Growth comparison of Arthrospira platensis in different vessels: standard cylinder vs. enhanced surface area at low light
Growth comparison of Arthrospira platensis in different vessels: standard cylinder vs. enhanced...
Gal, Jonathan L.; Cole, Nolan R.; Eggett, Dennis L.; Johnson, Steven M.
2023-12-31 00:00:00
British Phycological APPLIED PHYCOLOGY Society 2023, VOL. 4, NO. 1, 1–14 Understanding and using algae https://doi.org/10.1080/26388081.2022.2125829 Growth comparison of Arthrospira platensis in different vessels: standard cylinder vs. enhanced surface area at low light a b b a Jonathan L. Gal , Nolan R. Cole , Dennis L. Eggett and Steven M. Johnson a b Department of Microbiology and Molecular Biology, College of Life Sciences, Brigham Young University, Provo, UT, USA; Department of Statistics, College of Physical and Mathematical Sciences, Brigham Young University, Provo, UT, USA ABSTRACT ARTICLE HISTORY Received 14 March 2022 A laboratory scale prototype of a novel tube called AlgaTube™ (patent pending) was evaluated for Accepted 9 September 2022 growth of Arthrospira platensis (commonly referred to as Spirulina) in a controlled, 10-day batch trial at low light intensity. The two cultures (control and prototype) were grown in identical KEYWORDS conditions inside a closed incubator space at 32°C with a mean light intensity of 55 µmol AlgaTube™; Arthrospira; −2 −1 photons m s LED light, consisting mostly of red and blue wavelengths with some full spectrum biomass; cyanobacteria; −1 background radiation. OD was measured daily and converted to Concentration (C , g l ) using feed; fertilizer; nutrition; 590 d a standard curve. Four independent replicates of the trial were run. Six biomass growth metrics photobioreactor; wastewater; Spirulina were evaluated and compared. Across the board, all six growth metrics showed higher rates of biomass growth in the AlgaTube™. Maximum concentration was 34% higher (p = 0.02), cumulative production was 41% higher (p = 0.02), specific growth rate was 17% higher (p = 0.02), maximum specific growth rate was 23% higher (p = 0.02), mean daily production was 41% higher (p = 0.001), and maximum daily production was 38% higher (p = 0.19). We conclude that the novel shape of the AlgaTube™ prototype increased biomass growth rates. We believe that this study constitutes successful proof-of-concept for the AlgaTube™, but further studies are needed to optimize its performance. Introduction Industrial applications can be divided into three Photosynthetic microorganisms have been studied as steps: (1) growth and production, (2) harvest and pro- a feedstock for many commercial and industrial appli- cessing, and (3) marketing and delivery. The present cations, including: CO sequestration, biofuels, human study is focused exclusively on the production step, nutrition, fish and livestock feed, air purification, ferti- which is common to all the applications listed above. lizer, cosmetics, pharmaceutical manufacturing, waste- Thus, the present study (and the associated product) water remediation, and bioplastics (Stanley & Jones, could have a broad impact on all of the applications 1976; Benemann, 1979; Kumar, Dasgupta, Nayak, mentioned above. It is a platform technology. Lindblad, & Das, 2011; Priyadarshani & Biswajit, 2012; Production technology has historically been focused Rasala & Mayfield, 2015; Christaki, Karatzia, & Florou- on the open pond system, also known as the “raceway”, Paneri, 2010; Poonam & Sharma, 2017; Adeniyi, and some still use that technique (Cyanotech). More Azimov, & Burluka, 2018; Joshi, 2018; Liu, Pemberton, recently, closed production systems have been built Lewis, Scales, & Martin, 2020; Arora, Kumar, Bose, Li, & using clear, cylindrical piping (Phytobloom). These are Kulshrestha, 2021; Chong et al., 2021; Shahi et al., 2022). generally referred to as tubular photobioreactors The United States Department of Energy investigated (tPBRs). Closed systems are more costly to build, but photosynthetic microorganisms as a feedstock for bio- they offer advantages such as protection against con- fuels during the 1980s and 1990s (Sheehan, Dunahay, tamination, control of culture conditions, and increased Benemann, Roessler, & Weissman, 1998). Several com- production rates (Clippinger & Davis, 2019). panies are currently marketing such products [e.g., Different variables can impact the production rate of Cyanotech, TrueAlgae, and Algeternal] (Tacon & photobioreactors (PBRs). Temperature, light, pH, sali- Metian, 2008). During 2019, the US National nity, nutrients, and gas exchange can all be varied. Each Renewable Energy Laboratory published a review of variable can have a distinct impact on the culture growth the state-of-the-art for photosynthetic algae production rate (Hoseini, Almodares, Afsharzadeh, Shahriari, & (Clippinger & Davis, 2019). Montazeri, 2014; Kendirlioglu & Cetin, 2017; Olaizola & CONTACT Steven M. Johnson stevenj@byu.edu © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 J. L. GAL ET AL. Duerr, 1990; Soni, Sudhakar, & Rana, 2017, 2019; tubes that are widely used as state-of-the-art in tubular Srinivasan & Illanjiam, 2021a, 