates: The possibility and potential of its industrialization,” Biotechnol. Adv. 31(2), 129-139. DOI: 10.1016/j.biotechadv.2012.08.010

Hu, C. M., Wu, S. G., Wang, Q., Jin, G. J., Shen, H. W., and Zhao, Z. K. (2011). “Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum,” Biotechnol. Biofuel. 4, 25. DOI: 10.1186/1754-6834-4-25

Ioelovich, M. (2014). “Waste paper as promising feedstock for production of biofuel,” J. Sci. Res. Rep. 3, 905-916. DOI: 10.9734/JSRR/2014/8025

Kawaguchi, H., Vertes, A. A., Okino, S., Inui, M., and Yukawa, H. (2006). “Engineering of a xylose metabolic pathway in Corynebacterium glutamicum,” Appl. Environ. Microb. 72(5), 3418-3428. DOI: 10.1128/AEM.72.5.3418-3128.2006

Kim, J. H., Block, D. E., and Mills, D. A. (2010). “Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass,” Appl. Microbiol. Biot. 88(5), 1077-1085. DOI: 10.1007/s00253-010-2839-1

Koutinas, A. A., Chatzifragkou, A., Kopsahelis, N., Papanikolaou, S., and Kookos, I. K. (2014). “Design and techno-economic evaluation of microbial oil production as a renewable resource for biodiesel and oleochemical production,” Fuel. 116, 566-577. DOI: 10.1016/j.fuel.2013.08.045

Li, Y. H., Zhao, Z. K., and Bai, F. W. (2007). “High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture,” Enzyme. Microb. Tech. 41(3), 312-317. DOI: 10.1016/j.enzmictec.2007.02.008

Liang, Y. N., Tang, T. Y., Umagiliyage, A. L., Siddaramu, T., McCarroll, M., and Choudhary, R. (2012). “Utilization of sorghum bagasse hydrolysates for producing microbial lipids,” Appl. Energ. 91(1), 451–458. DOI: 10.1016/j.apenergy.2011.10.013

Liu, B., and Zhao, Z. K. (2007). “Biodiesel production by direct methanolysis of oleaginous microbial biomass,” J. Chem. Technol. Biot. 82(8), 775-780. DOI: 10.1002/jctb.1744

Liu, W., Wang, Y. M., Yu, Z. C., and Bao, J. (2012). “Simultaneous saccharification and microbial lipid fermentation of corn stover by oleaginous yeast Trichosporon cutaneum,” Bioresource Technol. 118, 13-18. DOI: 10.1016/j.biortech.2012.05.038

Liu, Z. J., Liu, L. P., Wen, P., Li, N., Zong, M. H., and Wu, H. (2015). “Effects of acetic acid and pH on the growth and lipid accumulation of the oleaginous yeast Trichosporon fermentans,” BioResources 10(3), 4152-4166. DOI: 10.15376/biores.10.3.4152-4166

Menon, V., and Rao, M. (2012). “Trends in bioconversion of lignocelluloses: Biofuels, platform chemicals & biorefinery concept,” Prog. Energ. Combust. 38(4), 522-550. DOI: 10.1016/j.pecs.2012.02.002

Miller, G. L. (1959). “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Anal. Chem. 31(3), 426-428. DOI: 10.1021/ac60147a030

Morgan, G. B., Lackey, J. B., and Gilcreas, F. W. (1957). “Quantitative determination of organic nitrogen in water, sewage, and industrial wastes,” Anal. Chem. 29(5), 833-835. DOI: 10.1021/ac60125a029

Nishimura, H., Tan, L., Sun, Z. Y., Tang, Y. Q., Kida, K., and Morimura, S. (2016). “Efficient production of ethanol from waste paper and the biochemical methane potential of stillage eluted from ethanol fermentation,” Waste. Manage. 48(8), 644-651. DOI: 10.1016/j.wasman.2015.11.051

Papanikolaou, S., and Aggelis, G. (2010). Yarrowia lipolytica: A model microorganism used for the production of tailor‐made lipids. Eur. J. Lipid. Sci. Tech. 112(6), 639-654. DOI: 10.1002/ejlt.200900197

Papanikolaou, S., and Aggelis, G. (2011). “Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production,” Eur. J. Lipid. Sci. Tech. 113(8), 1031-1051. DOI: 10.1002/ejlt.201100014

Qiao, K., Abidi, S. H. I., Liu, H. J, Zhang, H. R, Chakraborty, S., Watson, N., Ajikumar, P. K., and Stephanopoulos, G. (2015). “Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica,” Metab. Eng. 29, 56-65. DOI: 10.1016/j.ymben.2015.02.005

Ratledge, C. (1988). “Biochemistry, stoichiometry, substrate and economics,” in: Single Cell Oil, R. S. Moreton (ed.), Longman, London, pp. 33-70.

