Recovery of Fuel-Precursor Lipids from Oleaginous Yeast - ACS

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Recovery of Fuel-Precursor Lipids from Oleaginous Yeast Jacob S Kruger, Nicholas S Cleveland, Rou Yi Yeap, Tao Dong, Kelsey J. Ramirez, Nicholas J. Nagle, Andrew C Lowell, Gregg T Beckham, James D. McMillan, and Mary J. Biddy ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b01874 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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ACS Sustainable Chemistry & Engineering

Recovery of Fuel-Precursor Lipids from Oleaginous Yeast Jacob S. Kruger, Nicholas S. Cleveland, Rou Yi Yeap, Tao Dong, Kelsey J. Ramirez, Nicholas J. Nagle, Andrew C. Lowell, † Gregg T. Beckham, James D. McMillan, Mary J. Biddy* National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO, 80401 *Author to whom correspondence should be addressed. E-mail: [email protected]; Fax: +01-303-384-6363; Tel: +01-303-384-7904 †

Current Address: KBI BioPharma, Inc., 2500 Central Avenue, Boulder, CO 80301

Abstract: Bio-derived lipids offer a potentially promising intermediate to displace petroleum-derived diesel. One of the key challenges for the production of lipids via microbial cell mass is that these products are stored intracellularly and must be extracted and recovered efficiently and economically. Thus, improved methods of cell lysis and lipid extraction are needed. In this study, we examine lipid extraction from wet oleaginous yeast in combination with seven different cell lysis approaches encompassing both physical and chemical techniques (high-pressure homogenization, microwave and conventional thermal treatments, bead beating, acid, base, and enzymatic treatments) to facilitate lipid extraction from a model oleaginous yeast strain, Lipomyces starkeyi. Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields. Further exploration of acid treatment and integration with an economic model revealed that treatment at 170°C for 60 min at 1 wt% H2SO4 and 8 wt% yeast solids represents a viable option for both lipid recovery yield and process economics, enabling experimental lipid recovery yields of 88.5-93.0% to be achieved at a corresponding estimated minimum fuel selling price (MFSP) of $5.13-$5.61/gallon of gasoline equivalent (GGE). The same acid treatment conditions applied to two other strains of oleaginous yeast (Cutaneotrichosporon curvatus and Rhodotorula toruloides) resulted in similar lipid recovery yields. In pretreatment experiments scaled up to 300 mL, slightly lower temperatures or shorter pretreatment times, along with higher yeast solids loading, resulted in higher lipid yields than the conditions identified from the smallscale runs. Two replicate runs carried out at 170 °C for 30 min using 1 wt% H2SO4 and 19 wt% yeast solids achieved an average lipid recovery of 96.1% at a corresponding estimated MFSP of $4.89/GGE. In all cases, the lipids are primarily triglycerides and free fatty acids comprised mainly of palmitic, stearic, and oleic acids, with smaller fractions of polar lipids. The fatty acid composition of the lipids extracted from the wet treated cell mass is the same as that in freeze-dried whole oleaginous yeast cell mass, suggesting the acid treatment renders all lipids extractable. This work demonstrates that acid treatment is a robust and effective cell lysis technique in a microbial lipid-based biofuel scenario and provides a baseline for further scale-up and process integration.

Keywords: cell lysis, lipid extraction, single-cell oil, oleaginous yeast, acid pretreatment

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Introduction For the production of renewable biofuels, recent emphasis has been on molecular equivalents, or ‘drop in’ fuels, that do not require modifications in fuel delivery or consumption infrastructure. To that end, lipids are a leading candidate biofuels precursor because they are chemically similar to the alkanes that make up the major portion of gasoline, diesel, and jet fuel. The lipids most commonly considered for fuel production are accumulated in plant or microbial cell mass mainly as long-chain fatty acids (C16-C18) and acylglycerides that contain carbon in a highly reduced (i.e., energy-dense) form, often with one or more unsaturations. These lipids can be accumulated as carbon storage compounds by terrestrial plants, such as soybeans and sunflowers, or by microbes, such as algae, yeast, bacteria, and filamentous fungi. Microbially-produced oils have gained increasing interest due to fast growth and production rates, high lipid contents, and a lack of land-use concerns. These ‘single-cell oils’ are especially promising when produced in a two-stage system, in which polysaccharides accumulated photoautotrophically by a primary organism (e.g., switchgrass, agricultural residues, trees, or algae) are processed into a soluble carbohydrate stream, and then converted to lipids by a heterotrophic oleaginous organism. To that end, a number of oleaginous heterotrophic microbes have shown promise for the second stage in lipid production.1-3 Among these, oleaginous yeast are especially advantageous due to their fast growth and robust culture stability.3, 4 Extraction of accumulated lipids must be facilitated by some form of cell lysis or cell wall permeability that can enable substantial recovery of intracellular lipid products following subsequent liquid-liquid extraction. Many approaches have been evaluated in the context of extracting different intracellular components from various microbes.5-10 In general, cell lysis research for fuel lipid production has focused on algae; this research has similar priorities in targeting low cost/high efficiency recovery processes, but the trends may not be directly applicable to oleaginous yeast because of differences in cell wall chemistries in terms of polysaccharides, proteins, and other components.8, 9, 11-15 In contrast, cell lysis research in yeast (and other microbes) has generally focused on recovery of proteins and other high-value products; this research has considered different priorities in terms of lysis severity requirements and process economics. More recently, Bonturi et al.1 and Yu et al.16 investigated a range of treatments and solvent extractions for lipid recovery from oleaginous microbes. Both studies found that acid pretreatment effectively lyses multiple types of oleaginous organisms and facilitates high lipid recovery yields. However, industrially-feasibly approaches to cell lysis must use low loadings of chemicals and solvent systems that are scalable to be compatible with fuel production. Specifically, there is a need for development of a process for cell lysis of and fuel precursor lipid extraction from oleaginous yeast with scalability and economic favorability as primary objectives. Because the most favorable treatment approach is a complex function of the target product, integration of the extracted product with downstream processes, the organism of interest, and the culture conditions, we were motivated to explore a range of cell lysis techniques relevant to fuel lipid production from the robust oleaginous yeast strain, Lipomyces starkeyi. From the numerous cell lysis approaches described in the literature, we selected a slate of seven techniques that have been described as advantageous for process economics.5-10 In particular, we report examination of seven cell lysis techniques: two physical techniques (high-pressure homogenization, bead beating), two thermal techniques (microwave heating and conventional convective/conductive heating), and three chemical techniques (acid treatment, base treatment, and enzymatic treatment), as well as thermal and enzymatic treatments coupled to high-pressure homogenization. We have also performed a preliminary optimization of the most promising method and integrated our results with a techno-economic model. Materials and Methods Biomass Production Three different oleaginous yeast strains were used to produce cell biomass: L. starkeyi strain ATCC 12659, Rhodotorula toruloides strain DSMZ 4444 (formerly Rhodosporidium toruloides), and Cutaneotrichosporon curvatus strain ATCC 20509 (formerly Cryptococcus curvatus). L. starkeyi seed culture was generated as follows: 1 mL of concentrated L. starkeyi glycerol stock was used to inoculate a 250 mL shake flask containing 50 mL YP media (10 g/L yeast extract; 20 g/L peptone) supplemented with 20 g/L glucose. After 3 days of cultivation, the culture reached an optical density at 600 nm (OD600) of

