Journal of Applied Phycology 7: 85-95, 1995. 85( 1995 Kluwer Academic Publishers. Printed in Belgium.

A comparative study of four systems for tertiary wastewater treatment byScenedesmus bicellularis: New technology for immobilization

Valentino M. Kaya*, Joe1 de la Notie & Gaston PicardDepartement des Sciences et Technologie des Aliments, Groupe de Recherche en Recyclage Biologique etAquiculture (GREREBA), Universitd Laval, Sainte-Foy, Qu6bec, Canada GIK 7P4(*Authorfor correspondence)

Received 25 April 1994; revised 3 November 1994; accepted 5 November

Key words: immobilization, microalgae, Scenedesmus bicellularis, starvation, wastewater treatment, nutrientremoval

Abstract

Immobilization appears to be one of the best techniques to separate physically micro-algal cells from their culturemedium for the purpose of algal tertiary wastewater treatment. High operation costs and other drawbacks of large-scale physico-chemical methods of harvest led to a comparative study of biotreatment systems. Before treatmentbegan, Scenedesmus bicellularis cells were conditioned (starved) under four different sets of conditions: 1) non-immobilized cells with air bubbling (NCA); 2) cells immobilized in alginate beads (CBW) and 3) cells immobilizedon alginate screens (CSW), all conditioned in synthetic culture medium depleted in N and P; 4) cells immobilizedon alginate screens but conditioned in air at 100% relative humidity (CSA). Starvation was started under a light:darkphotoperiod of 16:8 h. Starved cells were then used to treat wastewater for a 2-h period. The performance of eachsystem was evaluated by determination of residual NH4-N and phosphate ions and by growth (dry weight, totalchlorophyll, cell count, protein content). We then tested the capacity of microalgae immobilized on screens toeliminate N and P from a secondary municipal wastewater effluent and examined the influence of temperature andstarvation. The quality of treated effluents was improved considerably with the system using CSA or CSW model.For CSA model, the protein content was 22.4 pg cell- ' compared to 12.9, 9.5, 9.1 pg cell- ' for NCA, CBWand CSW models, respectively. The CBW and CSW models were efficient for chlorophyll synthesis. The residualammonium content in natural wastewater after 2 h of treatment with CSA model was 39% at 6±2 "C and reached100% removal at 18±2 C. With the first 2 h, the removal of orthophosphate was inferior (53%) at 6+2 C, but88 to 100% at 18+2 "C depending on starvation times. Long starvation times (72 or 96 h) caused damage to cellsand uptake of nutrients was lower than with 54 h starvation. This work demonstrates that by using immobilizationon screens, removal of nutrients from wastewater was higher than with conventional biological tertiary wastewatertreatments (free cells or bead-shaped alginate particles).

Introduction For over half a century, numerous studies haveexamined microalgal nutritional needs and their cul-

The proliferation of algae downstream from waste- ture in controlled environments. Several authors (Cald-water treatment plants shows that secondary effluents well, 1946; Oswald & Gotaas, 1957; Matusiak, 1976)constitute a medium rich in nitrogen and phosphorus have suggested that some green algae and cyanobac-nutrients, favoring the development of algal blooms teria could remove nutrient ions effectively, depend-(Goldman, 1979; Tapie & Vermeglio, 1987). When ing on environmental conditions and the physiologicalabundant in secondary wastewater effluents, these inor- tolerance of the organisms. With respect to these envi-ganic nutrients are the major cause of degradation of ronmental problems associated with water pollutionreceiving waters (Metcalf & Eddy, 1979). by urban or food processing secondary effluents, the

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literature (de la Noiie et al., 1980; Lavoie & de laNoie, 1985; Picard et al., 1980; Pouliot & de la Nouie,1985; S6rodes et al., 1986) provides much informationconcerning systems for biological tertiary wastewatertreatment through controlled algal culture. However,the harvest of microalgae is still an impediment inmost cases.

The need for a practical and economic solution tothe problem of pollution by wastewaters is becom-ing more and more urgent. During the sixties, Hattoriand Mosbach (Navarro & Barbotin, 1985) studied thebehaviour of cells absorbed or included in syntheticsupports, inspired by immobilization techniques formicroorganisms developed for enzymology and bac-terial fermentation. However, the physical separationof microalgae from their culture medium limits sever-al procedures, due to their economic cost. The ener-gy requirement for biomass recuperation is too highand the technology too sophisticated despite the highremoval yield of nutrients. To overcome this constraint,Chevalier and de la Noiie (1985) established a wastew-ater treatment system with the help of Scenedesmusimmobilized in kappa-carrageenan gel, in the form of3-mm beads. Systems for tertiary wastewater treat-ment inspired by bacterial immobilization exist onlyon a laboratory scale or as demonstration units.

