Composition and Potential Products from Auxenochlorella protothecoides,Chlorella sorokiniana and Chlorella vulgaris

Abstract

The compositions of three microalgal strains that have potential as biofuel feedstocks were evaluated to investigate how their biomass could be best utilized in a portfolio of products in an algal biofuel biorefinery. These strains, Chlorella vulgaris, Chlorella sorokiniana and Auxenochlorella (Chlorella) protothecoides, can grow both heterotrophically and phototrophically. An underlying principle of this project was that a continuous and maximally productive growth system will be required for commercial-scale production of algal biomass. Alternatively, a two-stage, mixed trophic state production process could be utilized to achieve this goal. For this reason, our research focused on these strains grown phototrophically to produce biomass and grown heterotrophically with nitrogen stressing to induce lipid production. The compositions reported in this work should be reflective of what is possible in continuous phototrophic or heterotrophic systems or in a two-stage mixed trophic system that are envisioned for scaled production biorefineries.

Introduction

This study was an analysis of the composition of three algal strains that have potential as feedstocks for biofuel and bioproduct production grown under heterotrophic and/or photoautotrophic conditions. The authors' research was focused on identification of potential high-value products produced by these strains. This was done to develop a portfolio of potential products that could be produced in an algal biorefinery to allow production of algal biofuel(s) along with coproducts in a flexible, sustainable and commercially viable manner.

Most agree that sustainable production of algal biofuels will require the utilization of all or nearly all of the produced algal biomass in order to be cost competitive; one cannot discard a significant amount of the produced biomass without negatively impacting the overall process economics.^1
–3 The most direct approach would be to pyrolyze all of the residual biomass to generate additional biofuel, but this approach may ignore significant high-value products that could have a more positive impact on the overall production costs compared to the production of additional fuel by pyrolysis. This research project evaluated the composition of three potential microalgal strains and suggests potential paths to profitable product portfolios within the scope of an integrated algal biorefinery.

Algal Biofuels and Bioproducts

The real issue is not whether fuels can be made from algae, but rather how can these fuels be made in a manner that makes them commercially viable in a fluctuating marketplace. Production of biofuels from algae is possible by the direct production of biofuels from biomass⁴ or by extraction of algal lipids that can be further refined into biobased fuels with the generation of residual biomass.⁵

While direct production of fuel from biomass uses all of the available feedstock, it is possible that the production, dewatering, and downstream processing is too expensive to make such biofuels commercially competitive unless overall costs are minimal. When relying on the coproduction of high-value products to offset large-scale biofuel production (e.g., algal lipid to biodiesel), there is a possibility that the high-value products will be out of sync with their potential markets (e.g., production of β-carotene at a plant producing millions of barrels of oil will quickly flood the high value β-carotene product market), thereby reducing these coproducts' potential to positively impact the overall costs of the portfolio generated by the biorefinery. That is why a focus on highly productive strains (for biomass) or strong lipid-production strains (for biodiesel or drop-in replacement fuels) needs to also target products of higher value (but with broad usage, such as those identified by the Department of Energy on renewable chemicals).⁶ Examples of relevant potential coproducts that would not overwhelm existing markets are animal feeds, single-celled protein, and renewable chemical feedstocks.

Trophic State Discussion

Algae are generally thought to be photoautotrophic organisms, meaning they use light to produce fixed carbon from prototrophic chemicals, such as bicarbonate and carbon dioxide. However, algae are an extremely broad group of organisms that occupy varied environmental niches and apply a wide array of metabolic strategies to succeed. For this discussion, we will focus on microalgae and leave discussion of macroalgae (seaweeds) to other reviewers.^7–8 Within microalgae, there are algae that are obligate photoautotrophs, obligate heterotrophs, and facultative organisms that can use light or fixed carbon either together (mixotrophically) or separately.

This study focuses on three strains grown under purely phototrophic or heterotrophic growth conditions, which can also be envisioned as in-series unit processes for a mixed trophic state production process for algal lipids.⁹ A mixed trophic state production process is composed of two separate stages: i) the photoautotrophic stage, focused on biomass production; and ii) the heterotrophic stage focused on lipid production and accumulation in aerobic bioreactors using fixed carbon substrates (e.g., glucose or fructose). Therefore, the strains in this report were grown phototrophically to maximize biomass as well as heterotrophically to maximize lipids. The compositions of the strains were analyzed to identify potential coproducts from each stage.

