Utilization of Sweet Sorghum Juice for the Production of Astaxanthin

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Utilization of Sweet Sorghum Juice for the Production of Astaxanthin as a Biorefinery Co-Product by Phaffia rhodozyma Ryan Stoklosa, David Johnston, and Nhuan Nghiem ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03154 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018

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ACS Sustainable Chemistry & Engineering

Utilization of Sweet Sorghum Juice for the Production of Astaxanthin as a Biorefinery CoProduct by Phaffia rhodozyma Ryan J. Stoklosa*1, David B. Johnston1, and Nhuan P. Nghiem1 1

Sustainable Biofuels and Co-Products Research Unit, Eastern Regional Research Center,

USDA, ARS, 600 East Mermaid Lane, Wyndmoor, PA, 19038, United States †

Mention of trade names or commercial products in this publication is solely for the purpose of

providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. ARS is an equal opportunity provider and employer. *

Corresponding author:

Ryan J. Stoklosa, [email protected]; Phone: +1-215-233-6634 ABSTRACT This work investigates cultivating the red-pigmented yeast Phaffia rhodozyma in sweet sorghum juice (SSJ) to assess the production of astaxanthin as a potential biorefinery co-product. Shake flask cultures on defined sugar medium indicated that all three sugars (sucrose, glucose, and fructose) could be consumed with adequate nitrogen and nutrient supplementation. Only modest biomass growth and astaxanthin production could be achieved in SSJ without nitrogen supplementation; however, combining nitrogen supplementation with yeast extract in diluted SSJ could metabolize all sugars present in 168 hours. A 2 L bioreactor trial with full strength (i.e. undiluted) SSJ produced up to 29 g/L of biomass, 65.4 mg/L of astaxanthin, an overall cell astaxanthin content of 2.49 mg astaxanthin/g dry cell mass, and a volumetric astaxanthin productivity of 0.389 mg/L/hr after 168 hours of cultivation. Further process optimization is needed since glucose metabolism was incomplete in undiluted SSJ. KEYWORDS: Biorefinery; Co-products; Astaxanthin; Sweet sorghum INTRODUCTION The development of the corn ethanol industry has been successful by producing coproducts as a means to sustain economic profitability.1 In recent years second generation ethanol plants utilizing lignocellulosic or lesser grown agricultural feedstocks have come online to boost 1 ACS Paragon Plus Environment

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ethanol production in the United States to satisfy the Renewable Fuel Standard (RFS).2 However, added challenges associated with these feedstocks have prevented the full scale implementation of second generation ethanol plants. These challenges include feedstock recalcitrance,3 high capital costs,4 and feedstock transportation costs that could limit plant processing to less than 5000 dry MT per day.5 In order to reduce both capital and operational costs, co-products must be concomitantly produced alongside ethanol in a biorefinery. This allows a whole new portfolio of bio-based co-products to be generated from these feedstocks. Products can run the gamut from biofuels such as ethanol or butanol via fermentation,6-7 platform chemicals such as succinic acid or levulinic acid via fermentation or hexose degradation by a combination of dehydration and hydrolysis, respectively,8-9 or functional aromatic monomers and materials generated from recovered lignin.10-11 One underutilized feedstock in the United States that has the benefits of providing both structural and non-structural (i.e. soluble) carbohydrates for biochemical conversion to co-products is sweet sorghum. Sorghum bicolor, the principle modern species of sorghum grown today, contains a variety of cultivars and growing characteristics that make it an attractive feedstock to produce renewable co-products. Sorghum ranks as the fifth most grown cereal crop in the world, but has added advantages over other cereal crops by possessing drought tolerance and high-water use efficiency.12 The primary cultivars that are of agricultural interest include grain sorghum for ethanol or food production, forage sorghum for grazing pasture, biomass sorghum for bioenergy applications, and sweet sorghum for sorghum syrup production. Both grain sorghum and sweet sorghum can be utilized in processes similar to first generation ethanol. The high starch content contained in grain sorghum (~ 64-74% on a dry basis) has produced favorable ethanol yields in fermentations supplemented with protease.13 However, a primary drawback for grain sorghum is possessing a higher tannin content compared to most other cereal crops which can negatively impact starch hydrolysis yields and downstream ethanol fermentation.14-15 Alternatively, sweet sorghum contains a high content of non-structural sugars (primarily sucrose with lower amounts of free glucose and fructose) that can be extracted through similar processes used in the sugar cane ethanol industry.16 As opposed to sugar cane, sweet sorghum can be grown in wider agricultural hardiness zones. Within the continental United States, subtropical areas located within Florida and Louisiana can support both sugar cane and sweet sorghum cultivation while additional sweet sorghum growth can extend to temperate regions in the central Midwest and 2 ACS Paragon Plus Environment