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approximately 10, and it was used to inoculate a 2L baffled shake flask containing 500 mL of YP media supplemented with 50 g/L glucose at an initial OD600 of 0.5. After 3 days, this culture had reached an approximate OD600 of 10, and the culture was used to inoculate the bioreactors at an initial OD600 of 0.5-1. R. toruloides and C. curvatus seed culture were generated as follows: cells from glycerol stock propagated overnight in YP media supplemented with 20 g/L glucose were used to inoculate a 125 mL shake flask containing 25 mL YP media (10 g/L yeast extract; 20 g/L peptone) supplemented with 50 g/L glucose. This culture was then used to inoculate a larger baffled flask to generate enough cells to inoculate the bioreactor at an initial OD600 of 1.0. All shake flask cultivations were carried out in rotary shaker incubator where the speed was set at 250 rpm and temperature kept at 30˚C. Bioreactor cultivations were performed using either 500 mL vessels (BioStat Q-Plus, Sartorius, Goettingen, Germany) with 300 mL working volume or 10 L vessels (BioFlo New Brunswick, Eppendorf, Hauppauge, NY, USA) with 5 L working volume. The temperature was maintained at 30˚C, and the pH was kept constant by addition of 1N NaOH. L. starkeyi cultivations were carried out for approximately 96 hours (24 hours after glucose depletion) on YP media supplemented with 100 g/L glucose, and the pH was maintained at 5.1. Aerobic conditions were maintained by a continuous air supply at 0.5 vvm, and the dissolved oxygen level was set at 20% by adjusting the agitation speed. R. toruloides and C. curvatus cultivations were performed using minimal salts media supplemented with ~100 g/L total monomeric sugars (58 g/L glucose, 34.5 g/L xylose, 2.0 g/L galactose, 5.1 g/L arabinose, concentrations that mimic corn stover hydrolysate), and the pH was maintained at 5.2. Aerobic conditions were maintained by a continuous air supply at 1.5 vvm, and the dissolved oxygen level was set at 25% by adjusting the agitation speed. Cultivation broth was harvested upon sugar depletion. All cultivations were inoculated with pelleted cells at an initial OD600 of 1.0. In most cases, the L. Starkeyi broth was centrifuged, the cells were washed with deionized (DI) water, and the resulting pellet was resuspended in just enough DI water to make it transferable by pipette, which corresponded to 16 wt% solids. The resuspended slurry was divided into 3 mL aliquots and frozen at -70°C. In preliminary experiments with a different batch of yeast, the resuspended slurry was divided into only two aliquots; one was frozen at -70°C, and one was treated immediately. Also, in some experiments with high pressure homogenization, the broth, which was 6.65 wt% solids as received, was used directly. All samples that had been frozen were thawed directly before lysis experiments, and were never frozen more than once. Analysis of the cell mass for fatty acid methyl ester (FAME) content17 showed that the lipid content of the centrifuged and resuspended cell mass was 56.5% FAME on a dry weight basis, and the crude broth was 33.7% FAME on a dry weight basis. All experimental conditions were run in triplicate except for high-pressure homogenization, where only one sample was collected at each condition. However, homogenization runs at one condition at the end of one day, and at the same condition at the beginning of the next day gave similar results (Runs 10 and 10a below). Error bars are reported as the standard deviation of replicate measurements. For optimization experiments, a second batch of L. starkeyi 11557 was grown under identical conditions; FAME analysis indicated that this second batch had a lipid content of 66.3% on a dry weight basis. The R. toruloides broth was harvested and stored the same way, and diluted to 8 wt% solids for smallscale acid treatment. Due to production of extracellular polymeric substances (EPS), the C. curvatus broth could not be concentrated by centrifugation, and was processed as a dilute broth at 5.6 wt% solids. Lipid content of the lyophilized samples from these species were 64.9% for R. toruloides and 27.2% for C. curvatus by FAME analysis. The lower lipid content for C. curvatus reflects the presence of media salts, nutrients, and EPS present along with the cell mass in this sample. The overall composition of the recovered yeast material used in the cell lysis experiments is shown in Figure S1. High-Pressure Homogenization (HPH) HPH was conducted in collaboration with BEE International, Inc., using a Nano DeBEE homogenizer. Details of the technology are available at www.beei.com. In these experiments, the unwashed fermentation broth, which had been frozen at -70°C in a 500 mL bottle and thawed overnight, was used directly for homogenization in 14 mL aliquots. Bead Beating Bead beating was performed on a Digital Disruptor Genie from Scientific Industries, which was located in a cold room maintained at 4°C. 100 mg of beads, either ZrO2 or glass, were used to lyse 1.5 mL of slurry at 16 wt% solids.