2021b; Tayebati, Shariati, photobioreactors. The novel shaped culture vessel tested Soltani, & Tehrani, 2020; Uslu et al., 2009; Wang, Fu, & here was shown to significantly improve the growth Liu, 2007). rate. The increase was most likely due to changing the Cultures of photosynthetic algae and cyanobacteria shape of the sidewalls of the cylindrical culture vessel have been observed to experience growth inhibition by significantly reducing the self-shading effect by increas- the shadow effect, also referred to as the self-shading ing the surface area of the sidewalls of the culture vessel. effect (Frontasyeva et al., 2009; Olaizola & Duerr, 1990; This new configuration may help to improve the eco- Soni, Sudhakar, & Rana, 2019). This occurs when the nomics of any industrial process that utilizes photosyn- cell density is high enough that the cells shade each thetic microorganisms such as cyanobacteria and algae. other, thus blocking each other’s access to needed light. At particularly high concentrations, light penetra- Materials and methods tion is severely restricted. (González-Camejo et al., 2019; Shigesada & Okubo, 1981). Culture vessels Other studies have successfully shown that increasing The surface area of the sidewalls of the AlgaTube™ was the surface-area-to-volume ratio (SA/V) improves the the primary variable that we isolated and evaluated in growth rate by exposing greater numbers of cells to this experiment. Here, we use the term Photon Surface direct irradiance, thus reducing the shadow effect Area (PSA) to label this key variable and to differenti - (Converti, Lodi, Del Borghi, & Solisio, 2006; da Silva ate it from the cross-sectional surface area (CSSA), et al., 2016; Fremento et al., 2013). In those studies, SA/ which is a different measure altogether. In the present V was increased by reducing the diameter of cylindrical −1 study, PSA Vol is equivalent to the “SA/V” metric tubes, an effect that is inherent in the Euclidean geome- used by Converti, da Silva and other referenced try of standard cylindrical tubes. In the present study, authors. The control flask was a standard, cylinder- a different approach was taken to increasing the SA −1 −1 shaped vessel. Both vessels were custom made by 3- Vol ratio of culture vessels. The SA Vol was chan- D printing, using the same resin and the same manu- ged, not by reducing the diameter, but by using corru- facturing technology (DMS Somos® Watershed XC gated sidewalls in lieu of conventional sidewalls. 11,122 resin and Stereolithography (SLA)). The other The purpose of the present study was to evaluate dimensions of the two flasks were chosen such that the a prototypic tube with enhanced, corrugated surface remaining flasks were chosen such that the remaining area, the AlgaTube™, for its ability to reduce the shadow functional dimensions of the two flasks were identical effect and, thereby, improve the rate of photosynthetic (Fig 1 and Table 1). production, when compared to the standard, cylindrical Figure 1. Representation of the cross sections of the two flasks. The control flask (a) on the left, and the prototype flask (b) on the right have identical cross-sectional areas, but the sidewall configuration of the two flasks is different. These diagrams are intended for illustrative purposes only. They are not presented to scale, nor are they intended for manufacturing or engineering purposes. The key geometric measures of the respective flasks are presented in Table 1. APPLIED PHYCOLOGY 3 Table 1. Key Geometric Measures of the Flasks. included. The mean intensity applied to each culture ™ −2 −1 Geometric Measure Control AlgaTube Units was 55 µmol photons m s and was identical between Capacity Volume 1441 1441 ml the two cultures. The lights were maintained 24 hours b 2 Photon Surface Area 743 1641 cm a day with no period of darkness. Relative Photon Surface Area 100% 220% % of control d 2 Cross Section Surface Area 47 47 cm The two cultures were exposed to a continuous mag- −1 e −1 PSA Vol Ratio 0.52 1.13 cm −1 f netic field of approximately 300mT (milliTesla) each Relative PSA Vol Ratio 100% 220% % of control a 3 (Fig 2). The magnets (K&J Magnetics) were Capacity Volume: total volume of the flask when full to brim (ml = cm ). b 2 Photon Surface Area: total surface area of the sidewalls of the flask (cm ). Neodymium Grade N52 with a Br rating of 14 800 max ac Relative Photon Surface Area: expressed as %, with control = 100%. d Gauss and a BH rating of 52MGOe. Pure gaseous CO max 2 Cross Section Surface Area: area across which gas exchange and evapora- tion occur (cm ). was supplied from a pressurized tank through a regulator e −1 2 3 −1 PSA Vol Ratio: ratio of PSA to Volume for each flask (cm /cm = cm ). (FZone Model#FZ-2020PRO), a pH controller f −1 Relative PSA Vol Ratio: expressed as % with control = 100%. (Milwaukee MC122Pro), and a sparger stone (CR Brewbeer, 0.5um Diffusion Stones). The CO was dosed Culture species and growth conditions as needed using the automated pH controller set for 9.0 ± 0.2. Ambient air was continuously bubbled through both Seed culture was purchased from Algae Research Supply cultures using a