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drier regions in the Southwest.17-18 Aside from feedstock utilization, this expanded region for growing sweet sorghum can also be beneficial by developing double-cropping systems that can increase dry matter production and reduce soil erosion.19 Before the utilization of less expensive corn syrup as a sweetener additive, sweet sorghum was primarily grown for syrup production in the United States.20 However, with less emphasis on syrup production, sweet sorghum utilization has transitioned to biofuel and co-product applications.21-22 Astaxanthin (3,3’-dihydroxy-β,β’-carotene-4,4’-dione) is a lipophilic carotenoid derivative of β-carotene that has potential value as an important product in a biorefinery. The carotenoid is produced as a secondary metabolite in substantial quantities by two primary organisms: the microalga Haematococcus pluvialis and the red pigmented yeast Phaffia rhodozyma (also known as Xanthophyllomyces dendrorhus).23 Astaxanthin has two primary market applications: aquaculture and nutraceuticals. Astaxanthin is commonly supplemented into the food source for farm raised salmon so that the fish can obtain their characteristic color.24 Additionally, studies with salmon have confirmed astaxanthin’s antioxidant capability in vivo.25 The high antioxidant capability of astaxanthin has shown the ability to promote antiinflammatory responses along with general heart and liver health benefits.26-27 Most astaxanthin is produced via synthetic routes that do not allow direct price competition with natural astaxanthin production, however, smaller niche markets can support astaxanthin generation from biochemical processes.26 Other red yeasts (e.g. Cryptococcus or Rhodosporidium) can produce carotenoids such as β-carotene, γ-carotene, or torulene in similar composition, but P. rhodozyma biosynthesizes astaxanthin in quantities up to 84% of its total carotenoid composition output.23, 28-29

. Prior research has indicated P. rhodozyma has been able to be cultivated on a wide range of

agricultural feedstocks. This range of feedstock includes corn wet milling co-products such as thin stillage or corn condensed distillers solubles (CCDS) that provide a low cost medium for cultivation to hydrolyzates rich in xylose from hardwoods that achieve high astaxanthin titers and biomass growth with a fed-batch process.30-32 Co-product astaxanthin generation has been achieved with barley straw through aqueous ammonia pretreatment followed by generating two separate hydrolyzate streams; the first stream was rich in xylose after enzymatic hydrolysis with a commercial hemicellulase and the second stream contained the remaining insoluble fraction rich in cellulose that was subjected to simultaneous saccharification and fermentation (SSF).33 In this dual process ethanol could be fermented to 70 g/L, and the yield of astaxanthin in the 3 ACS Paragon Plus Environment


hemicellulose hydrolyzate stream reached 0.81 mg of astaxanthin per g sugar consumed, which was a similar yield obtained from corn fiber hydrolyzate.33-34 The research presented here combines sweet sorghum juice (SSJ) utilization with P. rhodozyma to determine the conditions needed to produce appreciable quantities of carotenoids (primarily astaxanthin). Feedstocks containing high sugar content have been tested with P. rhodozyma previously, but research on culturing Phaffia with juice from sweet sorghum is currently not prevalent.35-36 Defined sugar medium was prepared to mimic the sugar concentrations found in SSJ to assess biomass growth and carotenoid production in shake flasks with different levels of nitrogen and nutrient supplementation. The best performing conditions were then tested on SSJ at the same scale. SSJ was then cultured in 2 L bioreactor units with pH and dissolved oxygen (DO) control to achieve higher biomass and carotenoid yields. EXPERIMENTAL SECTION Yeast Strain Phaffia rhodozyma ATCC 74219 (UBV-AX2) was obtained from the American Type Culture Collection (Manassas, VA, USA). This strain was chosen based upon its ability to be the best astaxanthin producer amongst other strains.34, 37 Feedstock and Chemicals Sweet sorghum juice (SSJ) was obtained from Delta BioRenewables (Memphis, TN, USA). The SSJ was supplied frozen and stored in a freezer at -20°C. In preparation for experiments, SSJ was thawed by placing the storage containers in a warm water bath. The SSJ was divided equally into 1 L centrifuge bottles and centrifuged at 15,484g for 15 minutes in a Sorvall Instruments (Newtown, CT, USA) RC-3B refrigerated centrifuge. The SSJ was recovered and centrifuged a second time at the same condition to remove any further insoluble material. The centrifuged SSJ was then filtered through Whatman (GE Healthcare Life Sciences, Marlborough, MA, USA) glass microfiber filters. After filtering the SSJ was transferred to storage bottles and placed in a freezer at -12°C until use. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were suitable for cell and microbiological cultures. Yeast extract utilized in experiments had a grade level for molecular biology and a premium quality level. Sulfuric acid was purchased from Thermo Fisher Scientific (Pittsburgh, PA). 4 ACS Paragon Plus Environment

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Phaffia Culturing A stock culture in 10% (v/v) glycerol was reactivated from storage at -80°C. About 1 mL of the stock culture was added to a sterilized Erlenmeyer flask containing 25 mL of yeast malt broth solution. The strain was incubated for two days at 22°C and 200 RPM in a New Brunswick (Edison, NJ, USA) Innova 4230 incubator. After two days, the strain grew adequately to produce a slightly pink solution. Yeast malt agar plates were streaked and incubated for two days at 22°C until colonies appeared. The plates were removed, wrapped in Parafilm, and placed in refrigeration for storage. New liquid cultures of yeast malt broth were inoculated from a single colony on the stored plates. New plates were streaked every 2-3 weeks to maintain an active strain. Shake Flask Experiments Batch shake flask cultures were conducted in 250 mL Erlenmeyer flasks. Table 1 outlines the conditions for culturing performed on defined sugar medium. The experiments were conducted aerobically by utilizing foam plugs to cover the flask opening. The sugar concentrations were chosen to mimic the average concentrations found in SSJ.38 The defined sugar medium solutions were prepared in the presence of a sodium phosphate buffer to maintain the initial pH at 5.25. The defined sugar medium was sterilized by vacuum filtration by passing through a VWR (Radnor, PA, USA) vacuum filter cup with 0.2 micron filter. Each defined sugar medium shake flask experiment was conducted at 50 mL volume. To each flask 0.5 mL of inoculum was administered. The flasks were placed in a New Brunswick (Edison, NJ, USA) Innova 44 shaking incubator at 22°C and 200 RPM. Samples were taken at 24 hour time points to determine sugar consumption. Each sample was frozen until HPLC analysis. Evaluation of astaxanthin production and biomass growth was determined only at the end of each experiment. Sample preparation is outlined in Analytical Methods and Cell Mass Growth sections. Each experiment condition was conducted in duplicate. Similar shake flask cultures were next conducted on SSJ. Table 2 outlines the conditions utilized for SSJ shake flask experiments. The same procedure for sterilization and inoculation utilized for the defined medium samples was adapted for SSJ shake flask experiments. Instead of utilizing sodium phosphate buffer to maintain the initial pH of 5.25, the SSJ was adjusted to the desired pH by the addition of acid or base prior to filter sterilization. To test the effect of mineral 5 ACS Paragon Plus Environment