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Conventional (Sand Bath) Thermolysis Conventional convective/conductive thermolysis was carried out in 3 mL stainless steel microreactors, which consisted of a ½” Swagelok union capped at both ends, using a fluidized sand bath (Techne model 2D). 3 mL of slurry at either 16 wt% or 8 wt% solids was sealed in the microreactor, which was then immersed in a preheated sand bath for the desired length of time, after which the reactor was quenched in room-temperature water. The heatup time for the microreactors in the sand bath was less than five minutes. Microwave Thermolysis, Acid, and Base Treatment Microwave thermolysis was carried out in a CEM microwave reactor in a capped glass reactor tube. 4 mL of slurry at either 16 wt% or 8 wt% solids was sealed in the reactor and heated to the desired temperature for the desired amount of time. For acid and base treatments, 2 mL of 16 wt% slurry was added to the reactor, followed by 1 or 2 mL of 10 wt% H2SO4 for 2 wt% acid or 4 wt% acid, respectively, or 1 or 2 mL of 10 wt% NaOH for 2 wt% base or 4 wt% base, respectively. For the 2 wt% cases, 1 mL of DI H2O was added to make up the total reactor volume to 4 mL and the yeast solids content to 8 wt%. The heat-up time for the reactor tubes in the microwave reactor was less than two minutes. Enzymatic Lysis Two enzyme cocktails were mixed to deconstruct each component of the yeast cell wall: β-1,3 glucan, mannan, chitin, and protein. The enzymes for hydrolyzing these components were Lyticase from Arthrobacter luteus (a β-1,3 glucanase that also exhibits activity for other carbohydrate linkages), mannanase from Trichoderma longibrachiatum, chitinase from Streptomyces griseus, and papain protease from Papaya (Carica papaya) latex. All enzymes were purchased from Sigma Aldrich. The first cocktail consisted of 0.025 wt% chitinase, mannanase, and glucanase, and 2.5 wt% protease with respect to yeast dry weight. The second cocktail consisted of 0.025 wt% chitinase, mannanase, and glucanase, and no protease, to preclude the possibility that the protease may reduce the activity of the other enzymes. In these experiments, 2.5 mL of yeast slurry at 16 wt% solids was mixed with 1 mL enzyme cocktail and incubated in test tubes on a shaker stand at the desired temperature for the desired time. Scaled-up Acid Treatment Larger-scale acid treatment experiments were performed in a Zipperclave reactor with a 300 mL working volume. In this reactor, acidified yeast slurry was placed in a stainless steel canister and stirred by an impeller. The canister was positioned inside a heated, insulated jacket, which forms a pressure-tight seal with a reactor header around the canister. The reactor was operated in batch mode, and was primarily heated by introducing steam through a manual needle valve. The reactor generally reached targeted operating conditions within five minutes. After the reaction, excess steam was vented through a different needle valve to relieve pressure and the reactor was cooled to < 50 °C before removing the canister and placing it in a bucket of ice. During reactor operation, some of the introduced steam condensed inside the reactor. Additional heat and pressurization cycles were used to steam-clean the reactor after the reaction. The initial excess steam and the steam recovered from these cleaning operations were collected and extracted parallel to the yeast in the canister to calculate the total lipid yield. Temperatures during reaction were typically maintained within 2°C of the setpoint. Lipid Extraction and FAME Analysis After lysis treatment, the slurries were transferred to test tubes and a volume of hexane equal to the initial volume of the yeast slurry was added, or an equal volume of hexane to the volume of the broth in the case of HPH. The biphasic mixture was vortexed for 30 sec, and then stirred with a magnetic stir bar for 1 h, with vortexing for 30 sec at 15 min intervals. After extraction, the mixture was centrifuged at 2000g and the hexane layer was pipetted into pre-weighed vials. The hexane was evaporated at 30°C under nitrogen purge, and the vial was re-weighed to obtain the gravimetric yield of lipids. The crude lipids were analyzed for FAME content17 to obtain a final recovered lipid yield, which is calculated as the product of the gravimetric lipid yield and the calculated FAME purity, divided by the FAME content of the freeze-dried yeast cell mass. If the calculated FAME content in the lipid extracts was greater than 100%, the lipid extracts were assumed to be 100% FAME. Compositional Analysis The composition of lyophilized yeast cell mass was analyzed for its lipid content, ash, and carbohydrate content using previously described NREL Laboratory Analytical Procedures.18 Protein content was approximated by detection of hydrolyzed amino acids using AOAC Official Method 994.12.19 Detailed lipid composition was determined by solid phase extraction using an aminopropyl cartridge by Agilent Technologies. About 25 mg of each


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lipid sample to be analyzed was loaded into preweighed GC vials prior to the experiment. The cartridges were first conditioned with 12 mL of hexane. After that, 100 µL of 95:3:1 hexane:chloroform:methanol was used to dissolve the preweighed lipid samples. The samples were subsequently transferred to the cartridges. Three successive washes with 2:1 chloroform:isopropyl alcohol, 98:2 diethyl either:acetic acid, and 6:1 methanol:chloroform were run to elute neutral lipids, fatty acids, and polar lipids, respectively. Each wash was collected in a preweighed test tube and then dried down overnight in a vacuum oven. The test tubes were then weighed to obtain gravimetric recoveries. Results and Discussion In developing a scalable process for lipid recovery from L. starkeyi, we began with a screening of cell lysis techniques. While dozens of cell lysis and lipid extraction approaches can be found for algae and/or yeast in the literature,5-10 we elected to explore only those that have been described as advantageous for process economics. In particular, we favored physical and thermal lysis techniques that do not require additional reagents, freezing of the cell mass, or drying of the cell mass. In selecting chemical techniques, we also favored those that do not require freezing or drying, and techniques that use only reagents targeted to specific moieties of the yeast cell wall (i.e., reagents that hydrolyze polysaccharides and/or proteins). We stress that the goal of the initial screening was not to find optimal conditions for each technique, but rather to screen and understand trends for a wide range of process conditions, and establish a general range of attainable lipid recovery yields. We use the lipid recovery yield as a primary surrogate of overall post-harvest fuel production process economics because, as described in the technoeconomic analysis section below, it is one of the most important factors for minimum fuel selling price (MFSP), with changes in MFSP in the range of $0.06/gallon of gasoline equivalent (GGE) to ~$0.30/GGE per percentage point change in lipid recovery yield. 20, 21For all of the techniques screened, FAME analysis of the lipids (when sufficient lipids for the analysis were recovered) showed the extracts to be ~100% FAME. High Pressure Homogenization (HPH) We targeted HPH as a baseline scenario because it has several advantages as a treatment approach, including a wellestablished track record on the industrial scale and no need for chemical reagents. We selected a homogenizer design developed by BEE International, Inc., which combines features of traditional homogenizers and microfluidizers. Briefly, the technology functions by pressurizing the sample and sending it through a nozzle, and then through a variable-length flow path consisting of a series of “reactors” that are annular and cylindrical in shape, ~1 cm in length, and separated by a washer with a larger inner diameter than the inner diameter of the reactors. The sample is then either passed through the final reactor and recovered (parallel flow), or deflected and sent back through the reactors in the opposite direction (reverse flow). A short path length (smaller number of reactors) and parallel flow configuration puts greater emphasis on shear forces through the nozzle and cell-on-wall collisions, and more closely approximates a traditional HPH design. A longer path length and reverse flow configuration enhances cell-on-cell collisions and more closely approximates a microfluidizer. Further details and instrument visualization are given at the manufacturer web site listed in the Materials and Methods section. The conditions and yields of each run are shown in Table 1. Table 1. Conditions of high-pressure homogenizer runs.a Run

