- Email: [email protected]
Pilot-scale production of phylloquinone (vitamin K1) using a bubble column photo-bioreactor
Biochemical Engineering Journal 150 (2019) 107243
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Pilot-scale production of phylloquinone (vitamin K1) using a bubble column photo-bioreactor Thomas D.C. Tarento, Dale D. McClure, Fariba Dehghani, John M. Kavanagh
T
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The University of Sydney, School of Chemical and Biomolecular Engineering, NSW, 2006, Australia
H I GH L IG H T S
(50 L) process for production of vitamin K developed. • Pilot-scale of medium, sparger, superficial velocity and day length examined. • Effect produced was rich in vitamin K (330 μg g ). • Biomass • Concentration is ten times more than other rich dietary sources. 1
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A R T I C LE I N FO
A B S T R A C T
Keywords: Photo-bioreactor Bubble column Vitamin K Phylloquinone Microalgae Cyanobacteria
Phylloquinone (vitamin K1) is an essential nutrient for both humans and animals; it plays a key role in blood coagulation amongst other processes. In this work we describe the scale-up of a biosynthetic process for phylloquinone production using the cyanobacterium Anabaena cylindrica. A 50 L bubble column photo-bioreactor was used to cultivate A. cylindrica and the effects of a wide range of operating conditions including superficial velocities (0.67–6.7 cm s−1), day lengths (12–24 h), sparger designs and medium compositions were examined. The column design and superficial velocity had minimal impact on phylloquinone production, while changes to the medium composition and day length had large impacts. By varying these factors we were able to achieve final phylloquinone titres of the order 280 μg L−1 and productivities of 40 μg L−1 day−1, which are approximately double the values previously obtained. The biomass produced has clear applications in human and animal nutrition as its phylloquinone concentration was approximately 330 μg g−1 (dry basis); this being ten times higher than rich dietary sources. Conclusions regarding the effect of photo-bioreactor design and operating conditions can be applied to the production of phylloquinone, as well as the scale-up of cyanobacterial cultures more broadly.
1. Introduction Vitamin K refers to a group of structurally similar compounds which play essential roles in both human and animal nutrition. Phylloquinone (vitamin K1) is typically found in leafy green vegetables and the menaquinones (vitamin K2) are found in fermented foods. Menadione and its derivatives are synthetic vitamin K analogues that have been banned for human consumption but are still used in animal nutrition [1–5]. Vitamin K is best known for its role in blood coagulation [6], however it is also involved in other biological processes including bone maintenance, modulation of inflammation and prevention of arterial hardening [7,8]. Phylloquinone participates in photosynthetic electron transport (as part of Photosystem I) [9]. In our previous work [10] we identified a strain of microalgae (the
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cyanobacterium Anabaena cylindrica) with a phylloquinone concentration of the order 200 μg g−1 (dry basis); substantially greater than other dietary sources, with one gram of the dry algae providing 2–3 times the recommended daily intake of phylloquinone [11,12]. Other authors [13] have quantified the concentrations of phylloquinone produced by different species of algae (6–750 μg g−1), however little information about conditions favouring phylloquinone production is available in the open literature. In our previous work [10] it was found that high nitrate levels favoured phylloquinone production, and that the phylloquinone titre was directly proportional to the biomass concentration. Given its role within the cell it is likely that the most favourable conditions for phylloquinone production are those in which the culture is undergoing active, photosynthetic growth. In addition to being a rich source of phylloquinone, the biomass was found to be rich in protein (69% (w/w)
Corresponding author at: School of Chemical and Biomolecular Engineering, Building J01, The University of Sydney, NSW, 2006, Australia. E-mail address: [email protected] (J.M. Kavanagh).
https://doi.org/10.1016/j.bej.2019.107243 Received 8 January 2019; Received in revised form 18 April 2019; Accepted 26 May 2019 Available online 01 June 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
Biochemical Engineering Journal 150 (2019) 107243
T.D.C. Tarento, et al.
on a dry basis) and vitamin B12 [10]. Such biomass could potentially be incorporated into nutraceuticals or fortified foods to ensure sufficient supply of phylloquinone. Similarly, it could be used in animal nutrition applications to replace or supplement menadione. Biosynthesis of phylloquinone has the advantage that it operates at ambient temperature and pressure and avoids the use of organic solvents. Additionally, biosynthesis produces only the active E-isomer, whereas chemical synthesis of phylloquinone produces 10–20% of the inactive Z-isomer [14,15]. A key challenge in microalgal bioprocessing is the scale-up of the production process. Key factors include preventing contamination of the culture, ensuring good mass transfer (to supply carbon dioxide and remove oxygen), ensuring good mixing and, especially, ensuring sufficient light supply. For bubble column and airlift photo-bioreactors (PBRs), these factors are related to the reactor design (i.e. the reactor diameter and the selection of sparger) as well as the operating conditions (i.e. the superficial velocity). Flow in bubble columns can be divided into the homogenous flow regime (characterised by a narrow bubble size distribution with relatively uniform rise velocities) and the heterogeneous flow regime (characterised by a broader bubble size distribution and increased mixing in the liquid phase) [16,17]. The transition between these flow regimes depends on the column diameter, sparger design and superficial velocity. For the column examined in this work, the flow transition is predicted to occur at superficial velocities of the order 0.05 m s−1 based on published regime maps [17]. Whilst it is generally recognized that the column hydrodynamics is a key factor in PBR operation, due to its impact on mixing and mass transfer [18,19], there is relatively little data available in the open literature quantifying the effect of the column design on photo-bioreactor performance, particularly for cultures of cyanobacteria. Scale-up of cyanobacterial cultures may become increasingly important as these microorganisms have desirable characteristics for the photosynthetic production of chemicals [20–22]. Hence, the aims of the work are to develop a pilot-scale production for phylloquinone using A. cylindrica and, as part of this process, quantify the effect of the reactor design and operating conditions (including the medium composition and day length) on culture performance. 2. Method 2.1. Culture conditions A schematic of the pilot-scale photo-bioreactor used in this work is shown in Fig. 1. The PBR was constructed from clear acrylic tubing having a height of 2000 mm and an internal diameter of 190 mm. Air was introduced either through a perforated stainless-steel tube (PST) or a ceramic air-stone (CAS). The PST consisted of a 12.5 mm diameter stainless-steel tube with three rows of 10 holes. Each hole was 2 mm in diameter; the centres of the holes were spaced 10 mm apart. One row of holes was drilled on the centreline of the tube facing down; the other two rows were either side of this row, with an angle of 45° between the centres of each row. The CAS was a cylinder 130 mm in length and 30 mm in diameter made from fused alumina having pores less than 0.3 mm in size and was sourced from AquaOne. The flow-rates of air and carbon dioxide were measured using RM series rotameters (Dwyer); air was sourced from the building supply while the food-grade carbon dioxide was supplied from a cylinder (Coregas). The back-pressure was measured at the outlet of the rotameters using a LPG3 pressure gauge (Dwyer), this ranged between 25–35 kPa for all air flow rates and sparger configurations examined. Air flow rates of 0.2, 0.5, 1 and 2 vvm were examined; these correspond to superficial velocities between 0.67 and 6.7 cm s−1 at standard conditions (101 kPa and 298 K). Here we have chosen to use a wide range of air flow rates as this should encompass conditions in both the heterogeneous and homogenous flow regimes.