HITOP Aquarium Air Pump (Haisen). in Carlsbad, CA, USA, who identify the strain only as Dissolved oxygen levels were consistently lower than Arthrospira platensis (A. platensis). Modified Schlosser 150% of ambient levels. The temperature was maintained Medium (da Silva et al., 2016) was prepared using deio- at 32°C. The Relative Humidity fluctuated mostly within nized water from the Brigham Young University (BYU) a range of 10–15%. Periodically, due to the passage of Life Science Building (LSB). Two YOHAYOH LED storm systems, the Relative Humidity fluctuated outside Light Panels (45W, 50-60 Hz, 300 mA, 110 V, 30.99 cm that range. The setup is shown in Fig 2. x 30.99 cm x 4.06 cm) were fixed to the inside of the incubator. They emitted narrow spectral peaks at 460 nm (blue) and 630 nm (red) and a substantially lower Assessment of biomass concentration level of full visible spectrum background radiation. The ratio of red to blue in the present study is calculated Prior to daily sampling, each culture was mixed manu- from the manufacturer’s data as approximately 60:40. ally to promote homogeneity. Each day, the cultures A 15-Watt incandescent bulb (General Electric) was also were tested for Dissolved Oxygen (RCYAGO Portable Figure 2. Configuration of the growth chamber. The control flask was on the left, and the prototype flask was on the right. The two light panels were affixed to the rear wall of the incubator. The incandescent light was attached to the door and centered. The magnets can be seen on the lower left side of the control flask and on the lower right side of the prototype flask. The picture was taken with the door open for convenience. 4 J. L. GAL ET AL. Table 2. Dilutions for % Transmittance vs. Concentration stan- Dissolved Oxygen, Model#DO9100), pH (Milwaukee dard curve. MC122Pro), Total Dissolved Solids and Electrical –1 Dilution # Dilution Rate (%) Concentration (g l ) %T ABS Conductivity (Vivosun TDS/EC Metre), and Light Stock 100 1.63 0.70 >2.02 Transmittance and Absorption (Biolog Turbidimeter, 1 61 1.00 0.90 >2.02 −1 Model#21907, 590 nm). Concentration (C g l ) was 2 55 0.90 0.95 2.02 3 49 0.80 1.00 2.00 calculated from %T using a standard curve. 4 43 0.70 1.20 1.95 The organisms were observed to form clumps of 5 33 0.60 1.99 1.80 6 31 0.50 3.10 1.52 filaments, a sign of their previously documented colo- 7 25 0.40 5.70 1.30 nial nature (Demoulin et al., 2019). The clumps were 8 18 0.30 10.50 0.98 9 12 0.20 23.50 0.64 a source of minor error for the OD readings because 10 6 0.10 47.50 0.33 they interfered with homogeneity. The clumps caused 11 3 0.05 69.50 0.16 12 1 0.02 85.50 0.07 some minor, but noticeable, drift in the turbidity read- ings, as they circulated in the culture samples. To reduce the margin of error associated with this imperfect The diluent for these dilutions was Zarrouk Media homogeneity, the turbidity metre was observed for (ZM), because ZM was being used at the time the a full minute for each reading. Time weighted means standard curve was prepared. Subsequent to the creation of the %T and ABS were recorded for each mea- 590 590 of this standard curve, a decision was later made to surement, rather than single spot readings. switch to MSM media. To examine the potential impact that the change in diluent might have on the OD read- ings, the OD for pure MSM was compared with the Standard curve preparation OD for pure ZM. It was determined that the differ - A stock of A. platensis ARS was taken from a well-mixed ence in %T readings between the two media (<2%) was culture that had previously plateaued in the incubator. sufficiently small to justify continued use of the standard Five samples of 10 ml each were pipetted from that curve, without preparation of a new standard curve stock, and each was measured using the dry weight using MSM as the diluent. protocol (supplementary material). Those five samples Table 2. The data for the standard curve presented in produced five different results for dry weight − 1.63 g Fig 3. These data establish a correlation between bio- −1 −1 −1 −1 −1 −1 l , 1.68 g l , 1.34 g l , 1.71 g l , and 1.57 g l – which mass concentration (g l ) of A. platensis and the optical reflected various sources of error in the assay (supple- density of the culture at 590 nm. “%T” stands for mentary material). The high and low values from the Percentage Light Transmission. “ABS” stands for range were discarded, and the mean of the remaining Absorbance in absorbance units. %T and ABS are inter- −1 three values was calculated to be 1.63 g l . The 95% related, according to the standard Beer-Lambert equa- confidence interval of the three sample dry weights was tion: ABS = 2 – log (%T × 100). calculated to be ±3.83%. This was determined to be sufficiently accurate for the present study. Calculation of production (P ) Another sample from the same stock was taken and measured in the turbidimeter, producing a %T reading P is calculated by subtracting the previous day’s C from of approximately 0.70%. From this sample, twelve dilu- the current day’s C as follows. −1 tions were made to produce values of 1.00 g l down to −1 P dðg per lÞ ¼ C dðg per lÞ