There is much experimental evidence showing theeffect of N and P starvation on nutrient removal bymicroalgae (Picard et al., 1980; Syrett et al., 1986) andsome studies have been done with free or immobilizedmicroalgae in beads, but such tertiary wastewater treat-ment by microalgae remains to be optimized. Becausethis work aims at fast nutrient removal from wastew-ater by microalgae, another immobilization configu-ration was used i.e. a flat surface (screens) and nutri-ent depletion in air, to show the effect of starvationand activation by light intensity on ions uptake in themedium. It seemed necessary to test the capacity ofmicroalgae immobilized on screens to remove nitro-gen and phosphorus ions from a natural effluent, whileexamining the effect of wastewater temperature andstarvation times, in order to verify if microalgae couldbe able to give the reciprocal results by using secondarymunicipal wastewater effluent.

The purpose of this study was to determine theperformance of four biotreatment systems, in whichScenedesmus bicellularis cells were immobilized orfree and conditioned (starved) according to differentmodes. This is the first time, an attempt has been madeto establish relations between the two most used forms

of immobilization and different models of microalgalstarvation.

Materials and methods

Microalgal strain and growth of cells

An isolate of Scenedesmus bicellularis, from Valcarti-er wastewater treatment plant (Quebec) was grown intwo pyrex carboys (Kaya & Picard, 1994). Cell countswere determined daily with a Neubauer ultra planehaemacytometer. Twelve days after inoculation, thecell concentration of the carboys was about 1.5x10 7

cells ml- '. Cells were harvested by centrifugation(20000 x g , 10 min, 4 C) and yielded a concen-trated biomass of about 4.7 x 108 cells ml- l assuming100% recovery.

Immobilization and starvation protocol

Four systems of wastewater biotreatment were opti-mized separately: 1) non-immobilized cells with airbubbling (NCA); 2) cells immobilized in alginatebeads (CBW) and 3) cells immobilized on alginatescreens (CSW), all starved in modified Dauta (1982)medium at 23 C, without nitrogen and phosphorus;4) cells immobilized on alginate screens but starvedin air at 100% relative humidity (CSA). To increasethe microalgal cell number comparatively with usingthe non-immobilized cells, the optimum cell loadingwas determined to be 1:1 cell to alginate (3% dryweight) ratio to obtain 1.5% solution final concen-tration. Alginate 3-mm beads, containing entrappedmicroalgal cells were obtained by adding the suspen-sion mixture of algae and alginate, drop by drop withsyringes in 2% (w/v) CaC12. The ratio between beadsvolume and wastewater to be treated was fixed at 1/8after a few preliminary tests. For screens immobiliza-tion i.e. CSA and CSW of 3 mm thickness of gel, thesuspension mixture was solidified inside the screensby pulverization of a 2 (w/v) % solution of CaC12.All wastewater systems optimized, were starved underthe same conditions of illumination (150 mol photonm- 2 s - ') and a light:dark photoperiod of 16:8 h.

In the second part of this work, we examined theeffect of starvation times of 36, 54, 72 or 96 h forcells immobilized on screens, starved in air or in waterand the effect of wastewater temperature (6±2 C,13+2 C, 18+2 C) on nutrient removal by microal-gae.

Experimental procedure

Nutrient removal experiments were made in the samebioreactor and analyses of orthophosphate, ammoni-um, nitrate and nitrite present in wastewater werecarried out on a Technicon Model II Autoanalyzer.However, in the wastewater treatment system usingfree cells, wastewater samples were separated frommicroalgae by filtration (Whatman filter 934-AH).After the treatment run, biomass samples were takento measure the growth parameters as cell counts, drymatter, total chlorophyll or protein content. The gelcontaining immobilized microalgae was dissolved in0.2 M Na3PO4 to facilitate the evaluation of biomass.Cell counts (cells ml- l) were taken at 1 to 2 h intervalsafter wastewater treatment on subsamples. Chlorophyllcontent of each system was determined by double boil-ing methanol extractions. Then 2 ml culture suspensionwas added to 2 ml methanol at 60 °C for 20 min. Aftercentrifugation, the supernatant from extraction waskept in the dark and the resulting pellet was used for thesecond extraction. The content of chlorophyll was cal-culated by the method of Porra (1990). The extractionof proteins with 0.5 N NaOH and bicinchoninic acidwas measured by the SIGMA TPRO-562 procedureand the standard curve was obtained using a commer-cial preparation of bovine serum albumin (Sigma, St.Louis, USA) dissolved in 0.5 N NaOH.