Fig. 1. Schematic diagram of the 50 L photo-bioreactor used in this work.
The temperature of the PBR was controlled by circulating water (28 °C) through a 12.5 mm diameter stainless steel U-tube mounted in the top of the reactor. The distance between the top of the column and the bottom of the U was 1570 mm. A Polyscience 6000 series chiller was used to control the temperature of the circulating water. Temperature, pH and dissolved oxygen levels were monitored using probes mounted in the top of the PBR. An InPro 3250i pH probe and an InPro 6850i DO probe (both sourced from Mettler-Toledo) were used. Data from both probes was logged using a M200 transmitter connected to a computer running M200 TCT Software (also from Mettler-Toledo). A total liquid volume of 50 L was used with losses due to evaporation or sampling being made up daily prior to any measurements. Tap water was passed through a depth filter (1 μm, Parker) and an activated carbon filter (Stefani) before entering the column. MLA medium [23] concentrates (without the addition of vitamins or selenium) were prepared and added to the column before each batch. We added the nutrient concentrates at either double or five times the values for normal MLA medium. The flow rate of CO2 was increased briefly while adding the nutrients for the 5 × MLA; this reduced the pH to approximately pH 7 and thereby avoided precipitation. 2
Biochemical Engineering Journal 150 (2019) 107243
T.D.C. Tarento, et al.
Table 1 Summary of experimental results. Ranges are given for experiments done in duplicate. Sparger designs have been abbreviated as CAS = Ceramic Air Stone and PST = Perforated Stainless Tube. Sparger
Air flow rate (vvm)
Medium
Duration of daylight (h)
Final DCW (g L−1)
Final phylloquinone concentration (μg g−1)
Final phylloquinone titre (μg L−1)
Phylloquinone productivity (μg L−1 d−1)
CAS CAS PST PST PST PST PST PST PST PST PST
0.2 0.5 0.2 0.5 1 2 0.2 0.2 0.2 0.2 0.2
2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 5 × MLA 5 × MLA
12 12 12 12 12 12 16 20 24 12 24
0.42–0.54 0.43–0.43 0.44–0.47 0.46–0.46 0.46 0.42 0.63 0.80 0.87–0.90 0.46–0.51 0.81–0.88
256–264 280–312 294–319 272–321 279 279 304 224 183–207 291–307 319–341
108–141 121–133 138–139 124–147 129 117 192 180 139–158 134–141 270-299
15.7–18.8 16.3–18.2 18.8–19.5 19.4–19.6 18.8 17.0 27.9 26.2 22.4–26.5 17.7–18.6 39.2–42.0
Fig. 2. Plots showing the effect of the day length on (a) the biomass concentration; (b) the nitrate concentration; (c) the specific phylloquinone concentration and (d) the phylloquinone titre. The 12 and 24 h runs were performed in duplicate, while the 16 and 20 h runs were not repeated.
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Biochemical Engineering Journal 150 (2019) 107243
T.D.C. Tarento, et al.
Fig. 3. Plots showing the effect of the day length and medium concentration on (a) the biomass concentration; (b) the nitrate concentration; (c) the specific phylloquinone concentration and (d) the phylloquinone titre. All experiments were performed in duplicate.
obtained from the Australian National Algal Culture Collection. Cultures were maintained in 50 mL flasks, using MLA medium, at a light intensity of approximately 70 μmol photons m−2 s-1. To prepare the inoculum for the 50 L PBR, 20 mL of these cultures were added to 200 mL of fresh, sterile MLA medium in a 500 mL flask. These were grown for approximately one week. Culture from several flasks were pooled, and 500 mL was used to inoculate a 5 L flat-panel PBR (the design and operation of which is detailed elsewhere [10]). These cultures were grown for an additional week, using MLA medium and a light intensity of approximately 300 μmol photons m−2 s-1. This culture was then used as the inoculum for the 50 L PBR, sufficient culture (between 2.5-5 L) was added to the 50 L PBR such that the initial optical density (at a wavelength of 550 nm) was approximately 0.1.