Ratledge, C. (1993). “Single cell oils—have they a biotechnological future?” Trends. Biotechnol. 11(7), 278-284. DOI: 10.1016/0167-7799(93)90015-2

Ratledge, C., and Wynn, J. P. (2002). “The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms,” Adv. Appl. Microbiol. 51, 1-51. DOI: 10.1016/S0065-2164(02)51000-5

Ruan, Z. H, Zanotti, M., Wang, X. Q, Ducey, C., and Liu, Y. (2012). “Evaluation of lipid accumulation from lignocellulosic sugars by Mortierella isabellina for biodiesel production,” Bioresource Technol. 110, 198-205. DOI: 10.1016/j.biortech.2012.01.053

Ruan, Z. H, Zanotti, M., Zhong, Y., Liao, W., Ducey, C., and Liu, Y. (2013). “Co-hydrolysis of lignocellulosic biomass for microbial lipid accumulation,” Biotechnol. Bioeng. 110(4), 1039-1049. DOI: 10.1002/bit.24773

Ruan, Z. H., Hollinshead, W., Isaguirre, C., Tang, Y. J. J, Liao, W., and Liu, Y. (2015). “Effects of inhibitory compounds in lignocellulosic hydrolysates on Mortierella isabellina growth and carbon utilization,” Bioresource Technol. 183, 18-24. DOI: 10.1016/j.biortech.2015.02.026

Shi, A. Z., Koh, L. P., and Tan, H. T. W. (2009). “The biofuel potential of municipal solid waste,” Global Change Biology Bioenergy 1(5), 317-320. DOI: 10.1111/j.1757-1707.2009.01024.x

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., and Crocher, D. (2008a). Determination of Structural Carbohydrates and Lignin in Biomass (NREL/TP-510-42618), National Renewable Energy Laboratory, Golden, CO.

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., and Templeton, D. (2008b). Determination of Ash in Biomass (NREL/TP-510-42622), National Renewable Energy Laboratory, Golden, CO.

Tchakouteu, S. S., Kopsahelis, N., Chatzifragkou, A., Kalantzi, O., Stoforos, N. G., Koutinas, A. A., Aggelis, G., and Papanikolaou, S. (2017). “Rhodosporidium toruloides cultivated in NaCl‐enriched glucose‐based media: Adaptation dynamics and lipid production,” Eng. Life Sci. 17(3), 237-248. DOI: 10.1002/elsc.201500125

Tsigie, Y. A., Wang, C. Y., Truong, C. T., and Ju, Y. H. (2011). Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate,” Bioresource Technol. 102(19), 9216-9222. DOI: 10.1016/j.biortech.2011.06.047

Wang, X. S., Song, A. D., Li, L. P., Li, X. H., Zhang, R., and Bao, J. (2011). “Effect of calcium carbonate in waste office paper on enzymatic hydrolysis efficiency and enhancement procedures,” Korean J. Chem. Eng. 28(2), 550-556. DOI: 10.1007/s11814-010-0365-6

Xue, Y. P., Jin, M. J., Orjuela, A., Slininger, P. J., Dien, B. S., Dale, B. E., and Balan, V. (2015). “Microbial lipid production from AFEX^TM pretreated corn stover,” RSC Adv. 5(36), 28725-28734. DOI: 10.1039/c5ra01134e

Yu, X. C., Zheng, Y. B., Dorgan, K. M., and Chen, S. L. (2011). “Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid,” Bioresource Technol. 102(10), 6134-6140. DOI: 10.1016/j.biortech.2011.02.081

Yu, X. C., Zeng, J. J., Zheng, Y. B., and Chen, S. L. (2014). “Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus,” Process. Biochem. 49(3), 457-465. DOI: 10.1016/j.procbio.2013.10.016

Zikou, E., Chatzifragkou, A., Koutinas, A. A., and Papanikolaou, S. (2013). “Evaluating glucose and xylose as cosubstrates for lipid accumulation and γ‐linolenic acid biosynthesis of Thamnidium elegans,” J. Appl. Microbiol. 114(4), 1020-1032. DOI: 10.1111/jam.12116

Article submitted: March 10, 2017; Peer review completed: May 22, 2017; Revised version received and accepted: May 30, 2017; Published: June 6, 2017.

DOI: 10.15376/biores.12.3.5249-5263