Pressure

No. Reactors

Reactor Sizeb

No. Passes

Lipid Recovery Yield

1

41 kpsi

6

1 mm

1

27.8%

2

30 kpsi

6

1 mm

1

11.7%

3

15 kpsi

6

1 mm

1

4.2%

4

15 kpsi

11

1 mm

1

11.1%

5

30 kpsi

11

1 mm

1

29.1%

6

45 kpsi

11

1 mm

1

40.0%

7

30 kpsic

6

1 mm

1

20.2%

8

30 kpsid

6

1 mm

1

23.7%

9

30 kpsi

6

0.5 mm

1

34.4%

10

45 kpsi

6

0.5 mm

1

57.3%



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10a

45 kpsi

6

0.5 mm

1

61.1%

11

45 kpsi

6

0.5 mm

3

74.0%

12

45 kpsi

10

0.5 mm

1

67.3%

13

45 kpsi

10

0.5 mm

3

75.0%

14

45 kpsi

1

0.5 mm

1

67.3%

15

45 kpsi

1

0.5 mm

3

74.8%

a

All runs conducted in reverse flow configuration with Z5 nozzle and ~14 mL sample size. bReactor size is inner diameter of the annular cylinder reactors. c500 psi back pressure. d1000 psi back pressure. Runs 10 and 10a are replicate runs on different days.

We performed a series of experiments directly on harvested broth at 6.65 wt% solids, which had a FAME content of 33.7%. These experiments showed that a smaller orifice diameter and multiple passes through the instrument were beneficial, and that the highest pressure achievable (45 kpsi) gave the highest lipid recovery yields. Lipid recoveries reached a maximum of around 75%. Although each run represents a single experiment, a measure of the technique reproducibility can be seen by comparing runs 10 and 10a, which are identical experiments conducted on separate days. Although lipid recovery yields enabled by HPH plateaued around 75%, we hypothesized that though the cells may be fully lysed, some of the lipids could not be extracted because of emulsions that inhibited full recovery of the hexane phase. We confirmed a high degree of cell lysis using optical microscopy, (Figure S2). Thus, the homogenizer was functioning well as a lysis technique, but did not integrate well with the extraction protocol. Thus, we were motivated to explore modified extraction protocols to interface with HPH. In some cases, we were able to break the emulsions by freezing the biphasic extraction mixtures (hexane plus homogenized slurry) at 0°C overnight. In other cases, particularly those with high lysis yields, freezing was insufficient. Thus, we explored post-homogenization treatments in an attempt to improve the extractability. Because emulsions often form when amphiphilic cell components, such as proteins, are homogenized, we targeted breaking the emulsions with proteolytic enzymes and heat. We divided one of the homogenized slurries into two aliquots, and heated one at 140°C for 15 min and one at 160°C for 15 min. The slurries significantly darkened in color and did not form emulsions during extraction, but the extractability of the lipids did not improve. Similarly, adding papain to a homogenized sample and incubating at 45°C for 48 h, did not improve the lipid recovery yields. These results are summarized in Figure S3. It may be possible to improve lipid yields by other emulsion-breaking approaches, but the relatively strict economic limits on fuel production processes preclude consideration of many other techniques in tandem, such as salting out and pH modification (the latter when in combination with HPH). Other Lysis Approaches For the other cell lysis techniques, we continued with our original hexane extraction protocol, and employed a fractional factorial design-of-experiments approach. For each technique, we investigated three experimental parameters expected to have the greatest effect on lipid recovery yields based on preliminary experiments and literature results.5-10 The parameters were temperature, time, and chemical loading for the chemical techniques, temperature, time, and solids loading for the thermal techniques, and bead size, shake time, and shaker speed for bead beating. The specific conditions of each run are summarized in Table 2. The lipid recovery yields for each run are shown in Figure 1.

Table 2. Experimental conditions for cell lysis screening. Acid

Run

Temp (°C)

Time (min)

H2SO4 (wt %)

A1

100

60

2

Lipid Recovery Yield 7.4%

A2

100

20

4

9.8%


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Base

Sand Bath

Microwave

Enzyme

Bead Beater

a

A3

160

20

2

87.2%

A4

160

60

4

93.4%

Run

Temp (°C)

Time (min)

NaOH (wt %)

B1

100

60

2

0.3%

B2

100

20

4

0.0%

B3

160

20

2

0.5%

B4

160

60

4

0.0%

Run

Temp (°C)

Time (min)

Solids Loading (wt %)

SB1

170

30

16

2.5%

SB2

170

60

8

9.0%

SB3

220

30

8

43.7% 62.9%

SB4

220

60

16

Run

Temp (°C)

Time (min)

Solids Loading (wt %)

M1

160

60

16

16.5%

M2

220

60

8

27.6%

M3

160

120

8

34.4%

M4

220

120

16

34.3%

Run

Temp (°C)

Time (hr)

Enzyme Loading (wt %)

E1

22

48

2a

45.4%

b

49.8%

E2

22

24

1

E3

45

24

2

69.3%

E4

45

48

1

42.4%

Run

Bead Size (µm)

Time (min)

Speed (rpm)

BB1

100

5

3000

0.6%

BB2

212-300

5

1500

1.3%

BB3

100

15

1500

0.4%

BB4

212-300

15

3000

4.6%

BB5

100

15

3000

0.8%

BB6

100

30

3000

0.9%

0.025 wt% each of chitinase, mannanase, glucanase, 2.5 wt% protease. b0.05 wt% each of chitinase, mannanase, glucanase, no protease.



100% 80% Lipid Yield

60% 40% 20%

BB1 BB2 BB3 BB4 BB5 BB6

E1 E2 E3 E4

M1 M2 M3 M4

SB1 SB2 SB3 SB4

B1 B2 B3 B4

0% A1 A2 A3 A4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Run Number Figure 1. Lipid recovery yields obtained after applying one of six cell lysis techniques to L. starkeyi 11557. Labels on the x-axis correspond to run numbers in Table 2.