A sample port was located on the column centreline at a height of 1000 mm above the base of the column. This port had a 6.25 mm fitting with a valve, which was flushed thoroughly (with a volume of 50–100 mL) before samples were taken. Lighting was provided by LED light strips (9 W per strip, 6000 K colour temperature, Jaycar Australia). A total of 27 strips were used; as shown in Fig. 1 they were arranged in a 3 × 3 (H × W) grid. The distance from the base of the column to the bottom of the lights was 220 mm; the total height of the lights was 1520 mm and there was a distance of 110 mm from the lights to the PBR. The light intensity was measured using a Walz ULM-500 light meter with a US-SQS/L spherical sensor; measurements were made along the column centreline with the column full of water but without any aeration. The light intensity in the photosynthetically active region (400–700 nm) was of the order 300 μmol photons m−2 s−1. To quantify the effect of the lighting duration, experiments were performed using 12:12, 16:8 and 24:0 light:dark cycles. Based on our previous work [10], A. cylindrica was used for the production of phylloquinone. This strain (number CS-172) was
2.2. Analytical methods Microscopic examinations of the culture were performed using a Nikon ECLIPSE Ci-L microscope. The cell density was quantified by measurement of the dry cell weight. Samples (approximately 50 mL) 4
Biochemical Engineering Journal 150 (2019) 107243
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were taken and filtered through a pre-weighed Advantec GA-55 filter paper (Toyo Roshi Kaisha Ltd., Tokyo, Japan); the samples were washed with two volumes of 0.5 M ammonium bicarbonate and dried overnight at 105 °C. The dry samples were cooled in a desiccator and weighed, the dry cell weight was calculated as the difference between the initial and final weights divided by the volume of culture filtered. Uncertainty in the dry cell weight measurements is of the order 5% (as calculated using error propagation methodology). The nitrate concentration in the sample was measured using a standard spectrophotometric screening method (APHA-4500-NO3−-B) [24]. Samples were syringe filtered (0.22 μm) prior to analysis and measurements were made using a Varian Cary 50 spectrophotometer. To determine the concentration of phylloquinone in the biomass, samples (approximately 50 mL) were centrifuged (3000 RCF, 5 min) and the supernatant discarded. The samples were re-suspended in deionised water and the process was repeated to remove any salts; the samples were then freeze dried. Extraction and quantitation of phylloquinone was performed using a previously described method [10]. Values of the specific phylloquinone concentration are reported per gram of freeze dried biomass. In order to quantify the uncertainty in the extraction and analysis procedure it has been repeated twice, for three different species of algae [10]; it was found that the maximum coefficient of variation was 15%.
Overall, these results suggest that for the process and conditions examined, the effect of the sparger design and gas flow rate had a minimal impact on phylloquinone production. All subsequent experiments were therefore performed using the perforated stainless tube at an air flow rate of 0.2 vvm. These conditions were chosen as the perforated tube is easier to clean than the ceramic air stone and the lower air flow rate leads to a reduced energy consumption. 3.2. Effect of day length A potential method of increasing phylloquinone production is increasing the period of illumination (“day length”). However, excessive irradiation can also be damaging for the microalgae [19]. When grown in 5 L PBRs with 24 h of illumination per day it was found that the A. cylindrica cultures turned from blue/green to yellow in colour, potentially as a protective response to damaging high levels of irradiance. To quantify the effect of the day length, experiments were performed using 12, 16, 20 and 24 h day lengths; the results are shown in Fig. 2 and summarised in Table 1. It was observed that increasing the duration of lighting led to higher biomass concentrations. Increases in the day length also corresponded with an increase in the rate of nitrogen consumption; cultures grown with constant illumination exhausted the nitrate present in the medium after approximately four days while those grown with 12 h lighting still had nitrate present on day 7 (as shown in Fig. 2(b)). When examined under the microscope, cells from the nitrogen-exhausted cultures had changed morphology consistent with nitrogen limitation; specifically, heterocysts were observed (results not shown). Heterocysts are differentiated cells that allow filamentous cyanobacteria (like A. cylindrica) to fix atmospheric nitrogen [27]. No change in the colour of the culture was observed, most likely due to the greater path length in the 50 L PBR. Interestingly, this nitrogen limitation appeared to be correlated with a decrease in the specific phylloquinone concentration (as shown in Fig. 2(c)). The decrease in specific phylloquinone content is more than compensated for by the higher biomass concentrations at longer day lengths; as shown in Fig. 3(d) phylloquinone titres of 160–190 μg L−1 were achieved by increasing the day length; these values being greater than those for 12 h lighting (140 μg L−1). To determine whether or not the phylloquinone titre could be further improved, experiments were repeated with 12- and 24 -h lighting with further enriched medium (5 × MLA). The results are shown in Fig. 3. It was found that supplementing the medium led to nitrate replete conditions; as shown in Fig. 2(b) the nitrate concentration at the end of the experiment was 200–400 mg L−1. Supplementing the medium led to a higher specific phylloquinone concentration, this in turn led to higher phylloquinone titres, with values being of the order 280 μg L−1 for the PBR with constant illumination. These results indicate that it is likely the biomass growth could be enhanced by further increases in the light intensity above 300 μmol photons m-2 s−1. Unfortunately, testing the effects of higher light intensities was not possible with the current experimental set-up.
3. Results and discussion 3.1. Effect of PBR design and operating conditions As summarised in Table 1, there was relatively little variation between batches performed using the same experimental conditions, with this this being true for all of the conditions examined. The maximum variation between dry cell weights was of the order 30%, while it was of the order 20% for the specific phylloquinone concentrations. Microscopic examination of the culture showed that the morphology was also consistent for all conditions examined, indicating that for the range of gas flow rates evaluated (0.2 – 2 vvm, corresponding to superficial velocities of 0.67 – 6.7 cm s−1) the shear stress in the liquid phase was not damaging to the cells. It is clear that the effect of shear stress is species specific, as other authors [25,26] have noted that increasing the superficial velocity had negative effects on microalgal growth in bubble columns. It was found that both the rate of biomass growth and nitrate consumption were linear and comparable (Table 1) for the different spargers and superficial velocities examined. Such results demonstrate that varying these factors had negligible effects on the growth of A. cylindrica. Specific phylloquinone concentrations were in the range 250–330 μg g−1, with this value not being affected by the sparger design. Interestingly, the specific phylloquinone concentrations measured in the 50 L PBR were greater than those previously measured [10] in 5 L flat-panel PBRs (approximately 150 μg g−1). This is again thought to be due to the cells compensating for the lower light availability (due to the longer path length in the larger reactor). The phylloquinone titre was observed to increase in a linear fashion, this being explained by the fact that the biomass growth was linear and the specific phylloquinone concentration was approximately constant. During periods of illumination, the DO was of the order 105% of saturation, this being consistent with the production of oxygen by the microalgae. During the dark period, the concentration of dissolved oxygen decreased to the saturation value (i.e. 100%). Due to the relatively high levels of mass transfer found in bubble columns, the maximum DO was always far below inhibitory values (200% of saturation) [19]. In all instances the pH of the PBR was between pH 7–8, indicating that the growth rate was not limited by the supply of carbon. This is one explanation for why the operating conditions had minimal impact on process performance; it is likely that the rate of carbon uptake by the algae was always less than the rate of transfer from gas to liquid.