Materials and Methods

Algal Strains

Auxenochlorella (Chlorella) protothecoides KRT1009, Chlorella sorokiniana UTEX 1230 and Chlorella vulgaris KAV1000 (an isolate from the Danforth Plant Science Center's Backyard Biofuels Program) were used in this study. The A. protothecoides strain was derived from the Culture Collection of Algae at the University of Texas at Austin (Texas) and received as the UTEX 25 isolate. Upon receipt from UTEX, the culture contained several different discrete populations of cells, and the A. protothecoides isolate was a clonal culture derived from one of those cell lines. The identity of KRT1009 as A. protothecoides was determined using a cleaved amplified polymorphic sequences (CAPS) assay¹⁰ based on the published 18S ribosomal sequence published for this alga.¹¹ The nomenclature for this species was revised recently (renaming it Auxenochlorella from the previous Chlorella genus), and this revised nomenclature will be used throughout this paper.¹¹

Algal Growth Media

The components of the phototrophic culture medium are as follows (per L): 0.5 g NH₄Cl, 1.44 g K₂HPO₄, 0.72 g KH₂PO₄, 50 mg tetra sodium EDTA, 20 mg MgSO₄•7H₂O, 10 mg CaCl₂•2H₂O, 10 mg FeCl₃•6H₂O, 5 mg H₃BO₃, 1.25 mg ZnSO₄•7H₂O, 0.38 mg MnSO₄•H₂O, 0.25 mg CoCl₂•6H₂O, 0.25 mg Na₂MoO₄•2H₂O, 0.08 mg CuSO₄•5H₂O. For growth of A. protothecoides, the medium was supplemented with 100 μg/L thiamine hydrochloride (Vitamin B₁). For heterotrophic seed culture scale-up, the NH₄Cl was omitted and 20 mM glucose added.

For heterotrophic growth in fermentors, the following medium was used (amounts per L): 4 g NaCl, 8 g Na•glutamate, 1 g MgSO₄•7H₂O, 0.2 g CaCl₂•2H₂O, 6 g Difco^® Yeast Extract, 40 g glucose, 0.3 g KH₂PO₄, 109 mg citric acid monohydrate, 20 mg FeSO₄•7H₂O, 30 mg ZnSO₄•7H₂O, 6 mg MnCl₂•4H₂O, 2 mg CuSO₄•5H₂O, 0.3 mg Na₂MoO₄•2H₂O, 0.3 mg CoCl₂•6H₂O, and 15 mg H₃BO₃. pH was adjusted to 6.9 with 25% NaOH.

Phototrophic

Biomass was maintained on plates, then scaled up in increasing volumes such that the cells were growing exponentially at each transfer. The strains were grown in four stages: 1) four 25 mL volumes in 125-mL flasks; 2) four 500 mL volumes in 2-L round bottom flasks; and 3) two 20 L volumes in carboys; and 4) two 40 L volumes in hanging bags.

Stages 1 and 2 were maintained at 28 ± 2°C, a constant 350 ± 10 μmol/m²/s light intensity from an Eye Hortilux metal halide bulb (model MT1000B-D/HOR/HTL-BL1, Iwasaki Electric, Tokyo, Japan) in a SunTube™ six-inch reflector (Sunlight Supply, Vancouver, WA), 5,000 ppm ambient CO₂ concentration, and 175 rpm shaking speed. Stages 3 and 4 were maintained at the same light and temperature levels, but were bubbled with a sterile-filtered air/CO₂ mix (approximately 5% (w/w) CO₂) at 0.94 L/m² (2 standard cubic feet per hour). The gas flow provided both the mixing and carbon source. Stages 1–3 were carried out aseptically, while Stage 4 was not. The Stage 4 hanging bags were maintained at the same temperature, light, and CO₂ conditions as Stage 3, but with a higher total gas flow rate that was set manually to provide optimal mixing. The cultures spent approximately four days in Stage 4 and were harvested just after the exponential growth phase, where typical algae concentrations were measured in a range of 0.7–0.9 g/L. At this point during algal growth, nitrogen stressing is minimal. Biomass was harvested and concentrated to approximately 50 g/L using a custom-built crossflow microfiltration system with a XUSP-143 filter (Pall, Port Washington, NY). In reference to the two-stage mixotrophic production process, this concentrated, healthy algal culture would be transferred to aerobic bioreactors for the heterotrophic stage. The ability to change from a unialgal (not axenic) phototrophic culture to a heterotrophic culture raises the possibility of bacterial overgrowth. Our experience with this shift is strain-specific. For A. protothecoides, little precaution was required in the shift from phototrophic to heterotrophic growth; this alga seems to outcompete the associated bacteria for the added glucose. Work with C. vulgaris was slightly more problematic, with the possibility of bacterial overgrowth if the autotrophic algal culture was not robust. However, we were able to grow this strain with a high inoculum to 80–90 g dry weight (gdw)/L with 50% lipid. No work was done with a C. sorokiniana under the mixed trophic state system during this study. For this report, the biomass in the filter retentate was further concentrated in 1-L bottles with a refrigerated centrifuge (Beckman J6-B) and lyophilized for analysis.

