Catalytic Decomposition of the Oleaginous Yeast ... - ACS Publications

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Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Catalytic Decomposition of the Oleaginous Yeast Cutaneotrichosporon Oleaginosus and Subsequent Biocatalytic Conversion of Liberated Free Fatty Acids Martina K. Braun,† Jan Lorenzen,‡ Mahmoud Masri,‡ Yue Liu,† Eszter Baráth,*,† Thomas Brück,*,‡ and Johannes A. Lercher*,†,§ Department of Chemistry and Catalysis Research Center and ‡Werner Siemens-Chair of Synthetic Biotechnology, Technische Universität München, Lichtenbergstrasse 4, D-85748 Garching bei München, Germany § Institute for Integrated Catalysis, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, Washington 99352, United States ACS Sustainable Chem. Eng. Downloaded from pubs.acs.org by LUND UNIV on 03/06/19. For personal use only.

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S Supporting Information *

ABSTRACT: A single step catalytic cell wall lysis and triglyceride hydrolysis combined with the enzymatic conversion of lipids using the oleaginous yeast Cutaneotrichosporon oleaginosus (ATCC 20509) as a model is described. Catalytic decomposition of yeast cells resulted in hydrolysis of about a third of cellular polysaccharides and all triglycerides. Enzymatic processing of the lipid fraction with an oleate hydratase from Stenotrophomonas maltophilia led to conversion of oleic acid to 10-hydroxystearic acid (10-HSA) (50%) without additional purification. Cell wall polysaccharides were depolymerized by in situ formed amino acids from cell protein fragments. The activity of the in situ generated, free amino acids was higher compared to that of additionally added acids. Studies with the cellobiose and β-(1→3)-glucan indicated that glutamic and aspartic acids, which are the dominant amino acids in yeast cells, are surprisingly more effective in hydrolysis in aqueous phase than sulfuric acid. This points to a concerted mechanism of glycosidic ether bond cleavage catalyzed by amino acids rather than to a pathway catalyzed by hydronium ions. The overall yield of the presented downstream process at 453 K resulted in the release of 80% of total lipids. KEYWORDS: Cellobiose, β-Glucan, Hydrolysis, Amino acid, Oleaginous yeast, Cutaneotrichosporon oleaginosus

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tially present from thermocatalytic biomass pretreatment.2 Its genetic accessibility3 and increasingly characterized physiology4,5 make this uncommon yeast a suitable candidate for sustainable production of microbial lipids. Transformation of yeast biomass to biofuels and chemicals requires the disruption of cell walls, consisting mainly of carbohydrates, to release the main cell components, lipids and sugars.6 Practically, disintegration of a rigid cell wall such as that of C. oleaginosus is achieved by harsh chemical treatments, including the use of acids and alkaline hydrolysis,7 or by application of mechanical high-pressure homogenizers.8 Conversely, less mechanically intense physical processing such as shear, osmotic shock, or sonication, which are typically applied for Saccharomyces cerevisiae (S. cerevisiae) or other oily microorganisms,7,9 are not really effective in the case of C. oleaginosus.10 Common methods, like the Bligh and Dyer11

INTRODUCTION The route to a sustainable bioeconomy requires the development of robust biobased processes, which enable mass- and energy-efficient transformation of complex biomass into value adding chemical building blocks. In this respect, the application of oleaginous yeast and microalgae for the generation of biofuels and oleochemicals gains significant momentum due to their efficient cultivation and bio-oil product yields in excess of 50% w/dwbiomass (w/dwbiomass = weight/dry weight of biomass). The nonconventional, oleaginous yeast, Cutaneotrichosporon oleaginosus (C. oleaginosus), is receiving increased attention in process development, as it can accumulate up to 70% w/dwbiomass intracellular lipids1 when cultivated on cost-efficient biomass hydrolysates. Most notably, C. oleaginosus can metabolize a wide range of neutral and highly modified sugars, such as mannose, xylose, galacturonic acid, glucuronic acid, and N-acetylglucosamine, without any remarkable catabolite repression. Moreover, this yeast can be efficiently cultivated even in the presence of conventional fermentation inhibitors, such as furfural, poten© XXXX American Chemical Society

Received: September 19, 2018 Revised: January 30, 2019 Published: February 20, 2019 A