4. Conclusions The aim of this work was to evaluate the effect of photo-bioreactor design and operation on process performance, with a particular focus on cyanobacterial culture for the production of phylloquinone. A summary of all of the experimental results is given in Table 1. Compared to the results from our previous work [10] using smaller, 5 L flat panel photo-bioreactors, we were able to approximately double the productivity, from 22 μg L−1 day to approximately 40 μg L−1 day. It was found that the primary determinants of the productivity were the composition of the growth medium and the day length. The sparger design and the superficial gas velocity used had minimal impact on the concentrations of phylloquinone and biomass. On a dry weight basis the concentration of phylloquinone in the A. 5
Biochemical Engineering Journal 150 (2019) 107243
T.D.C. Tarento, et al.
cylindrica biomass is of the order 330 μg g−1; this is approximately ten times the concentration of other rich dietary sources. One gram of the dry biomass can provide approximately five times the recommended daily intake of phylloquinone [11]. Hence, there is a clear potential for the biomass to be used in animal nutrition or functional foods as a source of vitamin K1.
Plant Mol. Biol. 52 (2001) 593–626. [10] T.D.C. Tarento, D.D. McClure, E. Vasiljevski, A. Schindeler, F. Dehghani, J.M. Kavanagh, Microalgae as a source of vitamin K1, Algal Res. 36 (2018) 77–87. [11] Australian Government National Health and Medical Research Council, Nutrient Reference Values for Australia and New Zealand Including Recommended Dietary Intakes - Vitamin K, (2014). [12] N. EFSA Panel on Dietetic Products, Allergies, D. Turck, J.-L. Bresson, B. Burlingame, T. Dean, S. Fairweather-Tait, M. Heinonen, K.I. Hirsch-Ernst, I. Mangelsdorf, H.J. McArdle, A. Naska, G. Nowicka, K. Pentieva, Y. Sanz, A. Siani, A. Sjödin, M. Stern, D. Tomé, H. Van Loveren, M. Vinceti, P. Willatts, C. LambergAllardt, H. Przyrembel, I. Tetens, C. Dumas, L. Fabiani, S. Ioannidou, M. NeuhäuserBerthold, Dietary reference values for vitamin K, EFSA J. 15 (2017). [13] Y.D. Roeck-Holtzhauer, I. Quere, C. Claire, Vitamin analysis of five planktonic microalgae and one macroalga, J. Appl. Phycol. 3 (1991) 259–264. [14] A.M. Daines, R.J. Payne, M.E. Humphries, A.D. Abell, The synthesis of naturally occurring vitamin K and vitamin K analogues, Curr. Org. Chem. 7 (2003) 1625–1634. [15] S.D. Van Arnum, K. Vitamin, Kirk-Othmer Encyclopedia of Chemical Technology, John Wiley & Sons, Inc., New York, NY, 2000. [16] N. Kantarci, F. Borak, K.O. Ulgen, Bubble column reactors, Process Biochem. 40 (2005) 2263–2283. [17] Y.T. Shah, B.G. Kelkar, S.P. Godbole, W.D. Deckwer, Design parameters estimations for bubble column reactors, AlChE J. 28 (1982) 353–379. [18] J. Pruvost, F. Le Borgne, A. Artu, J.-F. Cornet, J. Legrand, Chapter five – industrial photobioreactors and scale-up concepts, in: L. Jack (Ed.), Advances in Chemical Engineering, Academic Press, 2016, pp. 257–310. [19] C. Posten, Design principles of photo-bioreactors for cultivation of microalgae, Eng. Life Sci. 9 (2009) 165–177. [20] J. Ni, F. Tao, Y. Wang, F. Yao, P. Xu, A photoautotrophic platform for the sustainable production of valuable plant natural products from CO2, Green Chem. 18 (2016) 3537–3548. [21] A.L. Carroll, A.E. Case, A. Zhang, S. Atsumi, Metabolic engineering tools in model cyanobacteria, Metab. Eng. 50 (2018) 47–56. [22] C. van den Berg, M.H.M. Eppink, R.H. Wijffels, Integrated product recovery will boost industrial cyanobacterial processes, Trends Biotechnol. (2018). [23] C.J.S. Bolch, S.I. Blackburn, Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz, J. Appl. Phycol. 8 (1996) 5–13. [24] American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 22 ed., American Public Health Association, Washington, DC, 2012. [25] A. Contreras, F. García, E. Molina, J.C. Merchuk, Interaction between CO2‐mass transfer, light availability, and hydrodynamic stress in the growth of Phaeodactylum tricornutum in a concentric tube airlift photobioreactor, Biotechnol. Bioeng. 60 (1998) 317–325. [26] S. Takahiro, M. Takeshi, O. Kazuhisa, K. Kozo, Gas‐Sparged bioreactors for CO2 fixation by Dunaliella tertiolecta, J. Chem. Technol. Biotechnol. 62 (1995) 351–358. [27] I. Berman-Frank, P. Lundgren, P. Falkowski, Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria, Res. Microbiol. 154 (2003) 157–164.
