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EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC, AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF

CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE 1

Octavio Perez-Garcia

Environmental Microbiology Group, The Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa

Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico

Luz E. de-Bashan



The Bashan Foundation, 3740 NW Harrison Blvd., Corvallis, Oregon 97330, USA

Juan-Pablo Hernandez



and Yoav Bashan 2



The Bashan Foundation, 3740 NW Harrison Blvd., Corvallis, Oregon 97330, USA

Heterotrophic growth of microalgae presentssignificant economic advantages over the more com-mon autotrophic cultivation. The efficiency of growth and nitrogen, phosphorus, and glucoseuptake from synthetic wastewater was comparedunder heterotrophic, autotrophic, and mixotrophic

regimes of Chlorella vulgaris Beij. immobilized inalginate beads, either alone or with the bacterium

Azospirillum brasilense. Heterotrophic cultivation of C. vulgaris growing alone was superior to autotroph-ic cultivation. The added bacteria enhanced growthonly under autotrophic and mixotrophic cultiva-tions. Uptake of ammonium by the culture, yield of cells per ammonium unit, and total volumetricproductivity of the culture were the highest under heterotrophic conditions when the microalga grew without the bacterium. Uptake of phosphate washigher under autotrophic conditions and similar under the other two regimes. Positive influence of

the addition of A. brasilense was found only whenlight was supplied (autotrophic and mixotrophic), where affinity to phosphate and yield per phosphateunit were the highest under heterotrophic condi-tions. The pH of the culture was significantly reduced in all regimes where glucose was consumed,similarly in heterotrophic and mixotrophic cultures.It was concluded that the heterotrophic regime,

using glucose, is superior to autotrophic andmixotrophic regimes for the uptake of ammoniumand phosphate. Addition of A. brasilense positively affects the nutrient uptake only in the two regimessupplied with light.

Key index words: autotrophic growth; Azospirillum ;Chlorella ; heterotrophic growth; immobilization;microalgae; mixotrophic growth; wastewater treat-ment

Abbreviat ion s: A NOVA , a na ly si s of va ri ance ;MGPB, microalgae growth-promoting bacteria;OAB medium, Okon, Albrecht, Burris medium;SE, standard error; SGM, synthetic wastewater medium

Autotrophic microalgae Chlorella spp. can grow heterotrophically (light independent) if supple-

mented with a preferred carbon source (Chen 1996,Shi et al. 2000, Lee 2004, Chen and Chen 2006,Qiao et al. 2009) or mixotrophically, that is, a com-bination of the two (Kaplan et al. 1986, Ogbonnaet al. 1997, Lee 2004).

Chlorella spp. grown under autotrophic conditionscan remove nitrogen and phosphorous from waste- waters under extremely diverse environmental con-ditions with efficiency of nitrogen removal generally higher than that of phosphorus (de la Noüe and dePauw 1988, Gonzalez et al. 1997, de-Bashan et al.2002b, 2008a, Olguı́n 2003, Hernandez et al. 2006);

1Received 10 July 2009. Accepted 28 January 2010.2 Author for correspondence: e-mail [email protected].

J. Phycol. 46, 800–812 (2010) 2010 Phycological Society of America

DOI: 10.1111/j.1529-8817.2010.00862.x

800


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however, autotrophic tertiary wastewater treatment by Chlorella spp. poses two inherent major limita-tions. Wastewater is treated in large volumes in bior-eactors where penetration of light into a denseculture is limited, a fact requiring large exposed sur-face area of the culture to light (Apt and Behrens1999, Behrens 2005), and cultivation produces rela-tively low-cell densities in the wastewater culture(Lee 2001, Valderrama et al. 2002). Light-indepen-dent heterotrophic growth may solve these difficul-ties (Lee 2001, Chen and Chen 2006). It eliminatesthe need for light and may allow cultivation of den-ser concentrations of the microalgae (Chen 1996,Ogbonna et al. 1997). Singularly, elimination of light as a growth factor significantly reduces thecost of cultivation, the major parameter when large volumes of wastewater are treated (de la Noüe et al.1992).