Heterotrophic

Heterotrophic cultivation started with cultures kept on heterotrophic seed culture medium plates. Fifty mL of heterotrophic seed culture medium was inoculated with a 10 μL loopful of biomass and grown to mid- or late-log phase. The entire 50 mL volume was then transferred to 500 mL of fresh heterotrophic seed culture medium and grown again to mid- to late-log phase, after which the entire volume was transferred to a fermentor. Fermentation was performed in a 10-L Biostat B fermentor (Sartoris, Goettingen, Germany), starting with 5 L of algal culture, into which a 605 g/L glucose solution was added periodically to maintain the glucose concentration in the fermentor at ∼20 g/L by adjusting the peristaltic pump rate to match the glucose uptake rate. The glucose was measured using subsampling and a YSI glucose meter. Fermentation was conducted at 28 ± 1°C with temperature controlled by a water jacket. A pH of 6.5 was maintained by addition of 3 M NaOH as required. The reactor was constantly mixed to maintain a dissolved oxygen level of ∼50%; this was controlled in the Biostat B fermentor by automatically adjusting agitation speed (350–600 rpm) under a set aeration of 4 L/min. Manual sampling was used to withdraw 50 mL of culture solution. The strains were nurtured to increase biomass then nitrogen stressed to induce lipid production. For A. protothecoides, the initial cell concentration was 0.684 g/L, and grown to 89.85 g/L after 174 h (12.3 g/L/d). Likewise, the initial concentration of C. vulgaris was 0.235 g/L, and grown to 30.90 g/L after 332 h (2.2 g/L/d), and C. sorokiniana was 0.178 g/L, and grown to 39.2 g/L after 312 h (3.0 g/L/d). These strains were harvested for analysis during the stationary phase when the biomass concentration and the lipid content became constant (as evaluated by multiple daily samples). The harvested biomass was centrifuged at 2,500 × g for 5 min. The liquid phase was then separated, and the biomass lyophilized for analysis.

In the literature, Chlorella vulgaris UTEX 259 grown on 1% glucose in the dark grew at 0.151 g/L/d,¹² considerably slower than what was observed in our study. However, another recent paper reported C. vulgaris growth rates for three different strains between 0.721–1.054 g/L/d that approached those seen in the current study.¹³ While Shi and colleagues demonstrated growth of A. protothecoides CS-41 at 10.9 g/L/d, maximum growth and an average growth over seven days of 4.6 g/L/d,¹⁴ very similar yields to those obtained in the current study. A study on three C. sorokiniana strains observed growth rates that ranged from 0.409–1.026 g/L/d,¹³ considerably lower than those we observed. In general, the biomass productivities are within the range of published values.