Conflict of interest The authors have no conflict of interest to declare. Acknowledgements The authors would like to gratefully acknowledge funding from the ARC Training Centre for the Australian Food Processing Industry in the 21st Century (IC140100026). TT would like to acknowledge the Australian Government for its support through the provision of an Australian Postgraduate Award scholarship. References [1] T.D.C. Tarento, D.D. McClure, A.M. Talbot, H.L. Regtop, J.R. Biffin, P. Valtchev, F. Dehghani, J.M. Kavanagh, A potential biotechnological process for the sustainable production of vitamin K1, Crit. Rev. Biotechnol. (2018) 1–19. [2] M. Kamao, Y. Suhara, N. Tsugawa, M. Uwano, N. Yamaguchi, K. Uenishi, H. Ishida, S. Sasaki, T. Okano, Vitamin K content of foods and dietary vitamin K intake in Japanese young women, J. Nutr. Sci. Vitaminol. 53 (2007) 464–470. [3] L.J. Schurgers, C. Vermeer, Determination of phylloquinone and menaquinones in food, Pathophysiol. Haemost. Thromb. 30 (2001) 298–307. [4] T.J. Koivu, V.I. Piironen, S.K. Henttonen, P.H. Mattila, Determination of phylloquinone in vegetables, fruits, and berries by high-performance liquid chromatography with electrochemical detection, J. Agric. Food Chem. 45 (1997) 4644–4649. [5] M.J. Shearer, et al., Chemistry, nutritional sources, tissue distribution and metabolism of vitamin K with special reference to bone health, J. Nutr. 126 (1996) 1181S. [6] J.L. Zehnder, Drugs Used in Disorders of Coagulation, Basic & Clinical Pharmacology, McGraw-Hill Education, New York, NY, 2015. [7] C. Vermeer, Vitamin K: the effect on health beyond coagulation – an overview, Food Nutr. Res. 56 (2012) 5329. [8] S.L. Booth, Roles for vitamin K beyond coagulation, Annu. Rev. Nutr. 29 (2009) 89–110. [9] P.R. Chitnis, PHOTOSYSTEM I: function and physiology, Annu. Rev. Plant Physiol.
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Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Pilot-scale production of phylloquinone (vitamin K1) using a bubble column photo-bioreactor Thomas D.C. Tarento, Dale D. McClure, Fariba Dehghani, John M. Kavanagh
T
⁎
The University of Sydney, School of Chemical and Biomolecular Engineering, NSW, 2006, Australia
H I GH L IG H T S
(50 L) process for production of vitamin K developed. • Pilot-scale of medium, sparger, superficial velocity and day length examined. • Effect produced was rich in vitamin K (330 μg g ). • Biomass • Concentration is ten times more than other rich dietary sources. 1
1
−1
A R T I C LE I N FO
A B S T R A C T
Keywords: Photo-bioreactor Bubble column Vitamin K Phylloquinone Microalgae Cyanobacteria
Phylloquinone (vitamin K1) is an essential nutrient for both humans and animals; it plays a key role in blood coagulation amongst other processes. In this work we describe the scale-up of a biosynthetic process for phylloquinone production using the cyanobacterium Anabaena cylindrica. A 50 L bubble column photo-bioreactor was used to cultivate A. cylindrica and the effects of a wide range of operating conditions including superficial velocities (0.67–6.7 cm s−1), day lengths (12–24 h), sparger designs and medium compositions were examined. The column design and superficial velocity had minimal impact on phylloquinone production, while changes to the medium composition and day length had large impacts. By varying these factors we were able to achieve final phylloquinone titres of the order 280 μg L−1 and productivities of 40 μg L−1 day−1, which are approximately double the values previously obtained. The biomass produced has clear applications in human and animal nutrition as its phylloquinone concentration was approximately 330 μg g−1 (dry basis); this being ten times higher than rich dietary sources. Conclusions regarding the effect of photo-bioreactor design and operating conditions can be applied to the production of phylloquinone, as well as the scale-up of cyanobacterial cultures more broadly.
1. Introduction Vitamin K refers to a group of structurally similar compounds which play essential roles in both human and animal nutrition. Phylloquinone (vitamin K1) is typically found in leafy green vegetables and the menaquinones (vitamin K2) are found in fermented foods. Menadione and its derivatives are synthetic vitamin K analogues that have been banned for human consumption but are still used in animal nutrition [1–5]. Vitamin K is best known for its role in blood coagulation [6], however it is also involved in other biological processes including bone maintenance, modulation of inflammation and prevention of arterial hardening [7,8]. Phylloquinone participates in photosynthetic electron transport (as part of Photosystem I) [9]. In our previous work [10] we identified a strain of microalgae (the
⁎
cyanobacterium Anabaena cylindrica) with a phylloquinone concentration of the order 200 μg g−1 (dry basis); substantially greater than other dietary sources, with one gram of the dry algae providing 2–3 times the recommended daily intake of phylloquinone [11,12]. Other authors [13] have quantified the concentrations of phylloquinone produced by different species of algae (6–750 μg g−1), however little information about conditions favouring phylloquinone production is available in the open literature. In our previous work [10] it was found that high nitrate levels favoured phylloquinone production, and that the phylloquinone titre was directly proportional to the biomass concentration. Given its role within the cell it is likely that the most favourable conditions for phylloquinone production are those in which the culture is undergoing active, photosynthetic growth. In addition to being a rich source of phylloquinone, the biomass was found to be rich in protein (69% (w/w)
Corresponding author at: School of Chemical and Biomolecular Engineering, Building J01, The University of Sydney, NSW, 2006, Australia. E-mail address: [email protected] (J.M. Kavanagh).
https://doi.org/10.1016/j.bej.2019.107243 Received 8 January 2019; Received in revised form 18 April 2019; Accepted 26 May 2019 Available online 01 June 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
Biochemical Engineering Journal 150 (2019) 107243
T.D.C. Tarento, et al.