DOI: 10.1021/acssuschemeng.8b04795 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Figure 1. Concept of the combination of chemical and enzymatic transformation of yeast as crude material.

solvents such as MIBK and enables usage and recycling of sugar monomers. We further proved the process concept by a partial conversion of the resulting free fatty acids (FFAs) to 10hydroxysteric acid, used as a precursor for lactones, lubricant additives, waxes, plastics, biopolymers, and coatings.16−18 Monomeric sugars, the side products of the hydrothermal treatment, were converted to bioethanol via a fermentative process of S. cerevisiae (DSMZ 4266).

extraction, use nonfood compliant organic solvents (n-hexane, methanol, chloroform). The combination of these methods for cell disruption and lipid extraction lowers, therefore, the quality of the final product and leads to a cost-intensive and energetically inefficient downstream processing.12−14 According to United States Department of Health and Human Services (U.S. DHHS) and United States Food and Drug Administration (U.S. FDA), solvents like n-hexane and chloroform should not exceed a limit of 290 and 60 ppm (pharmaceutical products), respectively. Methyl isobutyl ketone (MIBK) is an alternative strong lysis component that is much less toxic having a limit of 4500 ppm. However, while these methods are technically feasible, they do not present a viable option for future economic operation to retrieve the components of oleaginous yeasts and microalgae. The mass fraction of the cell wall from C. oleaginosus accounts for 12% of the dry biomass.15 The cell wall of C. oleaginosus mainly consists of neutral carbohydrates (63% of cell wall dry mass), while proteins (11%), sugar acids (glucuronic acid: 13%) , and chitin (9%) make up a smaller fraction of the yeast cell wall.15 Neutral carbohydrates contain alkali-soluble (β-(1→3)(1→6)-glucan) and alkali-insoluble β(1→3)-glucan. The monomeric sugar fraction mainly consists of glucose and traces of mannose and galactose, respectively. To utilize these components requires processing at this stage of cell wall destruction. Thus, the economically and ecologically viable utilization of yeast lipids requires a cost-efficient and environmentally friendly extraction process. The applied method should minimize chemical treatment for cell disruption and the use of highly toxic solvents for lipid extraction and should allow for the use of the remaining yeast biomass. A potential concept is schematically depicted in Figure 1. In this study, we address and demonstrate the consecutive cell lysis and intracellular lipid hydrolysis of the oleaginous yeast C. oleaginosus by combining hydrothermal treatment in the presence of an endogenous amino acid acting as catalyst. The presented process allows the use of less toxic organic

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MATERIALS AND METHODS

Chemicals. All chemicals were purchased commercially: Lglutamic acid (≥99%), L-aspartic acid (≥98%), glycine (≥98.5%), (D-(+)-cellobiose, β-(1→3)-glucan (from Euglena gracilis), galacturonic acid (≥97%), D-(+)-glucose (≥99.5%), D-(+)-xylose (≥99%), L-rhamnose (≥99%), L-(+)-arabinose (≥99%), D-(+)-mannose (≥99%), D-(+)-galactose (≥99%), D-(−)-fructose (≥99%), formic acid (≥95%), acetic acid (≥99%), levulinic acid (≥97%), 5(hydroxymethyl)furan-2-carbaldehyde (HMF) (99%), furfural (≥99%), ninhydrin reagent solution (2%), CDCl3 (99.96% D, 0.03% (v/v) TMS), glyceryl stearate (≥99%), oleic acid (≥99%), and methyl isobutyl ketone (≥99.5%) are from Sigma-Aldrich; laminaripentaose, -tetraose, -triose, and -biose are produced by Santa Cruz Biotechnology; H-MFI-90 (H-MFI) zeolite was obtained from Clariant. Yeast. C. oleaginosus (ATCC 20509) was obtained from Deutsche Stammsammlung von Mikroorganismen and Zellkulturen (DMSZ in Braunschweig, Germany). Detailed cultivation procedure is described in the Supporting Information. Part of the yeast suspension was lyophilized to complete dryness for analysis of the biomass composition, and the results are given in Table 1. The rest of the yeast suspension was stored at 253 K. The cold suspension was defrosted prior to each experiment. The standard operating procedure by Kjeldahl et al. was used to determine the protein amount. Dry yeast biomass (2 g) was sent for digestion (InKjel M, Behr labor technic-Germany), followed by distillation (Vapodest 10, Gerhardt Germany). The remaining ash was determined by incineration of 1.5 g of sample at 1273 K for 3 h, and protein content was assessed by measuring total organic nitrogen content. Sugar Analysis. Biomass was treated with two steps of H2SO4 at concentrations of 1% and 2% at 120 °C for 1 h, subsequently. B


