EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC, AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE¹

Microorganisms and growth conditions. The unicellular microalga Chlorella vulgaris (UTEX 2714, Austin, TX) and the microalgae growth-promoting bacterium (MGPB) A. brasilense Cd (DMS 1843, Braunschweig, Germany) were tested. Experiments were carried out under autotrophic, heterotrophic, and mixotrophic conditions in inverted 1,000 mL conical glass bioreactors (17 cm tall, 14 cm width at top, and 5 cm at the bottom), each containing 750 mL synthetic wastewater, equipped with uplift bottom sterile aeration controlled by a peristaltic pump providing air at 30 mL air·min⁻¹ measured with a fluxometer (GF-2000, Gilmont Instruments, Barrington, IL, USA) that pass three microbial filters: one 1 μm pore bacterial air vent filter (Pall-Gelman Laboratory, Ann Arbor, MI, USA) and two 0.2 μm sterile filters (Acrodiscs, Pall Corp., Ann Arbor, MI, USA). Each bioreactor was equipped with air outflow protected from contamination by a 0.2 μm filter and a glass tube exit at the bottom for taking wastewater samples. All experiments were performed in an environmental chamber (Biotronette Mark III, Lab-Line Instruments, Melrose Park, IL, USA) with fluorescent lamps. Heterotrophic cultivation in total darkness used the same environmental chamber, but the chamber was sealed. Autotrophic experiments were subjected to continuous fluorescent light at 90 μmol photon·m²·s⁻¹ at 28 ± 1°C. Mixotrophic cultures were grown under a 12:12 light:dark (L:D) cycle. All microorganisms were cultivated in modified, sterile synthetic wastewater medium, hereafter called SGM (de-Bashan et al. 2002b), where ammonium and phosphate concentrations were adjusted to the concentrations found in municipal wastewater of the city of La Paz, Baja California Sur, Mexico (O. Perez-Garcia, Y. Bashan, and M. E. Puente, unpublished data). The synthetic wastewater at pH 6.7 contained the following ingredients (in mg·L⁻¹): NaCl, 7; CaCl₂, 4; MgSO₄·7H₂O, 2; K₂HPO₄, 21.7; KH₂PO₄, 8.5; Na₂HPO₄, 25; and NH₄Cl, 191. The culture was stirred by continuous bubbling of sterile air. Heterotrophic and mixotrophic growth media were supplemented with a d-glucose solution at a concentration of 10 g·L⁻¹. The glucose stock solution was sterilized by filtration through two 0.2 μm Whatman cellulose nitrate membranes (G.E. Healthcare, Piscataway, NJ, USA). The specific growth medium C30 for Chlorella sp. (Gonzalez et al. 1997), nutrient broth (Sigma, St. Louis, MO, USA), nitrogen-free OAB medium specific for Azospirillum sp. (Bashan et al. 1993), and saline solution (0.85% NaCl) was used to prepare the preinoculum for the experiments and as controls.

Cell counting and immobilization of microorganisms in alginate.

These procedures were performed according to the description in de-Bashan et al. (2004). Briefly, axenic cultures (either C. vulgaris or A. brasilense) were mixed with 2% alginate solution. Initial cell concentrations among experiments were similar: 1.0 ± 0.5 × 10⁶ for C. vulgaris and 2.5 ± 0.5 × 10⁵ for A. brasilense. Beads (2–3 mm in dia.) were automatically produced in a 2% CaCl₂ solidification solution (de-Bashan and Bashan 2010). To immobilize the two microorganisms in the same bead, after washing the cultures, each was resuspended in 10 mL 0.85% saline solution and then mixed together with the alginate. Because immobilization normally reduces the number of Azospirillum cells in the beads, a second overnight incubation of the beads in diluted (10% of full strength) nutrient broth was necessary. Each bioreactor contained 30 g of beads.

For cell counting in each experiment, three beads per bioreactor were solubilized by immersing them in 1 mL 4% NaHCO₃ solution for 30 min. A. brasilense cells were first stained with fluorescein diacetate (Sigma) described in Chrzanowski et al. (1984) and then directly counted under a fluorescence microscope (BX41, Olympus, Tokyo, Japan). Chlorella was counted under LM with a Neubauer hemocytometer (Gonzalez and Bashan 2000) connected to an image analyzer (Image ProPlus 4.5, Media Cybernetics, Silver Spring, MD, USA). Growth rate (μ) was determined as follows:

(1)

where Nt₁ is the number of cells at sampling time and Nt₀ is the number of cells at the beginning of the experiment (Oh-Hama and Miyachi 1992).

Analytical methods. Nitrogen and phosphorus were analyzed from 5 mL samples obtained from the bottom exit of each bioreactor, using standard water analysis techniques (Eaton et al. 2005). Orthophosphate was measured by molybdate assay carried out in an analyzer (Flow Injection Analysis, Lachat QuikChem series 8000 FIAS+, Loveland, CO, USA) according to the manufacturer’s manual. Ammonium was measured by the phenate colorimetric method (Solorzano 1969) and adapted to the microplate (Versa Max tunable microplate reader, Molecular Devices, Sunnyvale, CA, USA) (Hernández-López and Vargas-Albores 2003). Glucose was analyzed by the glucose oxidase-peroxidase (GOD-PAP) method using a kit (Randox Laboratories, Crumlin, UK) according to the manufacturer’s manual. The pH was measured by a pH-electrode (Horiba-twin pH, Irvine, CA, USA).