on a dry basis) and vitamin B12 [10]. Such biomass could potentially be incorporated into nutraceuticals or fortified foods to ensure sufficient supply of phylloquinone. Similarly, it could be used in animal nutrition applications to replace or supplement menadione. Biosynthesis of phylloquinone has the advantage that it operates at ambient temperature and pressure and avoids the use of organic solvents. Additionally, biosynthesis produces only the active E-isomer, whereas chemical synthesis of phylloquinone produces 10–20% of the inactive Z-isomer [14,15]. A key challenge in microalgal bioprocessing is the scale-up of the production process. Key factors include preventing contamination of the culture, ensuring good mass transfer (to supply carbon dioxide and remove oxygen), ensuring good mixing and, especially, ensuring sufficient light supply. For bubble column and airlift photo-bioreactors (PBRs), these factors are related to the reactor design (i.e. the reactor diameter and the selection of sparger) as well as the operating conditions (i.e. the superficial velocity). Flow in bubble columns can be divided into the homogenous flow regime (characterised by a narrow bubble size distribution with relatively uniform rise velocities) and the heterogeneous flow regime (characterised by a broader bubble size distribution and increased mixing in the liquid phase) [16,17]. The transition between these flow regimes depends on the column diameter, sparger design and superficial velocity. For the column examined in this work, the flow transition is predicted to occur at superficial velocities of the order 0.05 m s−1 based on published regime maps [17]. Whilst it is generally recognized that the column hydrodynamics is a key factor in PBR operation, due to its impact on mixing and mass transfer [18,19], there is relatively little data available in the open literature quantifying the effect of the column design on photo-bioreactor performance, particularly for cultures of cyanobacteria. Scale-up of cyanobacterial cultures may become increasingly important as these microorganisms have desirable characteristics for the photosynthetic production of chemicals [20–22]. Hence, the aims of the work are to develop a pilot-scale production for phylloquinone using A. cylindrica and, as part of this process, quantify the effect of the reactor design and operating conditions (including the medium composition and day length) on culture performance. 2. Method 2.1. Culture conditions A schematic of the pilot-scale photo-bioreactor used in this work is shown in Fig. 1. The PBR was constructed from clear acrylic tubing having a height of 2000 mm and an internal diameter of 190 mm. Air was introduced either through a perforated stainless-steel tube (PST) or a ceramic air-stone (CAS). The PST consisted of a 12.5 mm diameter stainless-steel tube with three rows of 10 holes. Each hole was 2 mm in diameter; the centres of the holes were spaced 10 mm apart. One row of holes was drilled on the centreline of the tube facing down; the other two rows were either side of this row, with an angle of 45° between the centres of each row. The CAS was a cylinder 130 mm in length and 30 mm in diameter made from fused alumina having pores less than 0.3 mm in size and was sourced from AquaOne. The flow-rates of air and carbon dioxide were measured using RM series rotameters (Dwyer); air was sourced from the building supply while the food-grade carbon dioxide was supplied from a cylinder (Coregas). The back-pressure was measured at the outlet of the rotameters using a LPG3 pressure gauge (Dwyer), this ranged between 25–35 kPa for all air flow rates and sparger configurations examined. Air flow rates of 0.2, 0.5, 1 and 2 vvm were examined; these correspond to superficial velocities between 0.67 and 6.7 cm s−1 at standard conditions (101 kPa and 298 K). Here we have chosen to use a wide range of air flow rates as this should encompass conditions in both the heterogeneous and homogenous flow regimes.
Fig. 1. Schematic diagram of the 50 L photo-bioreactor used in this work.
The temperature of the PBR was controlled by circulating water (28 °C) through a 12.5 mm diameter stainless steel U-tube mounted in the top of the reactor. The distance between the top of the column and the bottom of the U was 1570 mm. A Polyscience 6000 series chiller was used to control the temperature of the circulating water. Temperature, pH and dissolved oxygen levels were monitored using probes mounted in the top of the PBR. An InPro 3250i pH probe and an InPro 6850i DO probe (both sourced from Mettler-Toledo) were used. Data from both probes was logged using a M200 transmitter connected to a computer running M200 TCT Software (also from Mettler-Toledo). A total liquid volume of 50 L was used with losses due to evaporation or sampling being made up daily prior to any measurements. Tap water was passed through a depth filter (1 μm, Parker) and an activated carbon filter (Stefani) before entering the column. MLA medium [23] concentrates (without the addition of vitamins or selenium) were prepared and added to the column before each batch. We added the nutrient concentrates at either double or five times the values for normal MLA medium. The flow rate of CO2 was increased briefly while adding the nutrients for the 5 × MLA; this reduced the pH to approximately pH 7 and thereby avoided precipitation. 2
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Table 1 Summary of experimental results. Ranges are given for experiments done in duplicate. Sparger designs have been abbreviated as CAS = Ceramic Air Stone and PST = Perforated Stainless Tube. Sparger
Air flow rate (vvm)
Medium
Duration of daylight (h)
Final DCW (g L−1)
Final phylloquinone concentration (μg g−1)
Final phylloquinone titre (μg L−1)
Phylloquinone productivity (μg L−1 d−1)
CAS CAS PST PST PST PST PST PST PST PST PST
0.2 0.5 0.2 0.5 1 2 0.2 0.2 0.2 0.2 0.2
2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 2 × MLA 5 × MLA 5 × MLA
12 12 12 12 12 12 16 20 24 12 24
0.42–0.54 0.43–0.43 0.44–0.47 0.46–0.46 0.46 0.42 0.63 0.80 0.87–0.90 0.46–0.51 0.81–0.88
256–264 280–312 294–319 272–321 279 279 304 224 183–207 291–307 319–341
108–141 121–133 138–139 124–147 129 117 192 180 139–158 134–141 270-299
15.7–18.8 16.3–18.2 18.8–19.5 19.4–19.6 18.8 17.0 27.9 26.2 22.4–26.5 17.7–18.6 39.2–42.0
Fig. 2. Plots showing the effect of the day length on (a) the biomass concentration; (b) the nitrate concentration; (c) the specific phylloquinone concentration and (d) the phylloquinone titre. The 12 and 24 h runs were performed in duplicate, while the 16 and 20 h runs were not repeated.