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ACS Sustainable Chemistry & Engineering Table 1. Cell Components Analysis of C. oleaginosus component protein lipid ash sugar glucose xylose, mannose, galactose, fructose mannitol fucose rhamnose

amount of dry yeast or solid residue was heated in air at 723 K and ashed overnight.

biomass C. oleaginosus (% w/ dwbiomass)

Table 2. Elemental Composition of Yeast Dry Mass and the Three Separated Parts (Lipids (A), Solid Residue (B), and Hydrolysate (C))a

5.3 ± 0.55 51 ± 1.26 1.2 ± 0.11 42 ± 0.45 32 ± 0.15 7 ± 0.03 1 ± 0.09 0.7 ± 0.02 1.4 ± 0.15

Resulted sugars were measured by HPLC system, using Agilent 1100 series HPLC with a (Shodex, RI101) refractive index detector and Dioden Array Detector (DAD); injection volume was 10 μL. A Rezex ROA-Organic Acid H+ (8%, 300 × 7.8 mm, Phenomenex) column was used with the eluent (5.0 mM H2SO4) at a flow rate of 0.5 mL min−1. The column oven was set at 343 K, and the RI detector was set at 313 K. Representative HPLC chromatograms are given in the Supporting Information (Figure S1). Ethanol Production. Wild type baker’s yeast (S. cerevisiae) was obtained from the German Microbial Strain Collection (DSMZ, Braunschweig, Germany). S. cerevisiae (DSMZ 4266) was activated on YPD Media for 2 days. The starter culture was transferred to 500 mL Erlenmeyer flasks containing 250 mL of the yeast hydrolysate. Starting optical density (OD) at 600 nm was 0.1. Cultivation was carried out for 70 h, at 28 °C with 120 rpm. Biomass growth was monitored at OD600 nm. Ethanol was measured using the ethanol assay kit (alcohol dehydrogenase kit K-ETOH 02/17) from Megazyme (Bray, Ireland). Hydrothermal Treatment of Yeast Cells and Model Polysaccharides Representing Yeast Cell Wall Components. Hydrothermal transformation of yeast was carried out in a stainless steel closed batch reactor with a Teflon inset. Typically, the reactor was loaded with 40 mL of yeast suspension and a certain amount of acid catalyst and then sealed. Under 500 rpm agitation, the reactor was heated to the desired temperature within 10 min. After the desired reaction time, the reactor was quenched by cooling with a water/ice bath. Each value in the kinetic curve consists of one separate experiment. Small soluble sugar products (1−5 sugar units) in the hydrolysate were analyzed on the Agilent HPLC. Amino acid concentration in the hydrolysate was determined via ninhydrin reaction of the amine function using glutamic acid as a standard.19 For this measurement, 1 mL of 2% ninhydrin reagent solution was mixed with 2 mL of diluted aqueous phase sample and treated at 375 K for 10 min. Absolute ethanol (5 mL) was added to the sample, and it was analyzed on a UV/vis spectrophotometer from Hitachi (U-3000) using the absorbance at 570 nm. The lipids were first extracted with 20 mL of methyl isobutyl ketone (MIBK) several times and separated from aqueous phase by centrifugation. Then the lipids were obtained after vaporizing MIBK under reduced pressure. The reactions of cellobiose and β-(1→3)-glucan were performed in pressure-resistant glass tubes. Typically, the sugar reactant, acid catalyst, and deionized water were filled into the tube. Then the tube was placed into a preheated oil bath under agitation of 500 rpm. After the reaction, the tube was cooled to room temperature in a water/ice bath. A new reaction was started for every point in the kinetic curves. Glucose formation was analyzed with HPLC (see Sugar Analysis section). Elemental analysis of dry yeast and corresponding reaction products for C, N, S, and H was performed on EURO EA (HEKA tech) equipment. Samples for P analysis were heated in concentrated acid prior to measurement. The amount of P was detected as phosphate (in heteropoly vanadate) with a UV/vis spectrophotometer at 410 nm absorbance (Shimadzu UV 160). Ash content (Table 2) was determined according to the method of Pramer et al.,20 where an

a

elemental composition (% w/ dwbiomass)

yeast dry mass

lipids (A)

solid residue (B)

hydrolysate (C)