Analytics

The proximate analyses for moisture, protein, fat, fiber and ash, as well as fatty acid profiles and amino acid profiles, were performed by New Jersey Feed Laboratory (Ewing Township, New Jersey). Starch content analysis, a measurement of amylopectin, was performed by Cumberland Valley Analytical Services (Hagerstown, Maryland) using a previously described method to correct for free glucose.¹⁵

The non-polar lipid was extracted from algae samples with hexane. To do this, algal biomass was washed three times with 2 parts per thousand (ppt) Instant Ocean^® (Instant Ocean Spectrum Brands, Blacksburg, Virginia) solution and then centrifuged to remove dissolved solids. The pellet was resuspended in deionized water, frozen, and placed in a lyophilizer. After lyophilization, 1 g biomass was placed in a 50-mL centrifuge tube with five 6.35-mm glass beads, 1 mL of 0.5 mm silica beads, and 15 mL of hexane. The tube was vortexed at high speed for 1 h, then placed in a centrifuge to rotate at 1,100 × g for 5 min. The hexane was removed and placed in a weighed glass vial to be evaporated with dry nitrogen. Two additional washes of 10 mL of hexane were added to the remaining biomass, vortexed, then centrifuged to remove residual lipid. The hexane washes were added to the same weighed glass vial. Once the weight of the vial became constant, indicating the hexane was evaporated, the mass of the lipid was measured gravimetrically. The hexane extract percentage is reported as the mass of the remaining lipid in the glass vial divided by the ash free dry weight (AFDW) of the mass of the algae sample. All measurements were performed in triplicate with a standard deviation within 1%.

Much of the non-polar lipids, as well as polar lipids such as phospholipids and carotenoids, can be extracted with acetone. For total lipid extractions, the hexane extraction procedure above was followed, but the hexane was replaced with a solution of acetone plus 0.1% butylated hydroxytoluene (BHT). The BHT was added as an antioxidant to protect lutein for further analysis. Lutein and chlorophyll analyses were performed on a high-performance liquid chromatography (HPLC) system (Thermo Fisher, Waltham, Massachusetts) with a photodiode array detector. The column was a Hypersil Gold C-18 column (Thermo Fisher), and a three-solvent system was used consisting of 35% methanol/15% acetonitrile/50% HPLC water for Solvent A; 100% acetonitrile for Solvent B; and 99% isopropanol/1% methyl tert-butyl ether for Solvent C. Lipid samples were dissolved in a solution of 50% isopropanol and 50% tetrahydrofuran, then inserted into the autosampler. Lutein, Chl a, and Chl b absorption peaks were monitored as a function of time, then integrated and compared to pure standards to determine the amount present in the lipid sample.

Results and Discussion

The proximate measurements of the three production strains under both heterotrophic and phototrophic growth conditions are presented in Table 1. The ash content of phototrophically grown algae was always higher (by about 2- to 4-fold) than that for heterotrophically grown algae. For this study, the phototrophic algae were not open to the environment, so dirt and dust accumulation, which typically add to the ash content in open pond and raceway systems, was not a factor in the observed higher ash content.

Table 1.

Proximate Analysis of Heterotrophically and Phototrophically Grown Algal Strains

	A. protothecoides UTEX 25		C. vulgaris KAV1000		A. sorokiniana UTEX 1230
COMPONENT (%)	HETEROTROPHIC	PHOTOTROPHIC	HETEROTROPHIC	PHOTOTROPHIC	HETEROTROPHIC	PHOTOTROPHIC
Moisture	3.53	1.99	1.87	2.67	0.94	1.81
Protein (crude)	4.27	51.82	14.81	52.89	16.47	43.12
Fat (crude)	34.66	0.91	11.88	7.29	5.36	1.96
Fiber (crude)	0.4	0.28	2.55	6.01	0.24	0.66
Ash	2.04	8.64	3.10	8.42	4.23	7.24
Starch	0.5	0.6	36	12.9	45.9	23.9

Table 1 illustrates that the proximate analyses done by the outside vendors do not fully account for the contents of the biomass. The proximate analysis is designed to measure components most often present in animal feeds and may have therefore incompletely accounted for some of the less typical components of these microalgal strains. These undefined components are especially obvious for biomass of the heterotrophic A. protothecoides for which more than half of the mass is undetermined. In this case, the crude fat amount may be under reported, since other lipid measurements were much higher (Table 2) ; this may reflect the use of measuring crude fat by the AOAC 954.02 method applying acid hydrolysis with petroleum ether by the vendor, New Jersey Feed Labs, more suited to non-polar lipids. Although there may be inaccuracies in the absolute mass of components reported in Table 1, all strains were analyzed using identical methods, and direct comparisons strain-to-strain on materials found in a normal proximate analysis are valid.

Table 2.

Total Chlorophyll, Lutein, and Other Carotenoids Content in Heterotrophically and Phototrophically Grown Algal Strains (Lipids Reported as Percentage of AFDW and Chlorophyll Reported at mg/gdw).