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Fig. 3. Plots showing the effect of the day length and medium concentration on (a) the biomass concentration; (b) the nitrate concentration; (c) the specific phylloquinone concentration and (d) the phylloquinone titre. All experiments were performed in duplicate.
obtained from the Australian National Algal Culture Collection. Cultures were maintained in 50 mL flasks, using MLA medium, at a light intensity of approximately 70 μmol photons m−2 s-1. To prepare the inoculum for the 50 L PBR, 20 mL of these cultures were added to 200 mL of fresh, sterile MLA medium in a 500 mL flask. These were grown for approximately one week. Culture from several flasks were pooled, and 500 mL was used to inoculate a 5 L flat-panel PBR (the design and operation of which is detailed elsewhere [10]). These cultures were grown for an additional week, using MLA medium and a light intensity of approximately 300 μmol photons m−2 s-1. This culture was then used as the inoculum for the 50 L PBR, sufficient culture (between 2.5-5 L) was added to the 50 L PBR such that the initial optical density (at a wavelength of 550 nm) was approximately 0.1.
A sample port was located on the column centreline at a height of 1000 mm above the base of the column. This port had a 6.25 mm fitting with a valve, which was flushed thoroughly (with a volume of 50–100 mL) before samples were taken. Lighting was provided by LED light strips (9 W per strip, 6000 K colour temperature, Jaycar Australia). A total of 27 strips were used; as shown in Fig. 1 they were arranged in a 3 × 3 (H × W) grid. The distance from the base of the column to the bottom of the lights was 220 mm; the total height of the lights was 1520 mm and there was a distance of 110 mm from the lights to the PBR. The light intensity was measured using a Walz ULM-500 light meter with a US-SQS/L spherical sensor; measurements were made along the column centreline with the column full of water but without any aeration. The light intensity in the photosynthetically active region (400–700 nm) was of the order 300 μmol photons m−2 s−1. To quantify the effect of the lighting duration, experiments were performed using 12:12, 16:8 and 24:0 light:dark cycles. Based on our previous work [10], A. cylindrica was used for the production of phylloquinone. This strain (number CS-172) was
2.2. Analytical methods Microscopic examinations of the culture were performed using a Nikon ECLIPSE Ci-L microscope. The cell density was quantified by measurement of the dry cell weight. Samples (approximately 50 mL) 4
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were taken and filtered through a pre-weighed Advantec GA-55 filter paper (Toyo Roshi Kaisha Ltd., Tokyo, Japan); the samples were washed with two volumes of 0.5 M ammonium bicarbonate and dried overnight at 105 °C. The dry samples were cooled in a desiccator and weighed, the dry cell weight was calculated as the difference between the initial and final weights divided by the volume of culture filtered. Uncertainty in the dry cell weight measurements is of the order 5% (as calculated using error propagation methodology). The nitrate concentration in the sample was measured using a standard spectrophotometric screening method (APHA-4500-NO3−-B) [24]. Samples were syringe filtered (0.22 μm) prior to analysis and measurements were made using a Varian Cary 50 spectrophotometer. To determine the concentration of phylloquinone in the biomass, samples (approximately 50 mL) were centrifuged (3000 RCF, 5 min) and the supernatant discarded. The samples were re-suspended in deionised water and the process was repeated to remove any salts; the samples were then freeze dried. Extraction and quantitation of phylloquinone was performed using a previously described method [10]. Values of the specific phylloquinone concentration are reported per gram of freeze dried biomass. In order to quantify the uncertainty in the extraction and analysis procedure it has been repeated twice, for three different species of algae [10]; it was found that the maximum coefficient of variation was 15%.
Overall, these results suggest that for the process and conditions examined, the effect of the sparger design and gas flow rate had a minimal impact on phylloquinone production. All subsequent experiments were therefore performed using the perforated stainless tube at an air flow rate of 0.2 vvm. These conditions were chosen as the perforated tube is easier to clean than the ceramic air stone and the lower air flow rate leads to a reduced energy consumption. 3.2. Effect of day length A potential method of increasing phylloquinone production is increasing the period of illumination (“day length”). However, excessive irradiation can also be damaging for the microalgae [19]. When grown in 5 L PBRs with 24 h of illumination per day it was found that the A. cylindrica cultures turned from blue/green to yellow in colour, potentially as a protective response to damaging high levels of irradiance. To quantify the effect of the day length, experiments were performed using 12, 16, 20 and 24 h day lengths; the results are shown in Fig. 2 and summarised in Table 1. It was observed that increasing the duration of lighting led to higher biomass concentrations. Increases in the day length also corresponded with an increase in the rate of nitrogen consumption; cultures grown with constant illumination exhausted the nitrate present in the medium after approximately four days while those grown with 12 h lighting still had nitrate present on day 7 (as shown in Fig. 2(b)). When examined under the microscope, cells from the nitrogen-exhausted cultures had changed morphology consistent with nitrogen limitation; specifically, heterocysts were observed (results not shown). Heterocysts are differentiated cells that allow filamentous cyanobacteria (like A. cylindrica) to fix atmospheric nitrogen [27]. No change in the colour of the culture was observed, most likely due to the greater path length in the 50 L PBR. Interestingly, this nitrogen limitation appeared to be correlated with a decrease in the specific phylloquinone concentration (as shown in Fig. 2(c)). The decrease in specific phylloquinone content is more than compensated for by the higher biomass concentrations at longer day lengths; as shown in Fig. 3(d) phylloquinone titres of 160–190 μg L−1 were achieved by increasing the day length; these values being greater than those for 12 h lighting (140 μg L−1). To determine whether or not the phylloquinone titre could be further improved, experiments were repeated with 12- and 24 -h lighting with further enriched medium (5 × MLA). The results are shown in Fig. 3. It was found that supplementing the medium led to nitrate replete conditions; as shown in Fig. 2(b) the nitrate concentration at the end of the experiment was 200–400 mg L−1. Supplementing the medium led to a higher specific phylloquinone concentration, this in turn led to higher phylloquinone titres, with values being of the order 280 μg L−1 for the PBR with constant illumination. These results indicate that it is likely the biomass growth could be enhanced by further increases in the light intensity above 300 μmol photons m-2 s−1. Unfortunately, testing the effects of higher light intensities was not possible with the current experimental set-up.