Acid and Base Treatments Acid and base treatments were selected to catalyze hydrolysis of polysaccharides and proteins. These treatments are less advantageous than physical and thermal treatments because they require addition (and post-reaction treatment) of acid or alkali, but these reagents are relatively inexpensive. Treatment with either 2 wt% or 4 wt% H2SO4 resulted in low lipid recoveries at 100°C, but high recoveries at 160°C. At 2 wt% acid and 160°C for 20 min, a lipid yield of 87.2% was achieved, as shown for runs A1-A4 in Figure 1. Similarly, at the most severe conditions we explored, 4 wt% acid and 160°C for 60 min, a lipid yield of 93.4% was achieved. Thus, it appears that acid treatment is able to satisfactorily release lipids in a readily-extractable form. The efficacy is somewhat intuitive, given that the yeast cell wall is primarily polysaccharides that can be hydrolyzed by strong acids.12, 13, 22 The post-treatment solutions significantly darkened in color, suggesting that some of the solubilized polysaccharides may have been dehydrated to furanic compounds or reacted with proteins via Maillard chemistry. Additionally, the acid and/or heat may have denatured emulsifying proteins, further promoting lipid extractability. In contrast, base treatment using NaOH under similar conditions resulted in lipid recoveries < 1%, as shown for runs B1-B4 in Figure 1. The low recovery is likely due to formation of a gel from the residual solids, which caused these materials to be difficult to agitate during hexane extraction. The gel may have formed because of changing surface charges due to deprotonation or denaturation of cell wall components at high pH (both the 2 wt% and 4 wt% NaOH are close to pH 14). It may be possible to improve lipid recovery by altering the extraction protocol, but overall, base treatment does not appear to be a promising option. Microwave and Sand Bath Thermolysis Treatments Thermolysis approaches are especially promising because they require neither addition of reagents nor high-pressure reactors. Middelberg6 summarized several instances of bacterial and yeast cell lysis by treatment at elevated temperature (commonly 45-60°C, up to 90°C in some cases) for 24-48 h, suggesting that yeast can be induced to autolyse by activating cell wall-degrading enzymes under thermal stress. Additionally, Lee et al.23 demonstrated that microwave irradiation at ~100°C for ~5 min was effective at releasing lipids from multiple species of algae, and that autoclaving was in some cases equally effective and in other cases not as effective as microwave irradiation. In light of our results with acid treatment, we were motivated to explore higher temperatures than 100°C. Also, in light of the differences between microwave and conventional thermolysis observed by Lee et al.,23 possibly due to the different heating mechanisms of the two techniques (primarily radiative for microwave thermolysis, and primarily


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convective/conductive for conventional thermolysis) we were motivated to explore both thermolysis techniques to avoid extrapolating the effectiveness of the technique from the less-scalable microwave reactor. The thermolysis treatments exhibited lipid recovery yields in the range of 40-60% for the harshest conditions, as shown for runs SB1-SB4 and M1-M4 in Figure 1. Lipid recovery yields from conventional thermolysis in a sand bath reactor reached 43.7% and 62.9% for runs SB3 and SB4, respectively, while lipid recovery yields from microwave thermolysis reached 34.4% and 34.3% for runs M3 and M4, respectively. These results are comparable to those of Espinosa-Gonzalez et al., who obtained ~70% lipid recovery yield from hydrothermal treatment of C. curvatus cell mass after 60 min at 280 °C.24 Although the operating costs for conventional heating are lower than for acid treatment at similar conditions, lipid recovery yield is the strongest influencer of overall process economics, as discussed below. Thus, it is unlikely that acid-free thermolysis will be able to compete with acid treatment in a biorefinery setting. Enzymatic Treatment Enzymatic treatment is potentially beneficial in that neither high temperatures nor high pressures are required, which potentially can decrease both capital and operating expenses. Additionally, the mild conditions may enable recovery of the hydrolyzed carbohydrate and protein fractions of the yeast cells, which can then be recycled or further upgraded. Enzymes can potentially be immobilized to facilitate recovery and reuse, which may further decrease operating costs. The drawbacks to enzymatic lysis are the long incubation times required to effect cell lysis and high operating costs if the enzymes cannot be recycled. Although the summary provided by Middelberg suggests that some yeast can be induced to autolyse by activating endogenous cell wall-degrading enzymes,6 we were motivated to explore a best-case scenario by adding relatively high concentrations of non-immobilized enzymes to target the polysaccharides and proteins present in the yeast cell wall; the specific enzyme cocktails are described in the Materials and Methods. Enzymatic treatment also showed intermediate ability to improve lipid recovery yields, with maximum recovery yields ranging from 40-70%, as shown for runs E1-E4 in Figure 1. The best results were obtained at 45°C for 24 h with the protease papain, and resulted in a lipid recovery of 69.3%. Although the enzyme cocktails have not been optimized, the enzyme loadings in these experiments are relatively high; without lipid recovery yields comparable to those observed with acid treatment, it is likely that the costs of these enzymes would preclude an economically viable process unless they could be immobilized and/or easily recycled. Bead Beater Treatment Advantages to cell lysis by bead beating include no reagents, and low temperature and pressure requirements (although much of the energy imposed on the grinding beads and cell mass is dissipated as heat). Reviews of cell lysis approaches have shown that high lysis yields, especially for yeast, are achievable using bead beating and have favorably viewed the application of bead milling at large scale.5, 6 In the present experiments, however, bead beating gave lipid recoveries < 5%, even under the most severe conditions of 3000 rpm shaking for 15 min, as shown for runs BB1-BB4 in Figure 1. Optical microscopy of the treated cells showed minimal cell debris and mainly intact cells, suggesting that the cell walls in our organism are more recalcitrant than in the organisms explored in other recent work.16, 23 We increased the severity by using the 100 µm beads at 3,000 rpm for 30 min, but lipid recovery on these runs was < 1%. It may be possible to increase lipid recovery further in other instrument configurations, but ultimately, bead beating seems to be on par with base treatment in terms of lipid recovery and thus was not a candidate for further optimization. In comparing the results of this screening with those from the literature,5, 6, 16, 23 it is worth noting that that in multiple instances (i.e., the thermolysis and bead beating experiments), the oleaginous yeast used here appear to be more recalcitrant than the microbes considered in previous studies. This trend highlights two important points. First, that cell lysis approaches used for one microbe cannot be assumed to transfer to other species, and second, that the nutrient limitation required to effect maximum bioconversion of carbohydrates to lipids may be associated with changes in cell wall composition that make the cells more resistant to lysis.25-29 Indeed, in the absence of gels or emulsions that inhibited the extraction step, techniques targeting dissolution of the polysaccharide matrix in the cell wall (acid and enzyme treatments) facilitated relatively high lipid recovery yields at relatively mild conditions, while those selected to overcome the cohesive forces of the full-strength cell wall (bead beating, microwave and sand bath heating) required harsh conditions to achieve significant lipid recovery yields. Based on optical microscopy, HPH, which falls into the second category, also required harsh conditions to achieve a high degree of cell lysis.