Heterotrophic growth of Chlorella spp. has beenknown for decades (Droop 1974) and is based onthe addition of glucose, acetate, or glycerol as the

main carbon sources; other carbon sources such asmalate (Qiao et al. 2009) and several more werealso evaluated. In some heterotrophic cultures, thegrowth rate of Chlorella, the dry biomass (Ogbonnaet al. 2000, Behrens 2005), ATP generated by thesupplied energy, and the effect on ATP yield (mg of biomass generated by mg of consumed ATP) are sig-nificantly higher than in autotrophic cultures(Martı́nez and Orús 1991, Chen and Johns 1996,Shi et al. 2000, Yang et al. 2000) and are mainly dependent on the species and strain used. Mixo-trophic cultivation proved a good strategy to obtainhigh biomass and growth rates (Ogawa and Aiba1981, Lee and Lee 2002) with the extra benefit of

producing photosynthetic metabolites (Chen 1996).Our working hypothesis for addressing a specific

goal of potential wastewater treatment is that hetero-trophic and mixotrophic cultivation of C. vulgaris are superior to autotrophic cultivation in terms of growth rate, affinity to the substrates, and the capa-city of the developed high populations to uptakenutrients from wastewater. This has been carried out by employing a recently proposed technology for wastewater treatment (de-Bashan et al. 2004) andplant–bacteria interaction (de-Bashan and Bashan2008), using a specific microalga immobilized with abacterium that promotes growth in microalgae. We

compared the growth rate and population size of the microalgae, its nitrogen and phosphorus uptakecapacity from synthetic wastewater, and culturalfactors, such as glucose consumption and pH shifts.This has been carried out under the three types of cultivation, where the sole difference was the energy supplied by light or glucose.

MATERIALS AND METHODS

Microorganisms and growth conditions. The unicellularmicroalga Chlorella vulgaris (UTEX 2714, Austin, TX) and the

microalgae growth-promoting bacterium (MGPB) A. brasilense Cd (DMS 1843, Braunschweig, Germany) were tested. Exper-iments were carried out under autotrophic, heterotrophic, andmixotrophic conditions in inverted 1,000 mL conical glassbioreactors (17 cm tall, 14 cm width at top, and 5 cm at thebottom), each containing 750 mL synthetic wastewater,equipped with uplift bottom sterile aeration controlled by aperistaltic pump providing air at 30 mL air Æmin)1 measured

with a fluxometer (GF-2000, Gilmont Instruments, Barrington,

IL, USA) that pass three microbial filters: one 1 lm porebacterial air vent filter (Pall-Gelman Laboratory, Ann Arbor,MI, USA) and two 0.2 lm sterile filters (Acrodiscs, Pall Corp.,

Ann Arbor, MI, USA). Each bioreactor was equipped with airoutflow protected from contamination by a 0.2 lm filter and aglass tube exit at the bottom for taking wastewater samples. Allexperiments were performed in an environmental chamber(Biotronette Mark III, Lab-Line Instruments, Melrose Park, IL,USA) with fluorescent lamps. Heterotrophic cultivation in totaldarkness used the same environmental chamber, but thechamber was sealed. Autotrophic experiments were subjectedto continuous fluorescent light at 90 lmol photonÆm2Æs)1 at 28 ± 1C. Mixotrophic cultures were grown under a 12:12light:dark (L:D) cycle. All microorganisms were cultivated inmodified, sterile synthetic wastewater medium, hereafter calledSGM (de-Bashan et al. 2002b), where ammonium and phos-phate concentrations were adjusted to the concentrationsfound in municipal wastewater of the city of La Paz, BajaCalifornia Sur, Mexico (O. Perez-Garcia, Y. Bashan, and M. E.Puente, unpublished data). The synthetic wastewater at pH 6.7contained the following ingredients (in mgÆL)1): NaCl, 7;CaCl2,4; MgSO4Æ7H2O, 2; K 2HPO4, 21.7; KH2PO4, 8.5; Na2HPO4, 25;and NH4Cl, 191. The culture was stirred by continuous bubblingof sterile air. Heterotrophic and mixotrophic growth mediaweresupplemented with a d-glucose solution at a concentration of 10 gÆL)1. The glucose stock solution was sterilized by filtrationthrough two 0.2 lm Whatman cellulose nitrate membranes(G.E. Healthcare, Piscataway, NJ, USA). The specific growthmedium C30 for Chlorella sp. (Gonzalez et al. 1997), nutrient broth (Sigma, St. Louis, MO, USA), nitrogen-free OAB mediumspecific for Azospirillum sp. (Bashan et al. 1993), and saline