3. Results and discussion 3.1. Effect of PBR design and operating conditions As summarised in Table 1, there was relatively little variation between batches performed using the same experimental conditions, with this this being true for all of the conditions examined. The maximum variation between dry cell weights was of the order 30%, while it was of the order 20% for the specific phylloquinone concentrations. Microscopic examination of the culture showed that the morphology was also consistent for all conditions examined, indicating that for the range of gas flow rates evaluated (0.2 – 2 vvm, corresponding to superficial velocities of 0.67 – 6.7 cm s−1) the shear stress in the liquid phase was not damaging to the cells. It is clear that the effect of shear stress is species specific, as other authors [25,26] have noted that increasing the superficial velocity had negative effects on microalgal growth in bubble columns. It was found that both the rate of biomass growth and nitrate consumption were linear and comparable (Table 1) for the different spargers and superficial velocities examined. Such results demonstrate that varying these factors had negligible effects on the growth of A. cylindrica. Specific phylloquinone concentrations were in the range 250–330 μg g−1, with this value not being affected by the sparger design. Interestingly, the specific phylloquinone concentrations measured in the 50 L PBR were greater than those previously measured [10] in 5 L flat-panel PBRs (approximately 150 μg g−1). This is again thought to be due to the cells compensating for the lower light availability (due to the longer path length in the larger reactor). The phylloquinone titre was observed to increase in a linear fashion, this being explained by the fact that the biomass growth was linear and the specific phylloquinone concentration was approximately constant. During periods of illumination, the DO was of the order 105% of saturation, this being consistent with the production of oxygen by the microalgae. During the dark period, the concentration of dissolved oxygen decreased to the saturation value (i.e. 100%). Due to the relatively high levels of mass transfer found in bubble columns, the maximum DO was always far below inhibitory values (200% of saturation) [19]. In all instances the pH of the PBR was between pH 7–8, indicating that the growth rate was not limited by the supply of carbon. This is one explanation for why the operating conditions had minimal impact on process performance; it is likely that the rate of carbon uptake by the algae was always less than the rate of transfer from gas to liquid.
4. Conclusions The aim of this work was to evaluate the effect of photo-bioreactor design and operation on process performance, with a particular focus on cyanobacterial culture for the production of phylloquinone. A summary of all of the experimental results is given in Table 1. Compared to the results from our previous work [10] using smaller, 5 L flat panel photo-bioreactors, we were able to approximately double the productivity, from 22 μg L−1 day to approximately 40 μg L−1 day. It was found that the primary determinants of the productivity were the composition of the growth medium and the day length. The sparger design and the superficial gas velocity used had minimal impact on the concentrations of phylloquinone and biomass. On a dry weight basis the concentration of phylloquinone in the A. 5
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cylindrica biomass is of the order 330 μg g−1; this is approximately ten times the concentration of other rich dietary sources. One gram of the dry biomass can provide approximately five times the recommended daily intake of phylloquinone [11]. Hence, there is a clear potential for the biomass to be used in animal nutrition or functional foods as a source of vitamin K1.
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Conflict of interest The authors have no conflict of interest to declare. Acknowledgements The authors would like to gratefully acknowledge funding from the ARC Training Centre for the Australian Food Processing Industry in the 21st Century (IC140100026). TT would like to acknowledge the Australian Government for its support through the provision of an Australian Postgraduate Award scholarship. References [1] T.D.C. Tarento, D.D. McClure, A.M. Talbot, H.L. Regtop, J.R. Biffin, P. Valtchev, F. Dehghani, J.M. Kavanagh, A potential biotechnological process for the sustainable production of vitamin K1, Crit. Rev. Biotechnol. (2018) 1–19. [2] M. Kamao, Y. Suhara, N. Tsugawa, M. Uwano, N. Yamaguchi, K. Uenishi, H. Ishida, S. Sasaki, T. Okano, Vitamin K content of foods and dietary vitamin K intake in Japanese young women, J. Nutr. Sci. Vitaminol. 53 (2007) 464–470. [3] L.J. Schurgers, C. Vermeer, Determination of phylloquinone and menaquinones in food, Pathophysiol. Haemost. Thromb. 30 (2001) 298–307. [4] T.J. Koivu, V.I. Piironen, S.K. Henttonen, P.H. Mattila, Determination of phylloquinone in vegetables, fruits, and berries by high-performance liquid chromatography with electrochemical detection, J. Agric. Food Chem. 45 (1997) 4644–4649. [5] M.J. Shearer, et al., Chemistry, nutritional sources, tissue distribution and metabolism of vitamin K with special reference to bone health, J. Nutr. 126 (1996) 1181S. [6] J.L. Zehnder, Drugs Used in Disorders of Coagulation, Basic & Clinical Pharmacology, McGraw-Hill Education, New York, NY, 2015. [7] C. Vermeer, Vitamin K: the effect on health beyond coagulation – an overview, Food Nutr. Res. 56 (2012) 5329. [8] S.L. Booth, Roles for vitamin K beyond coagulation, Annu. Rev. Nutr. 29 (2009) 89–110. [9] P.R. Chitnis, PHOTOSYSTEM I: function and physiology, Annu. Rev. Plant Physiol.
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