addition on P. rhodozyma cultivation and astaxanthin generation, SSJ was diluted in half prior to inoculation. Table 3 lists the concentration of each mineral added. The mineral concentration was based upon prior results indicating optimal growth for P. rhodozyma ATCC 74219.39 The pH for the diluted SSJ was increased to 5.5 as this pH has been found to be within an optimal range for biomass growth.40 Each experiment condition was conducted in duplicate. 2 L Bioreactor Experiments A New Brunswick (Edison, NJ, USA) BioFlo/CelliGen 115 Fermentor/Bioreactor was utilized for experiments conducted at a total volume of 1.5 L. Full strength SSJ was utilized and supplemented with 2.0 g/L yeast extract, 0.4 g/L urea, all minerals outlined in Table 3 and 2 mL of Sigma Antifoam 204. The initial pH was adjusted to 5.5 through the addition of acid or base. A buffer was not used in bioreactor experiments since the SSJ medium would have needed a large volume of buffer to maintain pH. Not only would a large volume of buffer be needed and thus extensively dilute the SSJ, but also the volume capacity of the bioreactor (2 L) would have been breached. The bioreactor was inoculated with 150 mL of sterile filtered and supplemented SSJ that was previously inoculated with 3 mL of P. rhodozyma culture and incubated for 24 hours. Prior to inoculation, the bioreactor and its contents were sterilized by autoclaving at 121°C for 20 minutes. The cultivation temperature was maintained at 22°C and the dissolved oxygen (DO) was controlled at 40% of air saturation by adjusting the agitation rate. The air flow rate was set at 2.0 L/minute. A pH of 5.5 was maintained with 1 M sulfuric acid and ammonium hydroxide diluted to 7.5% (w/w). A deadband interval of 0.3 was set for pH control. Samples were taken at 24 hour time points and frozen until sample preparation. The experiment trial was performed in duplicate. Analytical Methods The extraction and quantification of astaxanthin was performed based on previous studies.34, 41-42 In brief, a 1 mL aliquot sample was added to glass test tubes along with 3 mL of water. Samples were centrifuged for 5 minutes and the supernatant removed by aspiration. Glass beads (0.5 mm diameter) were added to the test tubes along with 1.5 mL of acetone. Test tubes were vigorously vortexed for 1 minute followed by sonication for 5 minutes. The samples were centrifuged for 5 minutes and the supernatant was transferred to a quartz cuvette and read at 480 nm on a Shimadzu UV-1800 UV/Vis spectrophotometer (Shimadzu Scientific Instruments, 6 ACS Paragon Plus Environment

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Somerset, NJ, USA). Acetone was utilized as a blank measurement, and a calibration curve was prepared by dissolving astaxanthin in acetone at varying concentrations. Sugar quantification was performed on an Agilent (Santa Clara, CA, USA) 1100 Series HPLC equipped with a Bio-Rad (Hercules, CA, USA) Aminex HPX-87P column and de-ashing guard column. Detection was performed with a refractive index (RI) detector. The mobile phase was ultrapure water obtained from an EMD Millipore (Billerica, MA, USA) Simplicity filtration system. The ultrapure water was filtered through a Whatman 0.2 micron nylon filter prior to use. All fermentation broth samples prepared for HPLC sugar quantification were centrifuged in an Eppendorf (Hauppauge, NY, USA) Mini-Spin Plus centrifuge at 9,600g for 10 minutes. The samples were syringe filtered through a PALL Life Sciences (Westborough, MA, USA) Acrodisc 0.2-micron filter into sample vials. Calibration standards containing fructose, glucose, and sucrose were analyzed during each HPLC sequence. Cell Mass Growth Due to the red pigmentation, the yeast cell mass growth was assayed by gravimetric analysis. A 5 ml fermentation broth aliquot was placed in 15 mL conical centrifuge tubes. The tubes were centrifuged, the supernatant discarded, and the solid pellet washed with DI water. After a second centrifugation, the wash water was decanted and the solid mass transferred to a pre-weighed aluminum pan. The pan was dried in a 135°C oven for 2 hours based upon prior method recommendation.34, 42 The pans were removed from the oven and cooled to room temperature in a desiccator before being weighed. Each cell mass sample was corrected for the presence of astaxanthin. Yield, Cell Content, and Volumetric Productivity Calculations Total astaxanthin and biomass yields were calculated according to equations 1 and 2, respectively:

⁄ = ⁄ =

(1)

(2)

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Where YP/S is the astaxanthin yield and YX/S is the biomass yield. CP is the astaxanthin concentration, CX is the biomass concentration, and CS is the consumed substrate (i.e. total sugar) concentration. The cell astaxanthin content was determined according to equation 3:

⁄ =

(3)

Where YP/X is the cell astaxanthin content on a mass fraction basis. Equation 4 defines volumetric productivity as:

=

(4)

∆

Where QP is the maximum volumetric productivity for astaxanthin and ∆t is the total time elapsed for Phaffia cultivation. RESULTS AND DISCUSSION Defined Sugar Medium Shake Flask Cultures Initial growth experiments of P. rhodozyma, in defined sugar medium were carried out in shake flasks at different yeast extract loadings. Figure 1 shows the time course sugar consumption for each yeast extract level. The cultivation conditions are listed in Table 1. Increasing the concentration of yeast extract resulted in increased sugar consumption by the organism; especially the hydrolysis of sucrose into its constituent monomers. Yeast extract supplied at 1.0 g/L (Figure 1C) allowed for complete sucrose hydrolysis within 72 hours, while faster sucrose hydrolysis was obtained at 2.0 g/L yeast extract (Figure 1D). Invertase activity by P. rhodozyma has been documented previously and can contribute to a lag in monomer assimilation by the increase in both glucose and fructose concentrations.43-44 Figure 2 shows the astaxanthin product output and biomass growth at each yeast extract loading. At a yeast extract concentration of 3.0 g/L both the astaxanthin and biomass concentration (Figure 2A), and the product yields for each (Figure 2B) reach a maximum under the supplied conditions. Cell astaxanthin content reaches 1.7 mg astaxanthin/g dry cell mass at 2.0 and 3.0 g/L yeast extract loadings. This cell astaxanthin content is much lower than what is obtained at 0.1 and 0.5 g/L yeast extract concentrations.


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At low yeast extract loadings P. rhodozyma has difficulty growing as shown in Figure 2. However, higher titer astaxanthin generation by P. rhodozyma does not develop until later logarithmic growth and continuing into the stationary phase.45-46 Therefore, to obtain greater astaxanthin production, greater biomass growth must be achieved. Upon culturing completion after four days the final pH decreased for each sample. At 0.1 g/L yeast extract concentration the final pH was 5.1, while at 3.0 g/L yeast extract concentration the final pH was 4.35. This lower pH might have an effect on astaxanthin production since greater carotenoid production can be achieved at a pH of 4.0.40 Although a yeast extract loading of 3.0 g/L in the defined sugar medium produced the highest astaxanthin titers and biomass growth, further experiments were conducted at yeast extract loadings lower than 3.0 g/L. First, from an industrial processing point of view, lower chemical input (in this case yeast extract) can lead to lower operational costs. Secondly, as the next round of experiments focused on nitrogen supplementation, there was prior evidence that nitrogen addition could compliment the nutrient components in yeast extract to improve both astaxanthin generation and biomass growth.47 Lastly, Figure 2B shows that the cell astaxanthin content on a mass basis is relatively the same for yeast extract loadings at 2.0 and 3.0 g/L. The cell astaxanthin content is an important parameter in knowing how enriched the yeast is with the carotenoid. Achieving a similar astaxanthin content in the biomass with less yeast extract is beneficial to the economic potential for producing a naturally derived carotenoid. Figure 3 presents the data for defined sugar medium shake flask cultures with different nitrogen supplementation levels. The supplied concentrations of urea and ammonium sulfate are equal at 0.01% and 0.1% nitrogen on a molar basis in solution. Like the results displayed in Figure 1, the sugar content in the defined medium supplemented with urea or ammonium sulfate was not fully utilized after 96 hours of cultivation (data not shown). The primary sugar monomers remaining were glucose and fructose while sucrose was hydrolyzed fully after 48 hours for all samples analyzed. A control sample containing only sugar and buffer (without nitrogen and nutrient supplementation) produced a total10 g/L in sugar consumption with 47 g/L of sucrose remaining after 96 hours. In Figures 3A and 3B, the product and biomass concentrations are presented. A higher concentration of astaxanthin is obtained, albeit only an increase by 3.6 mg/L, in Figure 3B with a higher loading of ammonium sulfate but the overall biomass growth decreases when compared to the lower ammonium sulfate loading. The same observation is seen with urea loading as well. The samples cultured under these conditions 9 ACS Paragon Plus Environment


