Article pubs.acs.org/IECR
Cite This: Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Effects of Medium Composition and Gas Superficial Velocity on Mass Transfer during Microalgae Culturing in a Bubble Column Photobioreactor Manuel J. Luna-Brito,†,‡ Julio C. Sacramento-Rivero,† and Sergio A. Baz-Rodríguez*,† †
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Facultad de Ingenieria Quimica, Universidad Autonoma de Yucatan, Campus de Ciencias Exactas e Ingenierias, Periferico Norte Kilometro. 33.5, Tablaje Catastral 13615, Col. Chuburna de Hidalgo Inn, Merida, Yucatan CP 97203, Mexico ‡ Facultad de Ciencias Quimico-Biologicas, Universidad Autonoma de Campeche, Av. Agustin Melgar s/n entre Calle 20 y Juan de la Barrera, Col. Buenavista, San Francisco de Campeche, Campeche CP 24039, Mexico ABSTRACT: The effects of the concentrations of biomass, mixed salts, total carbohydrates, and proteins on atmospheric CO2 transfer in a bubble column photobioreactor during the photoautotrophic culture of Chlamydomonas reinhardtii were studied. Microalgae cultures were carried out in saline media at three superficial gas velocities and two initial salt concentrations in terms of ionic strength. The influence of the gas superficial velocity and the instantaneous concentrations on both the liquid side mass-transfer coefficient (kL) and the specific interfacial area (a) were analyzed using a multifactor analysis of variance. kL decreased as the gas velocity increased, but a increased; the reduction of kL was due to transition behaviors between those expected for isolated bubbles and bubble swarms. As the biomass concentration increased, a decreased and kL increased. In addition, at higher initial ionic strength, a was larger. The mass transfer was independent of the concentrations of total carbohydrates and proteins.
1. INTRODUCTION Microalgae cultivation has potential applications in biofuel production, wastewater treatment, and CO2 fixation.1,2 Under photoautotrophic mode in closed photobioreactors, microalgae growth depends on the uptake of an inorganic carbon source; carbon dioxide from a bubbled gaseous inlet is the most employed source.3 Also, the aqueous media for microalgae cultivation involve diluted solutes, cells, and usually smooth hydrodynamic conditions (low shear stress). Each of these factors individually influences the carbon-dioxide transfer from the gaseous phase in different ways. The combined effect of the physicochemical properties, the instantaneous conditions of composition (cells, substrate, and metabolite concentrations), and the hydrodynamics govern the supply of the gas solute. The prediction of the resulting behavior needs to be studied as a combined effect in actual cultures due to the complex interactions between the above mentioned factors. The interfacial mass transfer in gravity-driven bubbly flows is a complex phenomenon that depends on the transport properties at interfaces, the degree of turbulence (related to shear stresses and cell damaging), the dynamic topology of mobile interfaces, and the physicochemical properties and local velocity fields of both phases.4 In practice, these effects are empirically encompassed in the calculation of volumetric masstransfer coefficients (kLa).5 In accordance with this, the mass© XXXX American Chemical Society
transfer studies for microalgae systems are often focused on volumetric mass-transfer coefficients.3,6−8 The main interest has been to know how the mass transfer affects the cell growth. However, the mass transfer can be affected by the cell growth and the medium composition by modifying the phase and interface structures, physicochemical properties, and the hydrodynamics. Thus the dynamic medium composition and kLa are interrelated. Moreover, kLa involves two separate effects: the first associated with the local interfacial transfer, namely, the mass-transfer coefficient on the liquid side (kL) and the second related to the upscaling of such local effects, namely, the specific interfacial area (a). Determining the individual behavior of those components would improve the understanding about how the instantaneous conditions of composition and the hydrodynamics affect the mass transfer in microalgae cultures, which remains an open issue. Few studies deal experimentally with separated analyses of kL and a in submerged bubbled bioprocesses, including microalgae cultures.9−13 Moreover, their dependency on the Received: Revised: Accepted: Published: A
August 16, 2018 October 23, 2018 November 26, 2018 November 26, 2018 DOI: 10.1021/acs.iecr.8b03940 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