	Auxenochlorella protothecoides UTEX 25		Chlorella vulgaris KAV 1000		Chlorella sorokiniana UTEX 1230
COMPONENT	HETEROTROPHIC	PHOTOTROPHIC	HETEROTROPHIC	PHOTOTROPHIC	HETEROTROPHIC	PHOTOTROPHIC
Lipid hexane extract (% AFDW)	60.4	0.6	26.3	22.1	9.6	4.2
Lipid acetone extract (% AFDW)	63.0	11.0	25.6	15.9	11.3	16.0
Chlorophyll A (mg/gdw)	ND	12.33	1.59	7.28	1.86	12.28
Chlorophyll B (mg/gdw)	0.0078	2.69	0.37	1.47	0.37	2.47
Lutein (mg/gdw)	0.09	3.49	0.72	2.52	0.89	3.30

ND: Not detected.

The protein content of the phototrophically grown algae was much higher than the corresponding heterotrophically grown algae (Table 1). This can be accounted for due to the high carbon to nitrogen ratio (C/N) in the heterotrophic growth medium, which stresses the cells and induces lipid production. Additionally, the lipid-laden algae tend to grow in size, which would reduce the number of cells in the measurement, thereby reducing the amount of protein rich cell organelles and cell walls present. This is especially important for the plastids associated with photosynthesis, which tend to atrophy under heterotrophy and high C/N ratios. The heterotrophic cultures of the C. vulgaris and C. sorokiniana maintained some residual green color while the A. protothecoides completely degreened, as reported previously.¹⁶ This trend was most pronounced in A. protothecoides, where the protein content was reduced by >91%. This contrasts to the 72.0% and 61.8% reductions (ash-in basis) seen with C. vulgaris and C. sorokiniana, respectively.

Heterotrophically grown A. protothecoides had the highest lipid content and lowest percentage of protein. Conversely, phototrophically grown A. protothecoides had the lowest fat and highest protein. The percentage of protein in phototrophically grown C. vulgaris was similar to that of A. protothecoides. The high protein content in these algal strains was expected; high-growth-rate cultures tend to produce more protein.¹⁷ Additionally, independent of growth conditions, the A. protothecoides strain accumulated very little starch compared to the other two strains, and C. vulgaris contained more fiber compared to the other two strains. The heterotrophically grown A. protothecoides strain produced three times as much crude fat than the second highest strain, which was heterotrophically grown C. vulgaris. The lower fat content of the C. vulgaris and C. sorokiniana strains is countered by a much higher content of starch, with 36% and 45.9% of the mass in C. vulgaris and C. sorokiniana, respectively (Table 1).

Algal biomass grown phototrophically contained high protein content that could be useful as a food or feed supplement to elevate the nutritional value of food and animal feeds. In addition, the protein content of the residual biomass after lipid extraction may have a similar use. The amino acid profiles from the protein are presented in Table 3. Phototrophic A. protothecoides has the highest amino acid content, followed by phototrophic C. vulgaris and C. sorokiniana. Every algal strain contained all of the essential amino acids for human nutrition. In all strains, except phototrophic C. vulgaris, glutamic acid was the most abundant amino acid. In phototrophic C. vulgaris, arginine, which is a conditionally essential amino acid, was the most abundant amino acid at 14.47% dry wt—twice as plentiful as any other amino acid present in the algal strains. Careful modification of the production process will be necessary to maintain the value of these coproducts. For example, direct pyrolysis of photosynthetic biomass can only recover value by first extracting proteins and lipids of value.

Table 3.

Analysis of the Amino Acid Composition in Heterotrophically and Phototrophically Grown Algal Biomass from Three Green Unicellular Algal Strains