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Techno-economic Analysis (TEA) Using data from the screening study as a baseline, we constructed techno-economic models around the four technologies that showed greater than 60% lipid recovery yields, namely acid treatment, high-pressure homogenization, thermolysis, and enzymatic treatment. Costs outside of the yeast cell lysis and lipid recovery steps were taken from our previously-published model for production of diesel fuel from C6 lignocellulosic sugars via yeast-derived lipids, with the C5 portion of lignocellulosic sugars diverted to production of coproduct succinic acid.20 The general parameters of the model are given in Table 3, while a full description can be found in Biddy et al.20 Details of the model inputs for the four technologies considered here are described below. Table 3. General parameters for techno-economic analysis of lignocellulosic sugars to renewable diesel blendstock via oleaginous yeast lipids.

a

Parameter

Valuea

Total plant capital cost

1216$MMb

Total plant fixed operating cost

27 $MM/yrb

Total plant variable operating cost

135 $MM/yrb

Lignocellulose C6 sugar content

37.85%

Lignocellulosic biomass cost

$80/ton

C6 sugar yield through into bioconversion

82%

Lipid metabolic yield (C6 sugars to lipids)

0.27 g/g

Hydrotreating yield (lipids to fuel)

81%

Values taken from Biddy et al.

20

b

Includes cost for acid lysis operation.

Acid treatment and thermolysis The model uses a base case acid treatment of the oleaginous yeast at 150°C for 30 min at 1 wt% H2SO4, which at a lipid recovery yield of 90% led to a minimum fuel selling price (MFSP) of $5.28/gallon of gasoline equivalent (GGE).20 The cost and design of the reactor are also consistent with Davis et al.30 The reactor is designed using 304 stainless steel metallurgy and uses a yeast solids concentration of 8-10 wt%. The model inputs for thermolysis are identical, except no acid is needed, and no neutralization after the reaction. The parameters considered in this model were time, temperature, acid loading, and lipid recovery yield. Analysis of the model showed that increasing the temperature from 150°C to 170°C would increase the MFSP by roughly $0.02/GGE, and that extending the residence time from 30 min to 60 min would increase the MFSP by roughly $0.14/GGE. Changes in the acid loading would have a more pronounced effect, with increasing the acid loading from 1 wt% to 2 wt% and 4 wt% would increase the MFSP by $0.14/GGE and $0.40/GGE, respectively, due to both added acid cost and the need to purchase additional NaOH for neutralization. Reducing the acid loading to 0 wt% (thermolysis) would decrease the MFSP by about $0.12/GGE for temperatures between 150 °C and 170 °C. At the highest temperature explored here (220 °C), the MFSP at 90% lipid recovery yield would be $5.20/GGE. However, in every case, lipid recovery yield is the strongest influence on MFSP and the observed yields are much lower than 90%. Namely, with the two demonstrated yields obtained at 43.7% and 62.9%, the estimated MFSP was determined to be $10.77/GGE and $7.52/GGE, respectively. High Pressure Homogenization The HPH model is based on a similar analysis for recovering algal lipids.21 The major costs associated with HPH are the capital cost of the homogenizer and electricity import to power the homogenizer. The costs in this case are taken from vendor estimates, consistent with previous work,21 and assume a power demand of 0.183 kWh/kg dry yeast cell mass at a 5000 L/h throughput. With the reported yields of 67.3% for the one-pass system, we estimate a MFSP of $7.65/GGE. For the case of the three-pass system, we estimate that the plant will require additional homogenizers to maintain the baseline throughput. Thus, while the yield of this system is higher at 75%, the benefit is offset by the higher capital and operating costs, resulting in a MFSP of $7.84/GGE. The key drivers for this design are both the capital cost of the homogenizer system and the cost associated with the electricity consumed in the overall process.


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The estimated homogenizer installed capital cost of the equipment ($85MM) accounts for around 25% of the MFSP. Overall electricity consumption of the process accounts for about 8% of the MFSP. As with the other lysis approaches, these large contributions are highly influenced by the lipid recovery yield. Additionally, as discussed above, the yields observed for this process are likely due to the formation of emulsions in the product rather than the effectiveness of cell lysis. Thus, if these emulsion challenges could be overcome to improve the lipid recovery yield, the overall MFSP would decrease. Enzymatic Lysis Inputs for the enzymatic lysis step are taken from Biddy et al.20 in the upstream saccharification step for the lignocellulose feedstock. In these cases, we have utilized the loadings and yields demonstrated in the experiments above. Two of the experimental runs were considered: a loading of 0.025 wt% each of chitinase, mannanase, glucanase, 2.5 wt% protease, with a lipid recovery yield of 49.8%, and a loading of 0.025 wt% each of chitinase, mannanase, and glucanase, with a lipid recovery yield of 69.3%. As a first pass estimate, we assume the cost of each enzyme mixture is similar to the cost of the enzymes used for lignocellulosic sugar hydrolysis. This enzyme cost assumption represents a best case scenario, and results in a MFSP of $9.30/GGE for the first case and $6.30/GGE for the second case. Integrating the model with the results from the screening shows that acid lysis is the closest to meeting model targets. The MFSP is highly sensitive to lipid recovery yield for each of the technologies included in the analysis. Thus, the most promising run for each technology is the run with the highest lipid yield. As shown in Figure 2, the lipid recoveries obtained in the screening for the highest lipid result in MFSPs of $5.63GGE for acid lysis, $7.52/GGE for thermolysis, $12.96/GGE for HPH, and $6.30/GGE for enzymatic treatment. $8.00

$6.00 MFSP ($/GGE)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


$4.00

$2.00

$0.00

Figure 2. MFSP for best-case runs from lysis screening.

Acid Treatment Optimization From these screening experiments, it was clear that acid treatment was the most promising cell lysis technique among the limited number of conditions screened for each lysis and recovery technology. Thus, we were interested in further optimizing acid treatment to achieve high yields at conditions that would be most favorable for process economics. We performed a three-factor central composite design optimization. Design parameters and conditions for the runs are shown in Table 4 and Figure 3, respectively. Lipid recovery yields from the optimization are also shown in Figure 3.