C H N S P Ob ash O/C O/N C/H

63 ± 0.5 10 ± 0.4 1 ± 0.01 BDL 0.6 ± 0.02 25 ± 0.8 3 ± 0.04 0.4 ± 0.04 22 ± 0.9 6.3 ± 0.1

76 ± 1.1 12 ± 0.1 0.05 ± 0.01 BDL n.d. 13 ± 0.2 n.d. 0.2 ± 0.01 275 ± 59 6.3 ± 0.05

53 ± 0.2 6.8 ± 0.2 5 ± 0.05 0.3 ± 0.005 n.d. 32 ± 1.3 2 ± 0.03 0.6 ± 0.02 5.9 ± 0.2 7.8 ± 0.21

35 ± 0.2 5.9 ± 0.1 2.4 ± 0.1 1.2 ± 0.1 n.d. 56 ± 0.3 n.d. 1.6 ± 0.02 23 ± 0.7 5.9 ± 0.1

BDL = below detection limit, n.d.= not determined. amount calculated by 100 − all other elements.

b

Oxygen

ICP-OES of the trace metals in the hydrolysate was measured on an Agilent ICP-OES 700. Standards were prepared from ICP-multiple elements standard IV from Merck to give 50, 10, and 1 ppm solutions. Measurements were performed at five wavelengths to exclude signaling interactions. Oleate Hydratase Cloning. The oleate hydratase coding gene Smlt209321 from Stenotrophomonas maltophilia (S. maltophilia) was chosen as a template for a codon-optimized gene-synthesis (lifetechnologies, GeneArt) in an Escherichia coli (E. coli) host strain. The resulting synthetic gene was subcloned in a pET28a expression plasmid and transformed into chemically competent E. coli BL21DE3 cells. Protein Expression and Production of Cell Free Extracts. The expression of the oleate hydratase was carried out in E. coli BL21DE3 cells, grown in Lysogeny Broth (LB) medium. Precultures were inoculated from a cryostock and grown overnight in 100 mL of LB in a 500 mL baffled flask at 37 °C and 120 rpm. Main cultures were grown up to an optical density, measured at 600 nm (OD600), of 0.6−0.8 at 310 K, before the expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. After 16 h of incubation at 289 K, the cells were harvested, and the resuspended (20 mM Tris-HCl buffer + 25 mM imidazole pH 7.2) cell pellets were disrupted by high-pressure homogenization (EmulsiFlex-B15, AVESTIN). A subsequent centrifugation step at 20.000 g for 40 min at 277 K was applied for the separation of the cell debris from the soluble protein fraction. The soluble protein fraction was utilized as cell free extract for the conversion of the yeast-derived lipid fractions. By using the cell free extract for the conversion reaction, a cost-intensive purification of the protein can be prevented. Enzymatic Conversion of Yeast-Derived Lipids by the Oleate Hydratase from Stenotrophomonas Maltophilia. Three different yeast-derived lipid fractions, obtained from thermal treatment at 393 K (I), 433 K (II), and 453 K (III), were selected for the conversion reaction with the oleate hydratase. The lipid fractions were liquefied at 333 K. Subsequently, 10 mg of each lipid fraction was preincubated in 500 μL phosphate-citrate buffer (pH 6.5) for 2 h at 308 K. After the lipids dissolved completely, 500 μL of E. coli cell free extract, containing the expressed oleate hydratase (5 mg mL−1), was added, and the enzymatic conversion was carried out at 308 K for 90 min. The lipids were extracted from the reaction mixture by the addition of an equal volume of ethyl acetate (EtOAc), instant mixing, and subsequent separation of the organic phase, comprising the lipid C