Autotrophic, heterotrophic, and mixotrophic growth of C. vulgaris in synthetic wastewater alone and jointly immobilized with A. brasilense. In the absence of light and a carbon source (control treatment), very slow growth of C. vulgaris occurred (Fig. 1a, lowercase letter analysis and μ calculation) with small, but significant enhancing effect of immobilization with the MGPB A. brasilense Cd starting after 3 d of cultivation (Fig. 1a, capital letter analysis).

Application of light significantly enhanced growth (Fig. 1b, lowercase letter analysis and μ calculation), followed the same trend as joint immobilization, but started a day earlier (Fig. 1b, capital letter analysis). Growing alone, C. vulgaris reached the stationary phase after 3 d, while jointly immobilized with the MGPB steadily increased population numbers up to 5 d (Fig. 1b, lowercase letter analysis).

Under heterotrophic growth, C. vulgaris grown alone had slightly larger populations and had higher growth rates than under autotrophic growth (compare Fig. 1, b and c), while joint immobilization significantly inhibited microalgal growth (Fig. 1c, both types of analysis and μ calculation).

Mixotrophic growth yielded intermediate populations and lower growth rates compared to autotrophic and heterotrophic growth, reached stationary phase after 2 d for the microalga grown alone (Fig. 1d, lowercase letter analysis and μ calculation), and was declining after 4 d of cultivation, while joint immobilization prevented population decline but did not enhance growth further (Fig. 1d, capital letter analysis).

Analysis of growth showed that 3 d of incubation is the important time for most cultures, indicating that heterotrophic growth was similar to autotrophic growth, while joint immobilization enhanced growth only under autotrophic conditions, with lesser enhancement also in the control (no light and no glucose).

Autotrophic, heterotrophic, and mixotrophic growth of A. brasilense grown alone in synthetic wastewater and jointly immobilized with C. vulgaris. No growth of A. brasilense occurred in wastewater without a carbon source, with or without C. vulgaris (Fig. 2a). Under autotrophic conditions, A. brasilense grew only in the presence of C. vulgaris for 3 d, and then growth declined (Fig. 2b). Under heterotrophic conditions, A. brasilense grew for 2 d, but later growth ceased (Fig. 2c, lowercase letter analysis). When the bacteria were jointly immobilized with C. vulgaris, the bacteria grew linearly, producing significantly larger populations (Fig. 2c, capital letter analysis and linear regression analysis). Under mixotrophic conditions, the bacteria grew for 2 d (jointly immobilized) and 3 d (alone), but then entered the stationary phase in both treatments (Fig. 2d, lowercase letter analyses). Joint immobilization had, in general, no effect on the growth of A. brasilense (Fig. 2d, capital letter analysis).

Uptake of ammonium under autotrophic, heterotrophic, and mixotrophic growth conditions. Without light and a carbon source, controls of alginate beads and beads containing A. brasilense did not remove ammonium from wastewater similar to wastewater incubated without any treatment (Fig. 3a, lowercase letter analysis). Under these conditions, C. vulgaris immobilized alone uptakes a small, but statistically significant, amount of ammonium for only 1 d, and later the uptake ceased (Fig. 3a, lowercase letter analysis). Jointly immobilized microorganisms could uptake more ammonium for only 2 d (Fig. 3a, capital letter analysis) before uptake ceased (Fig. 3a, lowercase letter analysis). Although the statistical analysis indicated significant ammonium uptake, in several cases, <20% of ammonium was taken up in the best cases after 3 d of incubation (Fig. 3e).

Autotrophic, heterotrophic, and mixotrophic growth conditions did not induce ammonium uptake in the two control treatments or treatment with Azospirillum (Fig. 3, b, c, and d, lowercase letter analyses). Under autotrophic conditions, treatment with immobilized C. vulgaris increased ammonium uptake, and jointly immobilized microorganisms enhanced it further for up to 2 d of incubation (Fig. 3b, capital letter analysis) where these treatments removed <30% of the ammonium (Fig. 3e).

Under heterotrophic growth conditions, ammonium uptake was further enhanced by C. vulgaris immobilized alone for 3 d, reached 44% removal, and then stabilized (Fig. 3c, lowercase letter analysis, and Fig. 3e). Joint immobilization continuously took up ammonium for 4 d, but at lower levels, reaching a maximum uptake of 22% after 3 d of incubation. Uptake of ammonium under mixotrophic conditions was somewhat similar to uptake under heterotrophic conditions, where joint immobilization was the more efficient treatment (Fig. 3, d and e).

At the cellular level, a different ammonium uptake profile occurred when the affinity of the cells to the substrate was calculated, and this affinity varied with time. After 1 d of incubation under heterotrophic conditions, jointly immobilized microorganisms had significantly higher affinity, compared to all treatments followed by joint immobilization under mixotrophic conditions (Fig. 3f). Later, even though joint immobilization was superior, differences with the other treatments were small (Fig. 3f, days 1 and 2).