With the exception of biomass dry weight, all other analyses were made from the supernatant obtained by centrifugation (6000g for 5 min, Thermo Fisher SL16R). The polyatomic ions (ammonia and phosphates) were measured as the controlling salt ions, which define the values of the IS. Almost 99% of the mass of dissolved salts are species including phosphate and ammonia. Then, the monitoring of these ions gives a measure of the change in concentrations of the whole salt species. Total carbohydrates and proteins were measured as representative metabolites liberated into the growth medium.24 Each experimental case involving a combination of IS0 and vSG had a total cultivation time of 48 h; also, the pH was monitored during the entire duration of the culturing. (It remained between 7.1 and 7.3.) At every monitoring time, the dynamic gassing method was used to determine the kLa of atmospheric CO2, and two sequences of high-velocity video were captured for the determination of the Sauter mean diameters (d32) from digital image analysis. The gas holdup (εg) was also calculated as the change of total medium volume due to the sparging of gas through the initially stagnant liquid. The dynamic concentrations of CO2 for kLa experiments were measured at the gaseous inlet and outlet of the photobioreactor and registered by PC at regular time intervals until saturation of the media. For this, a carbon-dioxide meter with pump-aspirated sampling (CARBOCAP GM70, Vaisala) was employed. From the data of gas concentration at the gaseous inlet and outlet and the spatial time of the gaseous phase, the mass transferred to the liquid was calculated. This was done for each time step, and the calculated transferred mass was added to the previous cumulative from the preceding steps. The kLa was determined as a fitting parameter from the calculated liquid concentrations (dissolved CO2, ρCO2,L) and the following dynamic model
dynamic composition of cultures is scarcely studied and is of current relevance.9,10 The freshwater green algae Chlamydomonas reinhardtii is one of the most studied microalgae. It is a relatively simple microorganism easily cultivated in photoautotrophic mode and widely used as model strain for biochemical studies. It has promising potential for cell factory purposes and biofuel production.14,15 In this work, the effects of the superficial gas velocity and the dynamic concentration of various culture media constituents on the carbon-dioxide transfer during the photoautotrophic culture of microalgae were studied. For this purpose, kL and a were obtained experimentally from Chlamydomonas reinhardtii cultures in a bubble column. Finally, the variation in kLa against the composition of statistically significant components and the superficial gas velocity was discussed.
2. MATERIALS AND METHODS The bubble column photobioreactor used in the experiments consisted of a jacketed cylinder of Pyrex glass of 1 m height and 0.098 m internal diameter, with a porous plate as gas distributor at the bottom (pore diameter 160−250 μm). In addition, a 0.15 m in height glass cube was placed concentrically on the column at a height of 0.35 m above the gas diffuser; once filled with water, the cube helps to avoid the optical distortion due to the column curvature for image acquisition. The column jacket was connected to a circulating bath (AD07R-20 PolyScience) for temperature control at 298.15 K through a flowing water loop. Linear fluorescent lamp bulbs were used to provide 100 μmol/m2s of irradiance at the photobioreactor surface. Chlamydomonas reinhardtii was selected as a model microalgae strain. The cells have an average size of 10 μm,16 significantly lower than average diameters of the gas distributor pores and the expected size of bubbles. Sueoka’s high salt medium was used as model culture medium. Because the main components of autotrophic media for microalgae culturing are salts and many species of electrolytic salts are involved, the ionic strength (IS) was used as a measure of their effective concentration in the media. The original medium has an IS around 0.085 M, which was defined as the initial ionic strength (IS0). One additional ionic strength was evaluated (0.17 M) by adding proportional amounts of the main electrolyte species to the medium (K2HPO4, KH2PO4, NH4Cl, MgSO4, CaCl2). Inoculums of 10% in volume were added to the saline medium in the photobioreactor for a total working volume of 5.5 L. The microalgae were previously grown in 2 L airbubbled vessels until a cell density between (3 and 4) × 106 cells/mL was obtained. During the experiments, the gas phase (air or nitrogen) was supplied to the column, and its flow was controlled through rotameters (102-05-ST, Cole-Parmer). Three values of superficial air velocity (vSG) were evaluated: 0.012, 0.018, and 0.024 m/s, corresponding to 1.0, 1.5, and 2.0 vessel volume per minute (vvm), respectively. The microalgae cultures were monitored at 0, 6, 12, 24, 36, and 48 h, taking samples for measuring: biomass dry weight (ρX), ammonia (method of salicylate based on Reardon et al.17 using the kit and procedure by Hach18), phosphates (method of molybdovanadate based on Greenberg et al.19 using the kit and procedure by Hach20), total carbohydrates (ρch) (method of phenol-sulfuric acid by Dubois et al.21), and total protein (ρprot) (method of Lowry modified by Peterson22 using the kit TP0300 and the procedure reported by Sigma-Aldrich23).