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ACS Sustainable Chemistry & Engineering fraction. As a negative control the same reaction was carried out with E. coli lysate that did not contain recombinant oleate hydratase. Lipid Analysis. The direct transesterification of the converted yeast lipids was performed according to a modified protocol of Griffiths et al.22 with the following modifications: replacement of the C17-TAG (TAG/triacylglycerol) by a C12- TAG, replacement of BF3 methanol by a HCl−methanol solution, and the C19-ME (ME/ methyl ester) was omitted. The conversion rate of TAG to fatty acid methyl esters (FAMEs) was calculated to be about 90−95%. Subsequently, the resulting FAME extract was injected into a Thermo Scientific TRACE Ultra Gas Chromatograph coupled to a Thermo DSQ II mass spectrometer and the Triplus Autosampler injector. Column: Stabilwax fused silica capillary (30 m × 0.25 mm, film thickness 0.25 μm). Program: initial column temperature 323 K, increasing (4 K min−1) up to a final temperature of 523 K. Carrier gas: hydrogen, flow rate 3.5 mL min−1. Peaks were identified by comparison to a marine oil standard (Restek) or by specific molecular masses detected.

hydrolysate after the hydrothermal treatment was tested as cultivation media for ethanol production, as alternative for terrestrial biomass. The hydrolysate was used without further nutrient addition or optimization. S. cerevisiae showed ability to metabolize the sugar contained in the hydrolysate and convert it to ethanol (see Figure S3). The ethanol titer (7.5 g L−1) was comparable to other ethanol production procedures, using thermochemically processed biomass hydrolysates with wild type baker’s yeast as the fermentation organism.24 A similar thermal treatment (T = 215 °C) was applied prior to enzymatic hydrolysis of sugar cane bagasse and subsequent yeast fermentation to obtain ethanol.24 Ethanol production of the final hydrolysate showed a similar titer of 7.4 g L−1, while a charcoal-based detoxification step prior to yeast cultivation was not required in the present study. In this experimental setup, the obtained ethanol titer is relatively low compared to industrial bioethanol processes, which is due to the application of a quite diluted yeast hydrolysate and the utilization of a publicly available yeast strain (S. cerevisiae, DSMZ 4266). Therefore, the current experiment demonstrates that the applied yeast hydrolysate can in principle be used as a basemedium for ethanol production. In this particular process setup, a higher productivity can be achieved by adding an additional nutrients (i.e., by supplementation of yeast extract) and the use of an industrially optimized yeast production strain that is capable of tolerating high product titers. Elemental Composition of Yeast Dry Mass and Separated Fractions. The dry biomass of C. oleaginosus consists of 63 ± 0.5% w/dwbiomass carbon, 10 ± 0.4% w/ dwbiomass hydrogen, 25 ± 0.8% w/dwbiomass oxygen, 1 ± 0.01% w/dwbiomass nitrogen, and 0.6 ± 0.02% w/dwbiomass phosphorus (Table 2). Sulfur content was below the detection limit ( Glu/2.24 × 10−4 mol L−1. The calculated pH, pKa, and pKb values showed similar trend (pH: H2SO4/2.23 > Asp/3.45 > Glu/3.65; pKa: H2SO4/3.67 > Asp/ 4.58 > Glu/4.98; pKb: H2SO4/10.33 < Asp/9.42 < Glu/9.02) as was it presented in the case of the cellobiose. The higher catalytic activity of Glu and Asp is more evident in the hydrolysis of β-(1→3)-glucan. β-(1→3)-Glucan, the main component of the yeast cell wall, is an insoluble, solid sugar polymer consisting of glucose linked by (1→3)glycosidic bonds. Thus, the hydrolysis of β-(1→3)-glucan better mimics the real hydrolysis reaction at the yeast cell wall (Figure 6). The negative control (50 mg β-(1→3)-glucan, 5 mL of water, 433 K) experiment did not show hydrolysis of the substrate β-(1→3)-glucan. The amino acid catalysts Asp and Glu showed higher or comparable catalytic activities for the hydrolysis of β-(1→3)-glucan than H2SO4. The hydrolysis rate of β-(1→3)-glucan over aspartic acid (4.3 × 10−9 mol(C) s−1) is similar to that over sulfuric acid (5.7 × 10−9 mol(C) s−1). The hydrolysis rate over glutamic acid was 1 order of magnitude higher (1.03 × 10−8 mol(C) s−1) than that in the presence of the homogeneous acid. Both amino acids combined led to a slightly higher hydrolysis rate (1.2 × 10−8 mol(C) s−1) compared to H2SO4. The ratio of the amino acids was selected with respect to the presence of these amino acids in the model yeast (Glu:Asp = 3.5:1).41 The normalization of the hydrolysis rates to the amount of hydronium ion (Figure 6) indicates that the rate increase given by the amino acids (H2SO4/1.9 × 10−4 mol(C) mol(H3O+)−1 s−1 < Asp/2.5 × 10−3 mol(C) mol(H3O+)−1 s−1 < Glu/9.2 × 10−3 mol(C) mol(H3O+)−1 s−1, Figure 6) is proportional to the hydronium ion concentration, similarly as was it observed previously of the cellobiose dimer (Figure 5). The fact that a mineral acid is less active than the amino acids and that difference between amino acids with the same hydronium ion concentrations (see Figure S12) exists strongly points to the possibility of a concerted hydrolysis. In analogy to the enzyme action51−55 we conclude, therefore, that the transition state is stabilized by the amino acid enhancing so the rate of hydrolysis.