solution (0.85% NaCl) was used to prepare the preinoculum forthe experiments and as controls.Cell counting and immobilization of microorganisms in alginate.

These procedures were performed according to the descriptionin de-Bashan et al. (2004). Briefly, axenic cultures (eitherC. vulgaris or A. brasilense ) were mixed with 2% alginatesolution. Initial cell concentrations among experiments weresimilar: 1.0 ± 0.5 · 106 for C. vulgaris and 2.5 ± 0.5 · 105 forA. brasilense . Beads (2–3 mm in dia.) were automatically produced in a 2% CaCl2 solidification solution (de-Bashanand Bashan 2010). To immobilize the two microorganisms inthe same bead, after washing the cultures, each was resus-pended in 10 mL 0.85% saline solution and then mixedtogether with the alginate. Because immobilization normally reduces the number of Azospirillum cells in the beads, a secondovernight incubation of the beads in diluted (10% of full

strength) nutrient broth was necessary. Each bioreactor con-tained 30 g of beads.

For cell counting in each experiment, three beads perbioreactor 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 Chrza-nowski et al. (1984) and then directly counted under afluorescence microscope (BX41, Olympus, Tokyo, Japan).Chlorella was counted under LM with a Neubauer hemocytom-eter (Gonzalez and Bashan 2000) connected to an imageanalyzer (Image ProPlus 4.5, Media Cybernetics, Silver Spring,MD, USA). Growth rate (l) was determined as follows:

H E T E R O T R O PH IC, A UT O T R O PH IC, A ND MIX O T R O PH IC G R O WT H O F IMMO B IL IZ E D MICR O A L G A E 801


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l ¼ ðlnNt 1 lnNt 0Þ=ðt 1 t 0Þ ð1Þ

where Nt 1 is the number of cells at sampling time and Nt 0is the number of cells at the beginning of the experiment (Oh-Hama and Miyachi 1992).

Analytical methods. Nitrogen and phosphorus were analyzedfrom 5 mL samples obtained from the bottom exit of eachbioreactor, using standard water analysis techniques (Eatonet al. 2005). Orthophosphate was measured by molybdate assay

carried out in an analyzer (Flow Injection Analysis, Lachat QuikChem series 8000 FIAS+, Loveland, CO, USA) accordingto the manufacturer’s manual. Ammonium was measured by the phenate colorimetric method (Solorzano 1969) andadapted to the microplate (Versa Max tunable microplatereader, Molecular Devices, Sunnyvale, CA, USA) (Hernández-López and Vargas-Albores 2003). Glucose was analyzed by theglucose oxidase-peroxidase (GOD-PAP) method using a kit (Randox Laboratories, Crumlin, UK) according to the manu-facturer’s manual. The pH was measured by a pH-electrode(Horiba-twin pH, Irvine, CA, USA).