dρCO ,L 2
dt
* = kLa[ρCO − ρCO ,L ] − ,L 2
2
1 YCO2 − X
μρX
(1)
Here ρ*CO2,L is the saturation concentration of the CO2 in the liquid, and YCO2−X is the biomass yield with respect to the consumption of CO2. μ is the specific growth rate and was calculated as follows: (i) The data of dry-weight biomass for each experimental run were fitted to a polynomial equation (ρX vs t, r2 > 0.98), and (ii) the specific growth rate was calculated from its definition, namely, μ = (1/ρX)(dρX/dt), evaluated at the sampling time. Equation 1 assumes perfect mixing in the liquid phase. For the measurement of the Sauter mean bubble diameter, a Nikon 1 J5 digital camera with a Micro-Nikkor 60 mm f/2.8D lens was used for acquiring video sequences of 3 s at 400 frames per second and 800 × 296 pixels in every sampling time for each experiment. Image acquisition was carried out at a height of 0.4 m above the gas distributor, in the front side of the glass cube. Several images were extracted and processed from the video sequences using digital image processing. By enhancing the contrast among the dark and clear zones of the images, the contours of the bubbles were traced. At least 200 bubbles were isolated from the images for each experimental case and sampling time. The bubble shape was assumed to be oblate ellipsoidal. The geometric properties of isolated bubbles were measured, and their equivalent diameters with respect to equivalent spheres were calculated as follows B
DOI: 10.1021/acs.iecr.8b03940 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
dBi = (E2e)1/3
(2)
where E and e are the major and minor axes of bubbles, respectively. Hence, the Sauter mean bubble diameter was calculated as follows m
d32 =
m
∑ dBi3/∑ dBi2 i=1
i=1
(3)
where m is the total number of bubbles isolated from the image processing for each experimental condition. The specific interfacial area based on the liquid volume was obtained from the fractional gas holdup and the Sauter mean diameter (d32) with the following equation a=
6εg d32(1 − εg)
(4)
The turbidity that is gradually developed in microalgae cultures limited the applicability of the imaging technique up to first 2 days of cultivation. However, biomass concentrations on the order of magnitude of typical industrial conditions (0.3 to 0.5 g/L) are already reached at that time. Once the specific interfacial area was known, the mass transfer coefficient for the liquid side was estimated as kL =
kLa a
(5)
Because a and kL are calculated using equations and from values of experimentally measured variables, the combination of uncertainties in the reported data from eqs 4 and 5 was calculated according to Caria25 ÄÅ ÉÑ1/2 ÅÅ Ñ ÅÅ i ∂y y 2 ÑÑÑ ÅÅ jj i zz Ñ δR y = ÅÅÅ∑ jjj zzz Sx2j ÑÑÑÑ i ÅÅ j j ∂xj z ÑÑ ÅÅ k { xj ÑÑ ̅ (6) ÅÇ ÑÖ
Figure 1. Effect on kL of (a) gas velocity (IS0 = 0.085 M) and (b) biomass concentration.
where δRyi are the uncertainties of yi (a and kL )and Sxj2 are the variances of the measured variables xj (d32, εg, kLa). A multifactor analysis of variance (MANOVA) was carried out to determine which factors (vSG, ρX, IS, ρch, and ρprot) have statistical influence over a and kL. Minitab 18 (Minitab) was used for the statistical analysis with a significance level of 95% (p value