Uptake of phosphate under autotrophic, heterotrophic, and mixotrophic growth conditions. Without light and a carbon source, all controls and treatments took up small quantities of phosphate to a level of up to 15.7% after 3 d of incubation, where the treatments of C. vulgaris immobilized alone removed the most (Fig. 4, a and e). Although statistical differences among the treatments were detected in most sampling times (Fig. 4a, capital letter analysis), quantities were small. Under autotrophic, heterotrophic, and mixotrophic conditions, both controls and beads with A. brasilense took up meager amounts of phosphate or none at all in some samplings (Fig. 4, b–d), while C. vulgaris alone removed up to 20.8% (autotrophic), 17.8% (heterotrophic), and 20.9% (mixotrophic) after 5 d (Fig. 4c). When jointly immobilized with A. brasilense, uptake increased to 31.5% (autotrophic), 22.6% (heterotrophic), and 24.3% (mixotrophic) after 5 d of incubation (Fig. 4b). The most rapid uptake was in the first 2 d under light conditions (autotrophic and mixotrophic conditions) (Fig. 4, b and e). Under heterotrophic conditions, similar quantities of uptake by C. vulgaris alone and jointly immobilized with A. brasilense occurred for the first 4 d of incubation with an increase in uptake by C. vulgaris alone after 5 d (Fig. 4, c and e). Similar removal was measured under mixotrophic conditions, where the C. vulgaris immobilized with A. brasilense performed better and most of the uptake occurred in the first day (Fig. 4, d and e). Affinity of individual cells for phosphate after 1 d followed a similar trend, where jointly immobilized cells under heterotrophic conditions had the highest affinity, followed by the same treatment under mixotrophic conditions (Fig. 4f). The superior affinity of heterotrophic, joint immobilization lasted, at a reduced rate, for an additional day, diminishing afterward (Fig. 4f).

Consumption of glucose under different conditions of incubation. Glucose was added only to cultures growing under heterotrophic and mixotrophic conditions. Under those conditions, both controls and beads with A. brasilense did not consume or remove significant amounts of glucose from the medium (Fig. 5, a and b, lowercase letter analysis). C. vulgaris alone under both growth conditions consumed glucose at the end of the incubation period, up to 18.6% under heterotrophic and up to 23.7% under mixotrophic conditions (Fig. 5, a and b). Joint immobilization enhances consumption of glucose during the first 2 d, but this level of consumption was not maintained (Fig. 5, a and b).

Changes in the pH under different conditions of incubation. In the absence of light and a carbon source, the pH did not change with time for any treatment (Fig. 6a). Under autotrophic conditions, C. vulgaris alone or jointly immobilized with A. brasilense significantly and similarly reduced the pH to <5 (Fig. 6b). Under heterotrophic conditions, significant reduction in the pH of both treatments was recorded (Fig. 6c, lowercase letter analysis), where joint immobilization reduced it faster in the first 3 d (Fig. 6c, capital letter analysis), but the level was similar to C. vulgaris alone afterward. Under mixotrophic conditions, similar phenomena occurred, with the single difference that C. vulgaris alone lowered the pH faster than a culture with jointly immobilized microorganisms (Fig. 6d).

Productivity of the three incubation regimes. The number of cells grown per mg of ammonium per liter of culture after 3 d of incubation was the highest under autotrophic growth; the other treatments had no significant effect (Fig. 7a, lowercase letter analysis). Jointly immobilized microalga and bacteria under autotrophic and heterotrophic conditions produced significantly fewer cells for the quantity of ammonium consumed, where no effect occurred under mixotrophic and control conditions (Fig. 7a, capital letter analysis).

C. vulgaris immobilized alone under heterotrophic conditions had the highest yield for phosphate consumed, and the autotrophic and mixotrophic growth was less (Fig. 7b, lowercase letter analysis). Immobilization of the bacterium reduced the yield (heterotrophic condition) or had no effect (autotrophic and mixotrophic conditions). Only under the conditions of the control did joint immobilization have a significant effect on using phosphate (Fig. 7b, capital letter analysis).

C. vulgaris immobilized alone under heterotrophic conditions had the highest yield per glucose consumed; mixotrophic growth was less efficient (Fig. 7c, lowercase letter analysis). Immobilization under heterotrophic conditions had a negative impact on glucose yield and had no effect under mixotrophic conditions (Fig. 7c, capital letter analysis).

Volumetric productivity per liter of culture of the three systems indicates that microalga immobilized alone under heterotrophic conditions had the best performance, followed by autotrophic and the mixotrophic (Fig. 7d, lowercase letter analysis). Joint immobilization with the bacterium enhanced total volumetric productivity under autotrophic and mixotrophic conditions, but not under heterotrophic conditions (Fig. 7d, capital letter analysis); under autotrophic conditions, p volumetric productivity of jointly immobilized cultures was as high as under heterotrophic cultures solely of C. vulgaris.