Experimental design and statistical analysis. The setup of allexperiments was of batch cultures. Each experiment wasperformed in triplicate, where one bioreactor served as areplicate and each experiment was repeated twice. Eachexperiment had four regimes: controls (no light, no glucose),autotrophy (light, no glucose), heterotrophy (no light, glucoseadded), and mixotrophy (12:12 photoperiod, glucose added).Each regime contained five treatments: medium alone, beads

without microorganisms, beads with C. vulgaris , beads withA. brasilense , and beads with the two microorganisms jointly immobilized. Three samples were taken for all analyses at eachsampling time. The following variables were analyzed: Volu-metric productivity (Qp ) was calculated as:

Qp ¼ P 1 P 0: ð2Þ

where P 1 and P 0 are grams of product (as cells or biomass) ina defined volume during the time between initial and finalsampling (Cooney 1983). Affinity or specific activity (Sa ) of the microalgal cells to the substrate in a specific interval of time was calculated as:

Sa ¼ S t =N t ð3Þ

where S t is grams of substrate S consumed or product formedin a specific time interval t, and Nt is grams or number of cat-alysts (enzymes or cells) at the end of the time interval t (Cooney 1983). Yields (Y P ⁄ S) (number of cells produced permg substrate uptake in 1 L of culture during a defined incu-bation period) were calculated as:

Y P=S ¼ ðP 1 P 0Þ=ðS 1 S 0Þ ð4Þ

where P is the number of cells at the end (P 1) and at thebeginning (P 0) of a defined time interval, and S is the sub-strate (ammonium, phosphate, or glucose) at the end (S 1)and at the beginning (S 0) of this time interval (Bitton 2005).

As data in both repetitions of the each experiment were simi-

lar, data were combined for analysis by two-way analysis of var-iance (ANOVA) and then by Tukey’s post hoc analysis orStudent’s t -test, both at a significance level of P


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analysis). Under these conditions, C. vulgaris immo-bilized alone uptakes a small, but statistically signifi-cant, amount of ammonium for only 1 d, and laterthe uptake ceased (Fig. 3a, lowercase letter analysis). Jointly immobilized microorganisms could uptakemore ammonium for only 2 d (Fig. 3a, capital letteranalysis) before uptake ceased (Fig. 3a, lowercaseletter analysis). Although the statistical analysis indi-

cated significant ammonium uptake, in severalcases,


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to all treatments followed by joint immobilizationunder mixotrophic conditions (Fig. 3f). Later, eventhough joint immobilization was superior, differ-ences with the other treatments were small (Fig. 3f,days 1 and 2).

Uptake of phosphate under autotrophic, heterotrophic,and mixotrophic growth conditions. Without light anda carbon source, all controls and treatments tookup small quantities of phosphate to a level of up

to 15.7% after 3 d of incubation, where the treat-ments of C. vulgaris immobilized alone removedthe most (Fig. 4, a and e). Although statistical dif-ferences among the treatments were detected inmost sampling times (Fig. 4a, capital letter analy-sis), quantities were small. Under autotrophic,heterotrophic, and mixotrophic conditions, bothcontrols and beads with A. brasilense took up mea-ger amounts of phosphate or none at all in somesamplings (Fig. 4, b–d), while C. vulgaris aloneremoved up to 20.8% (autotrophic), 17.8% (het-erotrophic), 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 rapiduptake was in the first 2 d under light conditions(autotrophic and mixotrophic conditions) (Fig. 4,b and e). Under heterotrophic conditions, similarquantities 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). Simi-lar removal was measured under mixotrophic con-ditions, where the C. vulgaris immobilized withA. brasilense performed better and most of theuptake occurred in the first day (Fig. 4, d and e). Affinity of individual cells for phosphate after 1 dfollowed a similar trend, where jointly immobilizedcells under heterotrophic conditions had the high-est affinity, followed by the same treatment undermixotrophic conditions (Fig. 4f). The superioraffinity of heterotrophic, joint immobilization

Fig. 2. Populations of immobilized Azospirillum brasilense alone and jointly immobilized with Chlorella vulgaris growing under autotroph-ic, heterotrophic, and mixotrophic growth conditions in synthetic wastewater. Values of each curve denoted with a different lowercase let-ter differ significantly by two-way analysis of variance (ANOVA) and by Tukey’s post hoc analysis at P < 0.05. Values at each date denoted with a different capital letter differ significantly by Student’s t- test at P < 0.05. Bars represent standard error of the mean (SE). Absence of a bar indicates negligible SE. Linear regression under heterotrophic growth is for the joint immobilization treatment.