selected due to its similar activation energy with laminaribiose (1→3-type linkage) and gentiobiose (1→6-type linkage) under identical reaction conditions.49,50 Reactions with model substrates such as cellobiose (Figure 5) and β-(1→3)-glucan (Figure 6) were performed at low conversions ( Asp/2.75 × 10−4 mol L−1 > Glu/1.55 × 10−4 mol L−1, while the calculated pH values at the reaction temperature were H2SO4/2.45 > Asp/3.55 > Glu/3.81 (the corresponding pKa values were H2SO4/3.25 > Asp/4.46 > Glu/5.0). The pKb values were determined as H2SO4/10.75 < Asp/9.54 < Glu/ 9.00. With the increased concentration of the hydronium ions (Figure 3), the glycosidic ether bond-breaking rate increased as well indicating that the rate of the QC−O−CQ (Q = glucose units) bond cleavage is proportional to the hydronium ion concentration. Nucleophiles such as the deprotonated amino acids can interact as stabilizers of the highly active oxocarbenium ion.51,52 The charge stabilization capability of the corresponding amino acid nucleophile was proved via selective changes of the active site nucleophile, in Agrobacterium β-glucosidase.53−55 The replacement of the active site nucleophile Glu358 (glutamic acid at 358 position of the enzyme peptide chain) to Asp caused 2500-fold rate reduction, corresponding to ∼19 kJ mol−1 destabilization.53−55 The

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CONCLUSIONS In this study, we describe an overall pathway for the hydrothermal conversion of C. oleaginosus yeast biomass under mild conditions (453 K). The approach leads to three product fractions, a lipid fraction, serving a starting substrate for biofuels or lubricant additives production, an aqueous H