	A. protothecoides UTEX 25		C. vulgaris KAV 1000		C. sorokiniana UTEX 1230
AMINO ACID (%)	HETEROTROPHIC	PHOTOTROPHIC	HETEROTROPHIC	PHOTOTROPHIC	HETEROTROPHIC	PHOTOTROPHIC
Methionine	0.08	0.89	0.29	0.44	0.36	0.78
Cysteine	0.05	0.51	0.16	0.18	0.24	0.37
Lysine	0.26	2.84	0.83	1.49	1.58	3.68
Phenylalanine	0.19	2.41	0.54	1.11	0.63	1.77
Leucine	0.37	4.53	1.01	1.95	1.18	3.14
Isoleucine	0.17	1.7	0.31	0.6	0.48	1.22
Threonine	0.23	2.31	0.5	0.85	0.62	1.58
Valine	0.29	2.87	0.59	1.05	0.97	1.77
Histidine	0.09	0.96	0.23	0.47	0.32	0.76
Arginine	0.27	3.42	0.65	14.47	0.85	3.15
Glycine	0.25	2.85	0.72	1.31	0.8	2.06
Aspartic acid	0.44	5.16	1.31	4.02	1.38	3.68
Serine	0.27	2.4	0.55	1.08	0.57	1.45
Glutamic acid	0.56	6.88	1.65	6.76	2.07	4.47
Proline	0.26	2.93	0.92	1.76	1.24	2.15
Hydroxyproline	0.1	0.39	0.06	0.06	0.06	0.09
Alanine	0.37	3.95	1.18	1.76	1.11	2.7
Tyrosine	0.12	1.68	0.38	0.86	0.51	1.36
Tryptophan	0.03	0.38	0.37	0.36	0.14	0.39
Total % in algal biomass	4.4	49.06	12.25	40.58	15.11	36.57

In Table 2, the difference between the hexane extraction (non-polar lipid extract) and the acetone extraction (total lipid extract) indicates the amount of polar lipids, such as phospholipids and carotenoids, present in the algae. This difference was significant in the phototrophically grown A. protothecoides and C. sorokiniana, where more than 10% of the cell AFDW was composed of polar lipids. However, this trend was not congruent with phototrophically grown C. vulgaris. Phototrophically grown C. sorokiniana had a higher percentage of lipids than heterotrophically grown C. sorokiniana, which seemed to sink carbon in starch instead of lipid. Conversely, heterotrophic A. protothecoides and C. vulgaris have higher lipid contents than their respective phototrophically grown algal biomass. The heterotrophic A. protothecoides possesses more than twice as much lipid compared to heterotrophic C. vulgaris, the second highest lipid producer.

The chlorophyll and lutein contents in heterotrophically and phototrophically grown production strains are presented in Table 2. As expected, heterotrophically produced algal biomass grown in the absence of light does not contain large amounts of photosynthetic pigments, such as chlorophyll or carotenoid. The highest chlorophyll content was found in phototropic A. protothecoides. Phototrophic A. protothecoides produced the greatest quantity of lutein (3.49 mg lutein/gdw biomass) with phototrophically grown C. sorokiniana producing a similar quantity of lutein (3.30 mg/gdw) and total chlorophyll (14.75 mg/gdw). While lutein as a coproduct faces the challenge of potentially overwhelming the market when paired with commercial scale biofuel production, having it as a potential coproduct as both a highly purified nutraceutical and as a lower value feed additive would expand the product portfolio of any biorefinery.

The fatty acid profiles of the lipids extracted from the algal strains are shown in Table 4. This table was produced by normalizing the fatty acid profile on a relative sample basis, performed by New Jersey Feed Laboratory, to the lipid hexane extract percentages found in Table 2. Since the fatty acid profile was performed on the lipid hexane extract, normalizing to the lipid hexane extract AFDW% yields the percentage of fatty acids present in the algal strains, and direct comparisons can be made among the algal strains. Heterotrophic A. protothecoides was composed of 43.17% oleic acid followed by 8.23% linoleic and 5.06% palmitic acid. Both heterotrophic and phototrophic C. vulgaris possessed similar fatty acid profiles with a bit more lipid in the heterotrophically grown algae. In C. sorokiniana, 18:2ω6 linoleic was the highest fatty acid found in both heterotrophic and phototrophically grown algae, but less than that of heterotrophically grown A. protothecoides. The algal strains evaluated here tended not to produce fatty acids smaller than C:16 nor fatty acids chains longer than C:20.

Table 4.