804 O CT A V IO PE R E Z -G A R CIA E T A L .


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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 mixotrophicconditions. Under those conditions, both controlsand beads with A. brasilense did not consume orremove significant amounts of glucose from the

Fig. 3. Ammonium uptake (a–d), ammonium consumed (e) on the third day of incubation, and affinity for ammonium (f) of Chlo- rella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growthconditions in synthetic wastewater. All statistical analyses were performed using two-way analysis of variance (ANOVA) combined withTukey’s post hoc analysis or by Student’s t- test both at P


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medium (Fig. 5, a and b, lowercase letter analysis).C. vulgaris alone under both growth conditions con-sumed 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 con-sumption was not maintained (Fig. 5, a and b).

Fig. 4. Phosphate uptake (a–d), phosphate consumed at the third day of incubation (e), and affinity for phosphate (f) of Chlorella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growth condi-

tions in synthetic wastewater. All statistical analyses used two-way analysis of variance (ANOVA) combined with Tukey’s post hoc analysis orby Student’s t- test both at P < 0.05 as follows: In (a–d), values on each curves denoted with a different lowercase letter differ significantly by ANOVA. Values at each date denoted with a different capital letter differ significantly by ANOVA. In (e), columns of C. vulgaris aloneor jointly immobilized with A. brasilense separately denoted with a different lowercase letter differ significantly by ANOVA. Pairs of columnsat each treatment type denoted with a different capital letter differ significantly by Student’s t- test. In (f), values corresponding to eachday separately denoted with a different capital letter differ significantly by ANOVA. Bars represent standard error of the mean (SE). Absence of a bar indicates negligible SE. Some statistics letters were eliminated for clarity.



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Changes in the pH under different conditions of incubation. In the absence of light and a carbonsource, 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


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step for evaluating the potential of different cultiva-tion systems for wastewater treatment, this study used a defined synthetic wastewater in small labora-tory bioreactors and a common strain of C. vulgaris,previously shown to be an efficient wastewater treat-ment agent (de-Bashan et al. 2002b, 2004) andcapable of growing heterotrophically on municipal wastewater supplemented either with glucose or Na-acetate (O. Perez-Garcia, Y. Bashan, M. E. Puente,unpublished data). Glucose is known to support heterotrophic growth of several Chlorella sp., includ-

ing C. vulgaris (Lee 2004).Heterotrophic cultivation of C. vulgaris was supe-rior to autotrophic and mixotrophic growth,although each cultivation scheme has its advantagesunder specific conditions, depending on the endgoal of cultivation: biomass or nutrient uptake. Thehighest ammonium uptake (36%) occurred in het-erotrophic cultivation when the culture was loaded with a high concentration of ammonium, higherthan under autotrophic cultivation. This uptake by the entire heterotrophic culture contrasts with thesuperiority of autotrophic cultivation in terms of the

amount of growing cells, biomass produced, growthrate, and the yield of cells per quantity of ammo-nium consumed. Although even higher percentageuptakes of ammonium were previously recordedusing this immobilization system and this strainunder autotrophic conditions (de-Bashan et al.2002b, 2004), those were performed in a far lowerinitial ammonium concentration (


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and using glucose nutrition (Buchanan et al. 2000).The reason that heterotrophic cultures of C. vulgaris growing alone uptake more ammonium than cul-tures that are jointly immobilized with A. brasilense isbecause of the higher growth rate. Consequently,larger populations form, and this is not related tothe high affinity of the cells for ammonium. Undermixotrophic conditions, cultures of C. vulgaris (alone or with A. brasilense ) take up more ammo-nium than cultures under autotrophic conditionsbecause of higher affinity for ammonium under

these conditions. Higher affinity for ammonium inheterotrophic and mixotrophic cultures occursbecause more ATP and NAD(P)H for metabolicprocesses is available, which is unrelated to auto-trophic carbon assimilation (Calvin cycle) (Yanget al. 2000), such as for primary nitrogen assimila-tion by the enzymes of the nitrogen cycle in Chlo- rella spp. The difference between the Sa values fornitrogen and phosphorus (discussed later) might beexplained because during the first day of samplingthe largest uptake of both nutrients occurred withthe smallest population of microalgae, compared to

later samplings, where the population was larger but removal of the nutrients did not significantly increase or increased only to a small extent.