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basidiomycete yeast Trichosporon oleaginosus: Insights into substrate utilization and alternative evolutionary trajectories of fungal mating systems. mBio 2015, 6 (4), e00918−15. (4) Meo, A.; Priebe, X. L.; Weuster-Botz, D. Lipid production with Trichosporon oleaginosus in a membrane bioreactor using microalgae hydrolysate. J. Biotechnol. 2017, 241, 1−10. (5) Bracharz, F.; Redai, V.; Bach, K.; Qoura, F.; Brück, T. The effects of TORC signal interference on lipogenesis in the oleaginous yeast Trichosporon oleaginosus. BMC Biotechnol. 2017, 17 (1), 27. (6) Garcia Alba, L.; Torri, C.; Samorì, C.; van der Spek, J.; Fabbri, D.; Kersten, S. R. A.; Brilman, D. W. F. Hydrothermal treatment (HTT) of microalgae: Detailed molecular characterization of HTT oil in view of HTT mechanism elucidation. Energy Fuels 2012, 26 (1), 642−671. (7) Jacob, Z. Yeast lipids: extraction, quality analysis, and acceptability. Crit. Rev. Biotechnol. 1992, 12 (5−6), 463−491. (8) Dong, T.; Knoshaug, E. P.; Pienkos, P. T.; Laurens, L. M. L. Lipid recovery from wet oleaginous microbial biomass for biofuel production: A critical review. Appl. Energy 2016, 177, 879−895. (9) Dallies, N.; Francois, J.; Paquet, V. A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast 1998, 14 (14), 1297−1306. (10) Colombo, A. L.; Padovan, A. C. B.; Chaves, G. M. Current knowledge of Trichosporon spp. and Trichosporonosis. Clin. Microbiol. Rev. 2011, 24 (4), 682−700. (11) Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37 (8), 911−917. (12) Bonturi, N.; Matsakas, L.; Nilsson, R.; Christakopoulos, P.; Miranda, E.; Berglund, K.; Rova, U. Single cell oil producing yeasts Lipomyces starkeyi and Rhodosporidium toruloides: Selection of extraction strategies and biodiesel property prediction. Energies 2015, 8 (6), 5040−5052. (13) Jin, G.; Yang, F.; Hu, C.; Shen, H.; Zhao, Z. K. Enzyme-assisted extraction of lipids directly from the culture of the oleaginous yeast Rhodosporidium toruloides. Bioresour. Technol. 2012, 111, 378−382. (14) Li, Y.; Naghdi, F. G.; Garg, S.; Adarme-Vega, T. C.; Thurecht, K. J.; Ghafor, W. A.; Tannock, S.; Schenk, P. M. A comparative study: the impact of different lipid extraction methods on current microalgal lipid research. Microb. Cell Fact. 2014, 13, 14. (15) Depree, J.; Emerson, G. W.; Sullivan, P. A. The cell wall of the oleaginous yeast Trichosporon cutaneum. J. Gen. Microbiol. 1993, 139 (9), 2123−2133. (16) Naughton, F. C. Production, chemistry, and commercial applications of various chemicals from castor oil. J. Am. Oil Chem. Soc. 1974, 51 (3), 65−71. (17) Fonseca, F. L.; Frases, S.; Casadevall, A.; Fischman-Gompertz, O.; Nimrichter, L.; Rodriguez, M. L. Structural and functional properties of the Trichosporon asahii glucuronoxylomannan. Fungal Genet. Biol. 2009, 46 (6−7), 496−505. (18) Wichmann, H.; Bahadir, M. Bio-based ester oils for use as lubricants in metal working. Clean: Soil, Air, Water 2007, 35 (1), 49− 51. (19) Moore, S.; Stein, W. H. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 1954, 211 (2), 907−913. (20) Grant, C.; Pramer, D. Minor element composition of yeast extract. J. Bacteriol. 1962, 84 (4), 869−870. (21) Joo, Y. C.; Seo, E. S.; Kim, Y. S.; Kim, K. R.; Park, J. B.; Oh, D. K. Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia. J. Biotechnol. 2012, 158 (1−2), 17−23. (22) Griffiths, M. J.; van Hille, R. P.; Harrison, S. T. L. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids 2010, 45 (11), 1053−1060. (23) Yamada, E. A.; Sgarbieri, V. C. Yeast (Saccharomyces cerevisiae) protein concentrate: preparation, chemical composition, and nutritional and functional properties. J. Agric. Food Chem. 2005, 53 (10), 3931−3936.

fraction to be used as fermentation medium, and a fraction containing the insoluble solids that can be thermally utilized. Amino acids, originating from the yeast cell, act as endogenous catalysts increasing the hydronium ion concentrations for the hydrolysis of the yeast cell wall and stabilize the transition state. Supporting this hypothesis model reactions with cellobiose and β-(1→3)-glucan revealed glutamic acid and aspartic acid as hydrolysis catalysts, which gained higher hydrolysis rates than mineral sulfuric acid. The application of bioresourced amino acids as catalyst for repeated yeast decomposition reactions would provide a green solution without an additional catalyst, contaminating the resulting products. This work offers a green and economic way for the utilization of yeast and a new insight into the conversion of biomass in the presence of endogenous catalysts. This present study shows the synergy between chemical and biotechnological techniques to develop industrially relevant processes.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b04795. Detailed experimental procedures and characterization data; Figures S1−S13; Tables S1−S2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yue Liu: 0000-0001-8939-0233 Eszter Baráth: 0000-0001-8494-3388 Thomas Brück: 0000-0002-2113-6957 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support for this work by the Advanced Biomass Value project (FKZ 03SF0446A), sponsored by German Federal Ministry of Education and Research (“Bundesministerium für Bildung und Forschung”), is highly appreciated. Special gratitude is expressed to Martina Haack for her help in performing the HPLC and GC measurements.

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REFERENCES

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