Statistical analysis

The analyses of variance and the statistical differencesbetween the four models of biotreatment were realisedby the multiple comparison test using Super Anovaprocedure.

Results

Wastewater treatment parameters

The study was mainly focused on rapid removal ofNH4-N and P0 4-P. Orthophosphate contents of allmodels decreased slowly (Fig. 1A). Cell counts in theNCA system decreased from 1.4 x 107 cells ml- atthe beginning of starvation to 0.8 x 107 cells ml- oftreated wastewater i.e. a decrease of 42.8% where-as the counts of CSW and of CSA increased by 7and 21%, respectively. Although the best phospho-rus removal was obtained with CSA and CSW mod-els, its complete exhaustion required a longer retentiontime (>2 h). Figure 1B shows the ammonium removalcurves for each of the 4 models of wastewater treat-

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0 15 30 45 60 75 90 105 120

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Fig. 1. Removals of P0 4-P and NH4 -N from artificial secondaryeffluent by Scenedesmus bicellularis among wastewater treatmentsystems.

ment. A close examination of these curves shows thatCSA and CSW models are the most efficient treatmentsand were comparable, with marked decrease of ammo-nium ions removal after 75 and 90 min of incubation,respectively. Multiple variance comparisons show thatthere is a significant difference (P<0.05) between CSAand CBW, CSA and NCA, CSW and CBW, CSW andNCA systems. By contrast, there is no significant dif-ference (P>0.05) between CSA and CSW, CBW andNCA models.

Biomass growth parameters

Table 1 shows the variation of the growth parame-ters according to the different wastewater treatmentsystems (immobilization and conditioning). The dryweight in the NCA model was 8 to 10 times lowerthan in the systems using other 3 models. We note that

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total chlorophyll content increased when cells werestarved in water whereas the air phase favored proteincontent.

Effect of wastewater temperature and starvationtimes

The quality of Valcartier wastewater varied daily anddepended on the interaction of several factors such asthe effluent origin, the efficiency of primary and sec-ondary treatments to oxidize the organic matter. Duringthe summer period of this study, the concentrations ofvarious N sources, phosphorus nutrient and the N/Pratio were almost always different. The compositionof Valcartier secondary effluent was 423-794 /mol 1-'NH4 -N, 28-82 pmol 1-1 NO3-N, 14-77 amol 1- NO2-N, 32-68 mol 1- P0 4-P and the N/P ratio varyedfrom 10 to 21.

Figures 2 and 3 show the efficiency of immobi-lized microalgae, starved under two sets of condi-tions, in removing NH4-N from urban wastewater atdifferent temperatures. These figures also show thatScenedesmus bicellularis can adapt to temperaturevariations. It has been noted that there is an effectof temperature on nutrient removal which can best beshown by comparing Figs 2A to 2C and 3A to 3C,where the nutrient depletion was shown to be muchgreater at 18±2 °C than at 6±2 °C. For example, at awinter temperature of 6±2 °C when immobilized cellswere starved in air saturated at 100% relative humid-ity, the best removal of NH4 -N was 61.3% (Fig. 2A)after an incubation time of 2 h. At 18±2 C, 100%of ammonium nitrogen was removed after 75 minutes(Fig. 2C) with cells starved for 54 h.

Figures 4 and 5 show that the orthophosphateions were not completely removed within 120 min inboth starvation experiments when the temperature wasbelow 15 C, but a low fluctuation observed for NH4 -Nand P0 4-P at 6±2 °C. Depending on wastewater tem-perature, the disappearance of P0 4-P took a long time.However, after 2 h where the screens from CSA mod-el were incubated in wastewater, a P0 4-P removal of53.4% was obtained at 6±2 °C and 91.2% at 18+2 °Cfor 36 h of starvation duration, whereas 100% of phos-phorus was removed at 18±2 °C by cells starved for72 h.

Figure 6 shows the availability of N sources inValcartier wastewater and the ability of Scenedesmusbicellularis cells to remove preferentially ammonium,nitrate or nitrite ions. The removal of NO3-N and NO2-N did not exceed 10% at 6+2 °C and 13±2 °C but

at 18±2 C, the disappearance of these nutrients byScenedesmus bicellularis was NH4 -N>NO3 -N>NO2 -N.

Discussion

Measurement of nitrogen and phosphorus

The growth in a microalgal culture is normallydescribed by biomass increase expressed as cell num-ber, dry weight, chlorophyll or protein content (Dau-ta, 1982). The latter two parameters to express thebiomass content are more reliable and a lot more pre-cise because chlorophyll is one of the major pigmentsresponsible for the algal coloration and it is presentat all growth phases (Dauta, 1982; Fresnedo & Serra,1992), although the ratio of growth parameters as func-tion of cell mass changes with culture conditions.