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Table 4. Parameters used for optimization of acid treatment. Parameter 0

Temperature (°C) 160

Time (min) 20

H2SO4 (wt%) 2

-1

150

10

1

+1

170

30

3

-α

144

4

0.4

+α

176

36

3.6

Figure 3. Experimental conditions and lipid recovery yields for optimization of acid treatment.

Figure 3 shows that several treatment conditions achieved lipid recovery yields around 90%. Thus, it appears that, at least in the range of conditions studied, there is not a single set of conditions to maximize lipid recovery yield. Instead, it seems that a plateau is reached above a certain level of pretreatment severity, which can be achieved by any sufficiently severe combination of pretreatment time, temperature, and acid concentration (when the added acid concentration is greater than zero). This situation is amenable to analysis by condensing the three experimental variables into a single “severity factor,” R0’. For acid treatment, the severity factor can be defined as:

.

where [H+] is the acid concentration in mol/L, t is time in min, and T is temperature in °C.31 For the present experiments, time and temperature are straightforward. Acid concentration is approximated by converting the wt% H2SO4 (g H2SO4/g solution) to molarity using the density of H2SO4 solutions and the molecular weight of H2SO4. The density of sulfuric acid solutions is taken from Perry.32 This analysis neglects contribution of the yeast cell mass to the solution density, the contribution of H+ from the dissociation of HSO4- to H+ and SO42-, and any influence on [H+] by other components from the yeast, such as neutralization by ash and dissociation of sugars and proteins. These additional factors are not expected to significantly alter the results. The pretreatment severity factor is often analyzed as Log (R0’), which ranged from 1.5 to 3 in these experiments. As shown in Figure 4, a plot of lipid recovery yield as a function of Log (R0’) provides a useful trend that can be fit to a sigmoidal function.


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100% 90% Lipid Recovery Yield

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80% 70% 60% 50%

Experiment Fit

40% 1

2

3

Log Acid Teatment Severity Factor, R0' Figure 4. Lipid recovery yield from L. starkeyi 11557 as a function of acid treatment severity.

Because we are primarily interested in conditions that allow for high lipid recovery yields, it is more important to achieve a satisfactory fit at the higher-severity conditions. Thus, in the least squares error minimization, experimental points with a lipid recovery yield greater than 85% are weighted a factor of ten higher than the lower yield points. The “Fit” curve shown in Figure 4 is represented by the following equation:

( ) "

where Y is the lipid recovery yield and K1, K2, and K3 are adjustable parameters. The equation predicts that the maximum recovery yield is 92.9%, which is achieved at severity factors of 4.51 or greater, outside of our experimental range. However, a lipid recovery yield of 90% is achievable at a severity factor of 2.58 or greater (within the range of our experiments); the approach to 92.9% is thus slow, and achievable yields must be balanced with economic analysis to optimize process design. Integration of the TEA model with the sigmoidal model for prediction of the lipid recovery yield shows that the base case scenario has a severity factor R0’ = 1.96, which leads to a predicted lipid recovery of 73.6%, well below the 90% target. However, increasing the temperature to 170°C and the residence time to 60 min, still at 1 wt% H2SO4, increases the severity factor to R0’ = 2.85, which has a predicted lipid recovery yield of 91.7%. At the same time, the increases in temperature and residence time lead to an increase in the MFSP of $0.18/GGE, while the increase in the lipid recovery yield from 90% to 91.7% leads to a decrease in the MFSP of $0.10/GGE, for a modest net decrease of $0.08/GGE relative to the base case, or a MFSP of $5.36/GGE. We then validated the model under the proposed conditions (170°C, 60 min, 1 wt% H2SO4). Six replicate runs gave lipid recovery yields of 87.5%, 89.0%, 92.3%, 93.0%, 95.1%, and 92.3% (average = 91.5%). The corresponding MFSP for these runs is $5.61/GGE, $5.52/GGE, $5.33/GGE, $5.29/GGE, $5.13/GGE, and $5.33/GGE, respectively. Thus, the observed recovery yields span the model-predicted recovery, but because of the sensitivity of MFSP to lipid recovery yield, the variation in that outcome leads to uncertainty in the MFSP of about $0.50/GGE from the cell lysis and lipid extraction steps. Further improvements in process economics may be achievable by improving our extraction protocol to increase lipid recovery yields and by developing processes to isolate or produce valueadded coproducts from the non-lipid components.



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Extension to Other Yeast Strains We were also motivated to test the applicability of acid treatment on multiple species of oleaginous yeast to understand if the selected conditions were directly transferrable, or if the lysis treatment would need to be tailored for each strain. Thus, in addition to acid treatment of L. starkeyi 11557, we tested the proposed conditions (170°C, 60 min, 1 wt% H2SO4) with two other promising strains of oleaginous yeast: Cutaneotrichosporon curvatus 20509 and Rhodotorula toruloides 4444. The lipid recovery yields from these two strains were comparable to L. starkeyi 11557, as shown in Table 5. The variability of the C. curvatus yeast was higher than the other two, reflecting the production of EPS in this species and the concomitant harvesting and processing challenges. Table 5. Lipid recovery yields from three strains of oleaginous yeast.

a

Strain

Lipid Recovery Yielda

L. starkeyi 11557

91.5 ± 2.8%

C. curvatus 20509

99.7 ± 12.3%

R. toruloides 4444

88.5 ± 0.3%

Reaction conditions: 170°C, 60 min, 1 wt% H2SO4 (Log R’0 = 2.85), 4 mL slurry.

Preliminary Scale-up In light of the promising results for acid treatment at the 4-mL scale, we also performed a larger-scale (300 mL) acid treatment using a steam-heated reactor more representative of industrial implementation. Using this steam-heated semi-batch reactor and R. toruloides 4444 cell mass, we discovered that our optimized microwave conditions did not enable high lipid recovery yields. In initial runs, lipid recoveries varied widely between 30% and 80%. We considered that our reaction conditions may be too severe, and performed several more runs at lower reaction temperatures or shorter reaction times. We also increased the yeast loading from 8 wt% to 19 wt%. The less-severe conditions led to lipid recovery yields greater than 90%, and greater than 95% in some cases. These results are shown in Table 6. While the reason that the larger reactor performs better under less severe conditions is not yet clear, we note that the lower severity, higher solids loading (which results in less acid consumption per mass of yeast processed), and moderately higher yields are each likely to improve process economics. For example, the economic benefit of the lower severity, the nearly 60% decrease in acid consumption per mass of yeast processed and the nearly 5% increase in lipid yield would likely decrease the MFSP by roughly $0.14/GGE, $0.04/GGE, and $0.30/GGE, respectively, relative to the conditions proposed above. The TEA model suggests that the two runs at 170 °C for 30 min (average lipid yield of 96.1%) would give a MFSP of $4.89/GGE. Table 6. Lipid recovery yields from R. toruloides 4444 at the 300 mL acid treatment reactor scale. Temperature (°C)

Time (min)

Severity Factor (Log R’0) a

Lipid Recovery Yield

150

60

2.26

91.6%

160

60

2.55

95.6%

170

30

2.55

95.7%

170

30

2.55

96.5%

a

Reaction conditions: 1 wt% H2SO4, 19 wt% yeast solids, 300 mL slurry.