The presence of the MGPB A. brasilense enhancedammonium and phosphate uptake by the microal-gae when light was provided under autotrophic andmixotrophic conditions (de-Bashan et al. 2002b,2004, 2008c), but not under heterotrophic condi-tions. This occurred because the ‘‘helper’’ bacte-rium enhanced, in general, the growth rate of themicroalgae (Gonzalez and Bashan 2000, de-Bashan

et al. 2008a, this study) by affecting photosynthesispatterns. The specific role of Azospirillum had beenshown by producing phytohormones and affect-ing several cellular metabolisms of the microalga(de-Bashan et al. 2002a, 2008a). So far, previousstudies have shown that its role in removing nutri-ents is minimal (de-Bashan et al. 2002b, 2004).Furthermore, the two microorganisms significantly differ in individual cell size and their final popula-tion size in culture. We showed that microalgal pop-ulations are at least one order of magnitude largerthan those of the MGPB. While these microalgal

Fig. 7. (a–c) Yield (number of cells produced per mg substrate uptake in 1 L of culture during the first 3 d of incubation) of Chlo- rella vulgaris alone and jointly immobilized with Azospirillum brasilense growing under autotrophic, heterotrophic, and mixotrophic growthconditions in synthetic wastewater. (a) Ammonium, (b) phosphate, (c) glucose, and (d) volumetric productivity of all immobilized and jointly immobilized systems. In each panel, columns of C. vulgaris alone or jointly immobilized with A. brasilense separately denoted with adifferent lowercase letter differ significantly by two-way analysis of variance (ANOVA) and by Tukey’s post hoc analysis. Pairs of columns of each treatment type denoted with a different capital letter differ significantly by Student’s t- test, all at P


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populations are common for this species (Gonzalezand Bashan 2000), the levels of the bacterial popula-tion for this bacterial species are small (Bashanet al. 2004). This happened because the mediumsuitable for the microalga is less suitable for thegrowth of A. brasilense . Although A. brasilense canuse ammonium for growth (Van Dommelen et al.1997), the relatively small populations of A. brasilense in a jointly immobilized system removed only minute amounts of nitrogen and phosphate from wastewater (de-Bashan et al. 2002b, this study) ormay live also on exudates from the microalgae(Watanabe et al. 2006).

Under heterotrophic cultivation, joint immobili-zation significantly increased the affinity of C. vulgaris to ammonium and phosphate, compared toother treatments, but did not increase the popula-tion. The yield, a value composed of the number of cells per substrate (either ammonium or phos-phate), is very low. All of these findings indicatethat the cells are metabolically active but do not use

these nutrients for multiplication. Our experimentalevidence supports the claim by de-Bashan et al.(2005, 2008b) that the effect of the ‘‘helper’’ bacte-

rium is to increase ammonium consumption of eachcell and not that the elimination of ammonium is aresult of increase in the size of the population.

Removal of phosphate is far less affected by thedifferent treatments, where autotrophic cultivation was the best one and adding the MGPB into auto-trophic, heterotrophic, and mixotrophic conditionsenhanced this ability, similar to previous studies onthat topic (Hernandez et al. 2006). Differences inrates of removal between the regimes are not a con-sequence of the affinity of the cells for phosphate.In spite of the higher affinity in mixotrophic andheterotrophic cultures for phosphate, removal inautotrophic cultures was significantly superior. Supe-riority of C. vulgaris jointly immobilized with A. brasilense under autotrophic conditions could beexplained by accumulation of phosphorus aspolyphosphates (Hernandez et al. 2006) and ⁄ or ahigher efficiency in metabolizing inorganic phos-phorus to ATP via photo and oxidative phosphoryla-tion (Yang et al. 2000).