exhibited even greater pH drift during cultivation. After termination at 96 hours the final pH for the high loading of urea was at 7.08 while the high loading of ammonium sulfate was at 3.02. Phaffia rhodozyma is known to express urease to catalyze the hydrolysis of urea to carbon dioxide and ammonia.48 The high loading of urea (at 2.1 g/L) might indicate that the hydrolysis of urea generated an excess of ammonia that increases the pH above 7.0. The small increase in astaxanthin concentration (3.6 mg/L) at higher ammonium sulfate concentration (4.7 g/L) might be a contribution from the pH shift to 3.02 since it is known that more acidic pH can influence greater astaxanthin generation in P. rhodozyma.41, 49 The shift to a lower pH should not be unexpected since ammonium sulfate dissolved in water is naturally acidic. However, the shift to a pH around 3.0 might be more indicative that P. rhodozyma had access to an easily assimilable nitrogen source in ammonium sulfate as opposed to urea. With supplemented urea, the organism must first supply urease for hydrolysis to occur. A more assimilable nitrogen source could influence pH instability by the organism utilizing ammonia more quickly and while the ability to maintain system pH is lost. In Figures 3C and 3D the overall product yields are presented. Although astaxanthin yield increases at the higher nitrogen loadings, the overall cell mass yield is lowered at these conditions. The results obtained from defined sugar medium cultures gave a better indication for overall culture time and a nitrogen supplementation strategy. Sweet Sorghum Juice Shake Flask Cultures Sweet sorghum juice (SSJ) cultivation conditions are outlined in Table 2. Each flask was supplemented with either yeast extract, urea, ammonium sulfate, or without nitrogen and nutrients (as a control). Figure 4A presents the sugar consumption time course. The cultivation time for these experiments was increased to 168 hours to determine if more sugar could be metabolized along with greater astaxanthin accumulation and biomass growth. At some supplementation conditions (e.g. ammonium sulfate), sugar consumption is greater with less nitrogen addition when compared to defined sugar medium cultures. As expected each condition (except the control) could hydrolyze sucrose completely during fermentation. In Table 4 the first four rows present the overall product concentrations and yields, final pH, cell astaxanthin content, and volumetric productivity for each SSJ culture at the conditions listed. Only modest astaxanthin production and cell mass growth could be achieved without supplemented nitrogen. Compared to defined sugar medium, SSJ supplemented with yeast extract and urea achieved


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drastic improvements in astaxanthin accumulation while astaxanthin production decreased in SSJ with ammonium sulfate. Conversely, nitrogen supplementation produced an increase in biomass growth concentrations to around 12 g/L for all SSJ cultures as shown in Table 4. This is a higher concentration when compared to the defined sugar medium with similar supplementation levels that only produced biomass around 3 g/L. Astaxanthin concentration reached 19.51 mg/L and 10.18 mg/L, respectively, for SSJ cultures supplemented with either yeast extract or urea. Again, this concentration is higher than the defined sugar medium culture that only produced about 3.6 mg/L of astaxanthin. Although consistent biomass growth was achieved for each condition with a different nitrogen source, the concentration and yield of astaxanthin was lower for SSJ cultures supplemented with urea and ammonium sulfate as compared to yeast extract. The lower astaxanthin production also decreased the cell astaxanthin content from 1.516 mg/g to 0.83 and 0.375 for cultures supplemented with urea or ammonium sulfate, respectively. It is well known that carotenoid generation by P. rhodozyma and other organisms is affected greatly by nitrogen assimilation.50 It should be expected that an increase in biomass growth should generate more astatxanthin accumulation since the generation of the carotenoid by P. rhodozyma is growth associated. However, other variables can influence astaxanthin accumulation other than nitrogen assimilation. Low oxygen uptake that can decrease astaxanthin production, and the carbon-tonitrogen ratio (C/N) that can influence both biomass growth (low C/N) and astaxanthin production (high C/N) within the first 24 to 48 hours.29, 51-52 It’s possible that the combination of the high carbon content of the SSJ culture (~ 120 g/L) coupled with the lone nitrogen source of ammonium sulfate at 3.0 g/L had a detrimental effect on astaxanthin generation but not necessarily biomass growth. Overall, the sample with yeast extract performed more favorably towards astaxanthin generation and biomass growth since this complex nutrient can provide an organism not only with nitrogen, but also additional vitamins and minerals. Prior research on P. rhodozyma has indicated better biomass growth and astaxanthin production with the addition of minerals and vitamins to a given medium.39, 42 To improve biomass growth and product generation, SSJ was next supplemented with the minerals listed in Table 3 along with increasing the overall nutrient supplementation by combining yeast extract with urea or ammonium sulfate. It should be noted that the initial pH for SSJ medium at the



shake flask level was increased to 5.5 as opposed to an initial pH of 5.25 for the defined sugar medium prepared with phosphate buffer. Past research indicated that an initial starting pH of 6.0 was more beneficial for biomass growth while an initial pH of 5.0 favored astaxanthin accumulation.40 Figure 4B and Table 4 present the obtained results. Furthermore, this study utilized SSJ that was diluted in half (50% v/v) as a way to minimize inhibition that can be caused by high carbon loadings.51 Figure 4B shows that near complete sugar utilization could be achieved at all supplementation levels. Complete sucrose hydrolysis could be achieved in 48 hours at which time the concentration of glucose and fructose peaked. The introduction of minerals to the SSJ culture did appear to produce a positive effect in terms of sugar utilization and product generation. When compared to defined sugar medium supplemented with both yeast extract and urea or ammonium sulfate, as shown in Figure 3, incomplete sugar utilization was exhibited by the organism along with lower titers of astaxanthin and biomass growth when compared to the results in Table 4 for 50% (v/v) diluted SSJ. The improved sugar utilization and production of P. rhodozyma is most likely the result of SSJ dilution. As stated previously high carbon loadings can have an inhibitory effect on P. rhodozyma biomass growth by decreasing the amount of available nitrogen.52 The combinatorial effect of diluted SSJ, mineral addition, and coupling yeast extract with additional nitrogen was able to improve not only astaxanthin generation and biomass growth by P. rhodozyma, but also carbon source depletion in the SSJ culture. As shown in Table 4, astaxanthin production reached 39.38 mg/L and biomass growth reached 17.35 g/L for SSJ supplemented with yeast extract and urea. The high concentrations of astaxanthin and biomass also produced the highest product yield, cell astaxanthin content, and fastest astaxanthin volumetric productivity for all shake flask conditions listed in Table 4. When compared to the combination of yeast extract and urea, the yeast extract only condition and yeast extract with ammonium sulfate underperformed in terms of biomass growth and astaxanthin generation. Additionally, the yeast extract with urea combination produced a final pH of 5.51. While pH is expected to drift slightly during P. rhodozyma cultivation supplemented with urea, it was promising to observe that the initial pH of 5.5 was maintained after 168 hours of cultivation with the supplied nutrient and nitrogen source.