The different removals of ammonium andorthophosphate by cells (Figs 1, 2, 3, 4, 5) obtainedfor each model of starvation indicated that the rate ofremoval did not depend entirely on the immobiliza-tion configuration but also on the starvation mode withlight limitation being a factor. The light penetration inalgal culture is affected directly by incident light andinversely by the depth and density of the algal cul-ture. Indeed, better light penetration probably causedthe CSA and CSW models to be most efficient. Thiscould be due to the great activation of light intensity inthe case of microalgae immobilized on screens where-as for bead-shaped alginate particles, only the cells atthe surface are exposed to light. Results obtained withcells immobilized on screens and starved in air (CSA)or water (CSW) were almost identical with a slight-ly inferior capacity for the starving phase in nutrientdepleted water, although the difference was not sig-nificant (P>0.05). This observation leads us to assertthat accessibility and availability of light energy toimmobilized cells is better after conditioning in air thanin aqueous environment, where water could play therole of a shield, preventing deep penetration. Indeed,the starvation of systems using CBW or CSW mod-el oriented the growth towards chlorophyll productionbecause of light energy attenuation by water throughthe cultures whereas in air, it favored protein content(Table 1). Similar results of cellular chlorophyll a andintracellular concentrations of protein were reported inScenedesmus sp. and in Fragilaria crotonensis (Rhee& Gotham, 1980b). As shown in Fig. 6, the preferencefor ammonium ion is attributed to energy saving for

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Table 1. Relationship between residual growth parameters for different systems of wastewaterbiotreatment. Initial dry weight of microalgal cell in the NCA system was 0.29 g 1-1 and celldensity was 1.4x 107 cells ml- 1.

Wastewater Growth parameters

treatment Cell number Dry weight Protein Chl. Chl./d. wt

systems (107 cells ml - ) (g 1- 1) (pg cell- ) (pg cell - I) (%)

NCA 0.8 + 0.01 0.19 12.9 0.27 1.14

CBW 1.0 + 0.01 1.57 9.5 0.39 0.25

CSW 1.5 + 0.09 1.86 9.1 0.49 0.40

CSA 1.7 + 0.12 1.79 22.4 0.34 0.32

protein synthesis and for rapid cell growth (Bienfang,1975).

Several previous studies (Lavoie & de la Notie,1985; Pouliot & de la Noiie, 1985; Rao, 1986)have shown poor phosphorus removal by microalgaebecause of their limited physiological needs, the N/Pratio needed being 15 (S6rodes et al., 1986). Chevalierand de la Notie (1985) reported that nitrogen is a lim-iting factor in Valcartier wastewaters and that the N/Pratio is not optimal for Scenedesmus. This hypothesisis debatable since a N/P ratio lower than 7 is requiredfor nitrogen to be a limiting factor. Our results indicatea reverse tendency, Valcartier wastewaters during theperiod of this study being characterized by N/P>7, i.e.over 10, making phosphorus a limiting factor. Howev-er, depending on the pH and temperature, the removalof nitrogen in the form of ammonia 'NH3 stripping' canvary the ratio N/P. Reeves (1972) demonstrated that thestripping effect occurred only under strongly alkalineconditions and at an elevated temperature. During ourexperiment, the pH values usually fluctuated between6.5 to 7.3 in the starvation chamber and increased to8.5 when the starved cells were transferred to wastew-ater. The low pH values found in our study, suggestedthat NH3 stripping might not be significant.

The starvation approach was devised to depletemore quickly the dissolved nutrients in wastewater,especially ammonium and orthophosphate ions, whichare the major causes of microalgal and aquatic plantblooms. With mass algal cultivation in open ponds,NH 4-N or PO4-P removals usually take three to sixdays to obtain over 90% of efficiency depending onthe concentration and size of the inoculum (Pouliotet al., 1989; Tam & Wong, 1989). The retention timewith screens is clearly reduced to one half if we com-pare our results with those obtained by Chevalier and

de la Node (1985) where, Scenedesmus cells were notstarved. However, these differences will be improvedby the optimization of environmental conditions ofcell division during starvation such as enrichment ofC0 2, photoperiod duration, light intensity and degreeof starvation because if light, carbon dioxide and othernutrients are not limiting, the cells continue growingalthough less rapidly (Richardson et al., 1969). Sever-al other interactions of environmental factors such asphotoperiod, temperature, irradiance level and avail-ability of nutrients can influence cellular growth (Rhee& Gotham, 1980a, b).