Regardless of the cultivation conditions andbecause of the use of ammonium as the sole nitro-gen source, the pH of the culture always declined to

Table 1. General analysis of the best growth condition (autotrophic, heterotrophic, mixotrophic) and immobilization type(Chlorella alone or immobilized with Azospirillum brasilense ) for each parameter tested.

Parameter Per specific growth conditions Comparison among all growth conditions

Chlorella growth Control – jointly immobilized Autotrophic – jointly immobilizedHeterotrophic – Chlorella aloneMixotrophic – jointly immobilized

Heterotrophic – Chlorella alone

Azospirillum growth Control – similar

Autotrophic – jointly immobilizedHeterotrophic – jointly immobilizedMixotrophic – similar

Heterotrophic – jointly immobilized

Ammonium consumption Control – jointly immobilized Autotrophic – jointly immobilizedHeterotrophic – Chlorella aloneMixotrophic – jointly immobilized


Affinity for ammonium Not applicable Heterotrophic – jointly immobilizedPhosphate consumption Control – Chlorella alone

Autotrophic – jointly immobilizedHeterotrophic – jointly immobilizedMixotrophic – similar

Autotrophic – jointly immobilized

Affinity for phosphate Not applicable Heterotrophic – jointly immobilizedGlucose consumption Heterotrophic – jointly immobilized

Mixotrophic – jointly immobilizedSimilar

Ammonium yield Control – similar Autotrophic – Chlorella alone

Heterotrophic – Chlorella aloneMixotrophic – similar

Autotrophic – Chlorella alone

Phosphate yield Control – jointly immobilized Autotrophic – similarHeterotrophic – Chlorella aloneMixotrophic – similar


Glucose yield Heterotrophic – Chlorella aloneMixotrophic – similar


Volumetric productivity Control – jointly immobilized Autotrophic – jointly immobilizedHeterotrophic – Chlorella aloneMixotrophic – jointly immobilized

Autotrophic – jointly immobilizedHeterotrophic – Chlorella alone

Similar: treatment of Chlorella alone = treatment of jointly immobilized with A. brasilense.



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low values because consumption of ammoniumreleases protons and the decreased pH impairs cultivation (Kaplan et al. 1986, Grobbelaar 2004, thisstudy). When C. vulgaris grew on high concentra-tions of ammonium, the pH of the culture droppedas long as ammonium was present, even if the pH was adjusted daily (Tam and Wang 1996). All thesefactors imply that, in these cultures, there is no lossof ammonium via volatilization, because ammoniumis converted to volatile ammonia only at pH > 8.5. Joint immobilization with A. brasilense reduced thenegative effects of pH shifts, but at higher pH levelsthan the ones present in this study (de-Bashan et al.2005).

A general evaluation of the various growingregimes and immobilization parameters explored inthis study (Table 1) showed that heterotrophic cultivation of C. vulgaris yielded the highest uptake of ammonium from synthetic wastewater compared toautotrophic and mixotrophic growth. Heterotrophiccultivation did not enhance uptake of phosphate,

compared to the other treatments. Adding the MGPBto the culture had positive effects only for autotroph-ic cultivations (high ammonium and phosphateremoval and high cell populations of C. vulgaris ), but not for heterotrophic and mixotrophic cultures.

We thank the following personnel of CIBNOR: Iban Murillo(phosphate analysis), Jorge Cobos (bioreactor manufactur-ing), Roberto Hernandez (glucose analysis), and EstherPuente (advice concerning microbiological techniques). Thisstudy was mainly supported by Consejo Nacional de Ciencia y Tecnologı́a (CONACYT contract 23917), Secretaria de Medio

Ambiente y Recursos Naturales (SEMARNAT contract 23510),and time for writing by the Bashan Foundation, USA.O. P.-G. is a recipient of a fellowship from CONACYT (con-

tract 207021) with additional funding from SEMARNAT andthe Bashan Foundation.

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