Fatty Acid Profile as a Percentage of AFDW from Heterotrophic and Phototrophically Grown Algal Strains

FATTY ACID	C# : DOUBLE BONDS	HETEROTROPHIC KRT1009 (A. protothecoides)	PHOTOTROPHIC KRT1009 (A. protothecoides)	HETEROTROPHIC KAV1000 (C. vulgaris)	PHOTOTROPHIC KAV1000 (C. vulgaris)	HETEROTROPHIC UTEX 1230 (C. sorokiniana)	PHOTOTROPHIC UTEX 1230 (C. sorokiniana)
Lauric	12:0	0.00	NM	0.00	0.03	0.00	0.00
Myristic	14:0	0.32	NM	0.02	0.03	0.02	0.02
Myristoleic	14:1	0.00	NM	0.02	0.02	0.03	0.00
Pentadecanoic	15:0	0.00	NM	0.03	0.02	0.01	0.01
Palmitic	16:0	5.06	0.05	2.12	3.23	2.08	0.89
Palmitoleic	16:1	0.11	NM	0.28	0.24	0.31	0.12
Hexadecadienoic	16:2	0.00	NM	0.00	0.02	0.01	0.00
Hexadecatetraenoic	16:4	0.00	NM	0.01	0.04	0.01	0.02
Heptadecanoic	17:0	0.08	NM	0.02	0.06	0.01	0.00
Heptadecenoic	17:1	0.00	NM	0.00	0.00	0.00	0.00
Stearic	18:0	1.65	0.00	0.24	1.45	0.23	0.06
Oleic	18:1ω9	43.17	0.09	13.32	8.04	1.82	0.27
Oleic	18:1ω7	0.05	NM	0.36	0.10	0.28	0.05
Linoleic	18:2ω6	8.23	0.22	4.72	3.39	3.07	1.07
Linoleic	18:2ω4	0.00	NM	0.02	2.21	0.01	0.00
Linolenic	18:3ω3	0.55	NM	2.38	0.05	0.56	0.52
Arachidic	20:0	0.19	0.25	0.02	0.02	0.03	0.00
Eicosanoic	20:1ω9	0.08	NM	0.04	0.03	0.01	0.00
Eicosadienoic	20:2ω6	0.00	NM	0.01	0.02	0.01	0.00
Arachidonic	20:4ω6	0.06	NM	0.00	0.02	0.00	0.00
Lignoceric	24:0	0.00	NM	0.00	0.08	0.03	0.01
Other	n/a	0.83	ND	2.70	3.02	1.06	1.15
Total % ω3		0.55	NM	2.38	2.21	0.56	0.52
Total % ω6		8.29	0.22	4.73	3.41	3.08	1.07

NM: Not measured; ND: Not detected.

In general, protein isolate from these three species when grown phototrophically should be considered in cost model analyses due to its abundance (notably in delipidated biomass) and favorable amino acid composition which are well suited for use in animal feeds. However, protein is less abundant for the three species when grown heterotrophically with a focus on lipid production. In a mixed state production process, in which phototrophic growth (to build biomass) is followed by heterotrophic growth for high lipid accumulation, the protein value will necessarily decrease and becomes less practical as a coproduct. Additionally, specialty oils have potential as valuable coproducts and should be considered in cost model analyses. High oleic oil, found in the heterotrophically grown A. protothecoides, has utility in the food industry¹⁸ and can be used as a renewable chemical feedstock.^19,20

Conclusion

The composition of C. vulgaris, C. sorokiniana, and A. (Chlorella) protothecoides, grown both heterotrophically and phototrophically, were analyzed to develop a portfolio of potential products that could be produced in an algal biorefinery taking advantage of the unique abilities of these strains to grow autotrophically, mixotrophically and heterotrophically. They also lend themselves to a mixed trophic state production process for algal lipids.

Footnotes

Acknowledgments

The authors gratefully acknowledge the support from the US Department of Energy, National Energy Technology Laboratory (DE-FE-0001888 awarded to Phycal Inc., previously of Highland Heights, OH, but has now ceased operations). Appreciation also goes to Ben Kessler, Nick White, Willainia Studmire, and Anis Bakhit, who provided samples that were utilized in this study.

Author Disclosure Statement

Dr. Lane reports grants from Department of Energy (DOE), during the conduct of the study. Additional support for other projects was received from the National Science Foundation (NSF) to fund other research. Dr. Coury reports grants from DOE, during the conduct of the study. Dr. Allnutt reports grants from DOE, during the conduct of the study. Subsequently he has been funded by NSF and US Department of Agriculture grants that have no conflict with the submitted research.

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