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Bioreactor Culture with Sweet Sorghum Juice A 2 L bioreactor cultivation with dissolved oxygen (DO), agitation, and pH control was utilized on full strength SSJ supplemented with 2.0 g/L yeast extract and 0.4 g/L urea. Figure 5 shows the results from the cultivation while Table 4 reports final concentrations, yields, and productivity. As seen in Figure 5A, the time course sugar consumption shows that sucrose can be completely hydrolyzed within 72 hours while fructose consumption is nearly complete after 168 hours. Interestingly, the rate of glucose consumption stalls at 72 hours and is not completely metabolized by the organism. The final glucose consumption after 168 hours is around 24 g/L. Fructose utilization by P. rhodozyma can be delayed by the presence of glucose, but fructose consumption can finish before complete glucose utilization.53 In the glycolysis pathway, fructose enters a step below glucose which might contribute to the faster consumption of fructose.54 Moreover, glucose and fructose utilization by yeast strains is dependent on both yeast properties and respiration or fermentation condition.55 Saccharomyces cerevisiae has been well characterized to show that glucose and fructose uptake are controlled by both a low and a high affinity transport system.56 In S. cerevisiae hexokinases are known to catalyze the phosphorylation for both glucose and fructose, however, wine yeast strains of S. cerevisiae showed that a higher fructose-to-glucose phosphorylation ratio correlated to lower glucose preference and, in the case of a laboratory strain of S. cerevisiae, an overexpression of the Hxk1 enzyme favored more rapid fructose consumption over glucose.57-58 Another industrially relevant wine yeast strain of S. cerevisiae was shown to harbor a mutated Hxt3 transporter that resulted in higher fructose utilization.59 Taken together the underlying mechanisms by yeast strains to consume sugars can be limited by inherent molecular characteristics or growth environment. In the case with P. rhodozyma cultivated in SSJ, it is more likely that the bioprocessing conditions need further optimization to overcome incomplete glucose utilization. Figure 5B shows titers for astaxanthin and biomass. Although biomass growth slows after 48 hours, the growth continually increases until 168 hours. The overall biomass concentration reaches 28.8 g/L. On the other hand, astaxanthin concentration continually increases up to 168 hours where it reaches about 65 mg/L. Due to the increase in astaxanthin concentration, the overall yield for astaxanthin increases with time as shown in Figure 5C. Moreover, the cell astaxanthin content reaches 2.49 mg astaxanthin/g dry mass by 168 hours. Due to incomplete



glucose utilization, one of the fermentation trials progressed an additional three days to determine if any remaining glucose could be metabolized. Glucose consumption did occur but only decreased by an additional 3 g/L to 21 g/L at 240 hours. Although organic acid formation (e.g. acetic or formic acid) can be detrimental to the cultivation of this organism, it is unlikely that an appreciable amount of acid existed in the broth since the pH was maintained between 5.3 to 5.7 with no additional pH adjustment performed during cultivation of the organism. Additional 2 L bioreactor cultivations performed on 25% and 50% (v/v) diluted SSJ (Supporting Information Figures S1 and S2) gave additional insight into possible carbon loading inhibition on glucose utilization. Final concentrations, yields, and productivity for the bioreactor trials are included in Table 4. At a 50% (v/v) dilution all sugar contained in the SSJ could be utilized by the yeast strain, but at a 25% (v/v) dilution level 17.2 g/L of glucose remained after 168 hours. The 25% (v/v) dilution level has a starting total sugar concentration around 90 g/L while the 50% (v/v) dilution level started at 60 g/L total sugar. This indicates that a maximal initial sugar concentration might exist for P. rhodozyma to fully utilize a supplied carbon source during SSJ batch cultivation. This is also supported by control shake flask cultivations performed at initial sugar concentrations of 50 g/L, 75 g/L, and 100 g/L supplemented with 2.0 g/L yeast extract and 0.4 g/L urea (Supporting Information Figure S3 and Table S1). At 168 hours P. rhodozyma can utilize all sugar supplied at starting concentrations of 50 g/L and 75 g/L. An initial sugar concentration of 100 g/L inhibits complete sugar utilization at 168 hours leaving about 22 g/L of unutilized glucose. It is probable that at the cultivation conditions tested an initial sugar concentration between 75-90 g/L exists that will allow for complete sugar utilization by P. rhodozyma during batch bioprocessing. As indicated in Table 4 the biomass and astaxanthin production titers were lower for diluted SSJ cultivation when compared to undiluted SSJ. For the undiluted SSJ allowed to cultivate up to 240 hours no additional biomass growth was observed, but the concentration of astaxanthin did increase to 90 mg/L. This improved the astaxanthin yield to 1.08 mg astaxanthin/g sugar consumed and the cell astaxanthin content to 3.63 mg astaxanthin/g dry cell mass. These results obtained through cultivating P. rhodozyma on SSJ are encouraging when compared to other agricultural feedstocks. Date juice from Yucca fillifera supplemented with only urea was cultivated with a strain of P. rhodozyma for three days to produce an astaxanthin