Lipid composition In addition to lipid recovery yield, lipid composition is also an important parameter for downstream processing. For example, polar lipids can contain heteroatoms such as S and P that may poison hydroprocessing catalysts, and the presence of both FFAs and TAGs can impact the choice of upgrading technology. The purity of the extracted lipids by FAME analysis was, in each case, 98.2-104.1%. However, a more detailed analysis of lipid composition by solid phase extraction shows that the FAME components are split between TAGs, which is the primary component of the neutral fraction, and FFA. Less than 2 wt% of the hexane extract is comprised of polar lipids (Figure 5). Because storage lipids are predominantly TAGs, the prevalence of FFAs may suggest that some lipases are co-extracted with the lipids and slowly hydrolyze the TAGs to FFAs.33


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FAME analysis showed that the FFA fraction was ~100% FAME, while the neutral fraction was 80-90% FAME, likely due to the presence of minor components, such as sterols and pigments (e.g., carotenoids for R. toruloides). The FAME yields of the overall extracts are calculated at 100+% even in the presence of these other components because of an automatically-applied correction factor17 that likely underestimates the internal standard recovery in the current lipid extracts.

100%

1.8% 1.0% 7%

0.4%

Polar

80% Lipid Mass Fraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60%

38% 73%

FFA

91% 40% 60% 20% 26%

Neutral

0% R. toruloides

L. starkeyi

C. curvatus

Figure 5. Composition of lipids extracted from L. starkeyi 11557, C. curvatus 20509, and R. toruloides 4444 after acid treatment at 170°C for 60 min with 1 wt% H2SO4 as analyzed by solid phase extraction.

The fatty acid profile of the lipids shows that the lipids are primarily palmitic (16:0), stearic (18:0), oleic (18:1n9), and linoleic (18:2n6) acids, with < 10% of other fatty acids. The lipid profiles in the hexane extracts obtained from each strain are shown in Figure 6; FAME analysis of freeze-dried whole cell masses and solid residues after acid treatment and hexane extraction showed essentially the same profiles, suggesting that acid treatment is non-selective in rendering lipids extractable.



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Figure 6. Fatty acid profile of lipids extracted from L. starkeyi 11557, C. curvatus 20509, and R. toruloides 4444 after acid treatment at 170°C for 60 min with 1 wt% H2SO4. “Other” includes C12:0, C14:0, C15:0, C16:1n9, C17:0, C18:1n7, C18:3n3, C20:0, C22:0, and C24:0. Lipids included in the analysis but not detected for any of the three species include C8:0, C10:0, C14:1, C16:1n11, C16:1n6, C16:1n5, C16:2, C16:4, C17:1, C18:3n6, C18:4n3, C20:2, C20:3n6, C20:3n3, C20:4n6, C20:5n3, C22:1n9, C22:2, C22:4, C22:5 and C22:6n3.


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Overall, these results are consistent with previous work that found reaction with a strong acid to be an effective, robust treatment strategy for lysis of oleaginous microbial cell walls.1, 16 However, while Yu et al.16 and Bonturi et al.1 used hydrochloric acid in their experiments, we have shown that sulfuric acid, which may enable less expensive metallurgy in large-scale reactors than HCl, is also effective. Additionally, we have used less concentrated acid and lower acid-to-cell mass ratios. For example, both Yu et al.16 and Bonturi et al.1 used a ratio of roughly 0.025 mol acid per g cell mass; the ratio at our optimized conditions (1 wt% H2SO4, 19 wt% yeast solids, 170°C, 30 min) was much lower, approximately 5.4 x 10-4. Thus, we have achieved high lipid recovery yields while also decreasing acid use by nearly a factor of 50, compared to previous work. Decreasing acid consumption is an important parameter for both process economics and sustainability. Conclusions We screened seven cell lysis approaches and showed that acid treatment enables lipid recoveries above 90% at economically favorable treatment conditions of 170°C, 60 min, 1 wt% H2SO4, and 8 wt% yeast solids. In contrast, high pressure homogenization leads to lipid recoveries approaching 80%, enzymatic treatment allows for lipid recoveries up to 70%, conventional thermolysis gives lipid recoveries around 60%, and microwave thermolysis only achieves lipid recoveries around 30%, while bead beating and base treatment with NaOH lead to lipid recoveries < 5%. Additionally, acid treatment is effective on at least three different strains of oleaginous yeast, i.e., L. starkeyi, R. toruloides, and C. curvatus. An acid treatment-based lysis process can be readily scaled up to the 300 mL scale, at which lipid recovery yields > 95% are achievable from R. toruloides treated at 170 °C, 30 min, 1 wt% H2SO4, and 19 wt% yeast solids. The extracted lipids are relatively pure, primarily triglycerides and free fatty acids, and contain a high fraction of palmitic, stearic, and oleic acid chains, which can be readily upgraded to green diesel fuel.

Funding We thank the U.S. Department of Energy Bioenergy Technologies Office (DOE-BETO) for funding via Contract No. DE-AC36-08GO28308 with the National Renewable Energy Laboratory. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. Acknowledgements The authors are grateful to Thieny Trinh, Violeta Sanchez i Nogue, and Christine Singer for culturing the yeast used in the experiments, to Violeta Sanchez i Nogue and Brenna Black for valuable discussions, and to Brenna Black, Michelle Reed, and Paris Spinelli for assistance with detailed analysis of the lipids and yeast cell mass.

REFERENCES

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TOC/Abstract Graphic

TOC/Abstract Graphic Synopsis This paper demonstrates that lipids as renewable fuel precursors can be recovered from oleaginous yeast in high yield via acid treatment followed by hexane extraction.


Recovery of Fuel-Precursor Lipids from Oleaginous Yeast - ACS

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