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cell content of 1.203 mg astaxanthin/g yeast, which compares favorably here since at 72 hours a cell content of 1.17 mg astaxanting/g dry cell could be achieved.60 A more optimized and larger scale process utilizing molasses for cultivation of P. rhodozyma produced 36 g/L of biomass with 40 mg/L of astaxanthin and an astaxanthin cell content of 1.1 mg astaxanthin/g yeast within 200 hours.61 While biomass growth with SSJ was lower, the ability to obtain higher astaxanthin titers and greater astaxanthin content in a process not currently optimized or scaled up is encouraging for future process refinement. Even when comparing the same strain, SSJ cultivation with P. rhodozyma outperformed diluted corn fiber hydrolyzate where only 20.9 mg/L of astaxanthin was generated with a cell astaxanthin content of 0.82 mg astaxanthing/g dry cell.34 A compilation of the volumetric astaxanthin productivities are presented in Table 4 from shake flask and bioreactor experiments. As can easily be identified the yeast extract and urea combination in diluted SSJ at the shake flask level produced the best astaxanthin productivity. At the 2 L bioreactor scale with undiluted SSJ the astaxanthin productivity increased to 0.389 mg/L/hr after 168 hours of cultivation. This productivity is higher when compared to a mixedsugar medium (0.13 mg/L/hr) and glycerol medium (0.2 mg/L/hr) used for cultivation.42, 62 Although the cultivation of this yeast on SSJ is encouraging, more process optimization is necessary. More recent work has shown that a mutant strain of P. rhodozyma in a fed-batch process was able to produce astaxanthin up to 0.7 g astaxanthin/kg of culture broth during the maturation phase at a rate of 3.3 mg of astaxanthin/kg/hr by lowering the pH to 3.5 and increasing mineral and vitamin concentrations.49 One possible route for process optimization is to investigate fed-batch processing of SSJ by utilizing a concentrated form of SSJ to reduce fluctuations in reactor volume and improve astaxanthin titers and biomass growth. CONCLUSIONS Sweet sorghum juice (SSJ) was shown to be a viable feedstock to produce the carotenoid astaxanthin via Phaffia rhodozyma cultivation. Shake flask experiments performed with defined sugar medium indicated that yeast extract, urea, and ammonium sulfate could provide necessary nitrogen and nutrient supplementation to grow the yeast strain and produce astaxanthin as a secondary metabolite. Although P. rhodozyma could only modestly grow on SSJ without nitrogen supplementation, the combination of yeast extract and urea provided complete sugar 15 ACS Paragon Plus Environment


metabolism and adequate growth on 50% (v/v) diluted SSJ. Full strength SSJ cultivation with the same nitrogen supplementation in a 2 L bioreactor could produce up to 29 g/L of cell mass growth, 65.4 mg/L of astaxanthin, a cell astaxanthin of 2.49 mg astaxanthin/g dry cell mass, and a volumetric astaxanthin productivity of 0.389 mg/L/hr after 168 hours of cultivation. Although this is an encouraging result higher titer biomass growth, better astaxanthin generation, and larger production scale is needed for naturally produced astaxanthin to compete with synthetic astaxanthin economically. Further process optimization will be investigated since incomplete glucose metabolism was observed with full strength SSJ. Future studies will focus on improving growth through different nitrogen supplementation levels and employing fed-batch SSJ cultivations. ACKNOWLEDGEMENTS The authors would like to thank Delta Biorenewables for the supply of sweet sorghum juice used to conduct this research. Also, the authors would like to thank the following individuals for their experimental assistance: Renee Latona assisting with yeast culturing, experimental set up, and the analytical determination of both astaxanthin and dry cell mass; Jennifer Thomas assisting with propagating the initial stock culture; and Gerard Senske for assisting with setting up the bioreactor experiments. SUPPORTING INFORMATION Supplementary Figure S1: Sugar consumption, biomass and product yields, and cell astaxanthin content for 25% (v/v) diluted sweet sorghum juice Supplementary Figure S2: Sugar consumption, biomass and product yields, and cell astaxanthin content for 50% (v/v) diluted sweet sorghum juice Supplementary Figure S3: Sugar consumption on defined sugar medium shake flask cultivations with nutrient and nitrogen supplementation at varying dilution levels Supplementary Table S1: Biomass and product concentrations, yields, cell astaxanthin content, and volumetric productivities for defined sugar medium shake flask cultivations at varying dilution levels


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Table 1. Defined Sugar Medium Shake Flask Culture Supplementation Levels (Note: Sugar concentrations for all samples prepared at 75 g/L sucrose, 15 g/L glucose, and 10 g/L fructose; experiments conducted at 22°C and pH of 5.25) Nitrogen and Nutrient Source (g/L) Yeast Urea Ammonium Extract Sulfate 0.1 --0.5 --1.0 --2.0 --3.0 --2.0 0.21 -2.0 2.1 -2.0 -0.47 2.0 -4.7


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Table 2. Sweet Sorghum Juice Shake Flask Culture Supplementation Levels (Note: Experiments marked with a † had an initial pH of 5.25; experiments marked with a * had an initial pH of 5.5 and contained the listed minerals from Table 3; all experiments conducted at 22°C) Nitrogen and Nutrient Source (g/L) Strength Yeast Urea Ammonium Sulfate of Juice Extract Full† ---Full† 2.0 --Full† -0.4 -Full† --3.0 50% ---Dilution* 50% 2.0 --Dilution* 50% 1.0 0.4 -Dilution* 50% 1.0 -3.0 Dilution*



Table 3. Mineral Addition for Sweet Sorghum Juice Cultures Component Potassium Phosphate Monobasic Magnesium Sulfate Sodium Chloride Ferrous Sulfate Zinc Sulfate Citric Acid Manganese Sulfate Cupric Sulfate

Concentration (g/L) 5.71 5.71 1.0 0.078 0.021 0.0071 0.0042 0.0014


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Table 4. Collected Results for P. rhodozyma Cultivation in Sweet Sorghum Juice (SSJ) at Shake Flask and Bioreactor Volumetric Scales (Note: Experiments marked with * contained supplemented minerals listed in Table 3; Final pH, Concentrations, and Yields Are After 168 Hours of Cultivation) Experiment Scale

Shake Flask

Bioreactor

Experimental Condition No Supplementation

SSJ Strength Full

Yeast Extract (2.0 g/L) Urea (0.4 g/L) Ammonium Sulfate (3.0 g/L) No Supplementation* Yeast Extract (2.0 g/L)* Yeast Extract and Urea (1.0 g/L; 0.4 g/L)* Yeast Extract and Ammonium Sulfate (1.0 g/L; 3.0 g/L)* Yeast Extract and Urea (2.0 g/L; 0.4 g/L)* Yeast Extract and Urea (2.0 g/L; 0.4 g/L)* Yeast Extract and Urea (2.0 g/L; 0.4 g/L)*

pH 5.08

X (g/L) 2.38 ± 0.050

P (mg/L) 5.89 ± 1.174

YP/S (mg/g) 0.262 ± 0.051

YX/S (g/g) 0.106 ± 0.003

YP/X (mg/g) 2.49 ± 0.550

QP (mg/L/hr) 0.035

Full

4.83

12.83 ± 0.432

19.51 ± 2.766

0.329 ± 0.057

0.215 ± 0.014

1.516 ± 0.165

0.116

Full

5.11

12.24 ± 0.299

10.18 ± 1.036

0.215 ± 0.022

0.259 ± 0.006

0.83 ± 0.064

0.061

Full

3.98

13.68 ± 0.060

5.13 ± 0.239

0.067 ± 0.004

0.179 ± 0.003

0.375 ± 0.019

0.031

50% Dilution 50% Dilution 50% Dilution

5.34

1.31 ± 0.27

8.61 ± 0.61

0.476 ± 0.03

0.072 ± 0.014

6.76 ± 0.93

0.051

2.95

7.49 ± 1.30

14.83 ± 6.17

0.245 ± 0.101

0.124 ± 0.021

1.90 ± 0.494

0.088

5.51

17.35 ± 1.41

39.38 ± 11.65

0.681 ± 0.200

0.300 ± 0.024

2.23 ± 0.49

0.234

50% Dilution

3.82

12.29 ± 0.13

21.39 ± 3.27

0.348 ± 0.51

0.201 ± 0.001

1.74 ± 0.247

0.127

Full

5.46

28.8 ± 7.49

65.4 ± 5.23

0.766 ± 0.145

0.325 ± 0.049

2.49 ± 0.829

0.389

25% Dilution

5.51

27.3 ± 0.530

51.92 ± 0.935

0.820 ± 0.015

0.413 ± 0.008

1.903 ± 0.071

0.309

50% Dilution

5.76

23.7 ± 0.377

20.58 ± 3.26

0.348 ± 0.055

0.402 ± 0.006

0.865 ± 0.124

0.133

X is dry cell mass concentration, P is astaxanthin concentration, YP/S is astaxanthin yield (mg astaxanthin per g sugar consumed), YX/S is dry cell mass yield (g dry cell mass per g sugar consumed), YP/X is cell astaxanthin content (mg astaxanthin per g dry cell mass), and QP is the astaxanthin volumetric productivity (mg astaxanthin per L per hour).



Figure 1. Sugar Consumption Profiles for Defined Sugar Medium Shake Flask Cultures with (A) 0.1 g/L Yeast Extract, (B) 0.5 g/L Yeast Extract, (C) 1.0 g/L Yeast Extract, (D) 2.0 g/L Yeast Extract, and (E) 3.0 g/L Yeast Extract


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Figure 2. (A) Product Concentrations and (B) Product Yields and Cell Astaxanthin Content for Defined Sugar Medium Cultures at Varying Yeast Extract Supplementation Levels



Figure 3. (A & B) Product Concentrations and (C & D) Product Yields and Cell Astaxanthin Content for Defined Sugar Medium Cultures at Low and High Nitrogen Levels with Yeast Extract Loading of 2.0 g/L (Control Lacks Nitrogen and Nutrient Supplementation)


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Figure 4. Total Sugar Consumption in (A) Full Strength SSJ and (B) 50% Diluted (v/v) SSJ at Shake Flask Cultivation Scale



Figure 5. (A) Time Course of Sugar Consumption, (B) Product Concentrations, and (C) Product Yields and Cell Astaxanthin Content for Full Strength Sweet Sorghum Juice Culture at 2 L Bioreactor Scale


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TOC Graphic

Synopsis: Natural astaxanthin can be generated as a secondary metabolite from sweet sorghum juice cultured with Phaffia rhodozyma.


ACS Sustainable Chemistry & Engineering 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

Inoculation with Phaffia rhodozyma

Crushing

Juice

Bagasse for further biochemical conversion to fuels or co-products

Aerobic Fermentation

Natural astaxanthin for aquaculture or nutraceutical applications

ACS Paragon Plus Environment

Page 32 of 32

Utilization of Sweet Sorghum Juice for the Production of Astaxanthin

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