Results in Table 1 show that cell division occurredin CSA model as well as CSW or CBW models becauseat the end of the wastewater treatment, cell concentra-tion which was of 1.4 x 107 cells ml- l at the begin-ning, was up to 1.2 times higher after 48 h of star-vation followed by 2 h of incubation for the CSAmodel. On the contrary, the ratio variations betweenchlorophyll and the dry weight measured at the endof each wastewater treatment corresponded to that ofCapblancq (1982), which gave a chlorophyll percent-age between 0.3 and 2% of dry weight. The growth ofcells in CSA model and the removal rate of nitrogenand phosphorus ions indicated a good cell viability inair saturated in humidity. To avoid algal cell drying,relative humidity must be at its maximum to createoptimal conditions of condensation and to increasethe water film covering immobilized microalgae onscreens during starvation.

Among parameters that allow us to evaluate thebiomass, the dry weight was less precise for CBW,CSW or CSA because of the gel interference. The vari-ations in results obtained for this measurement implythat the gel would stay with cells for the treatmentwith CBW, CSW or CSA models. Under light limited

94

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0 15 30 45 60 75 90 105 120

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Fig. 6. Residual N-concentrations as function of time from sec-ondary municipal wastewater effluent at different temperatures inthe presence of Scenedesmus bicellularis immobilized on screensand starved in air at 100% relative humidity.

but nutrient sufficient conditions, the overall trend ofchlorophyll a content is to increase with decreasingintensity of light (Rhee & Gotham,1980b). Accord-ing to the extraction method used, the chlorophyll orthe protein content can be underestimated (Inskeep &Bloom, 1985). Numerous factors could account forthese result variations. For example, lipid synthesisis subject to nitrogen availability (Sansregret, 1986).Protein, chlorophyll and lipid production in microal-gae do not follow the same pathway in the light or darkphase. Eppley etal. (1969) and Terry etal. (1983) have

shown that protein levels remain stable during the darkphase but lipid levels decreased slowly. Setlik (1979)reported evidence that a high chlorophyll content cor-responds to an adaptation to weak light intensity. Ourresults show without ambiguity the same tendency forthe chlorophyll profile.

Temperature dependence of removal rates

The range of wastewater temperatures tested duringthis study was 4-20 C. Despite starvation times, ourresults illustrate that fluctuations in temperature hadan effect on the disappearance of nitrogen and phos-phorus. Nutrient removal was more effective for nitro-gen an for phosphorus regardless of temperature. Ter-ry et al. (1983) showed that at low temperatures, inmost cases, ammonium and orthophosphate removalrates were strongly dependent on temperature coef-ficient (Q1o), which implies little adaptation of thecells' ions removal systems to temperature. Rhee andGotham (1980a) found that in nutrient saturated con-ditions, temperature is a linear function of cell growthand it does not seem to influence the carbon fixation inScenedesmus sp.

The results obtained in this study demonstrated thatuptake of nutrients from CSA model was clearly supe-rior compared to CSW, CBW or NCA models. TheCSA model presents original characteristics and hasseveral advantages over conventional batch systemsbecause it can use maximum light energy. PossibilityOf low amount of CO2 enrichment and reduction ofscreens retention time in wastewaters to be treated arefavorable contributions to remember. Among numer-ous advantages of microalgal immobilization for ter-tiary wastewater treatment, microalgae immobilizedon screens where cells were starved in air saturatedat 100% relative humidity, can open the extrapola-tion and application at an industrial scale because ofthe possibility to harvest the biomass in a very simpleway. In fact, recovery of microalgae, one of the factorsthat slows down the development of several process-es can be done in this case by a scraping mechanicalsystem. Thus, the same screens can be used sever-al times after reconditioning. Another advantage ofscreens over bead-shaped particles is the non-existenceof gel breakage by collisions caused by air bubbling.Shading is reduced by spacing screens during starva-tion times, allowing maximal light activation of allcells.

From the results at different temperatures, it seemspossible to maximize the efficiency of nitrogen and

95

phosphorus elimination by selecting endogenous algaeto meet varying climatic conditions and physiologicalneeds. The duration of starvation depends on deter-mined objectives to eliminate all nitrogen or phospho-rus. The bioelimination of nitrogen takes place perfect-ly well with a conditioning of nutrient during 54 h at alltemperatures, that of phosphorus is dependent on tem-perature. The total elimination of phosphorus remainsto be improved. Considering these observations, theoptimization of a biotreatment system on screens willbest be performed with a gel able to immobilize themicroalgae on the support as long as possible.

Acknowledgments

We thank Mr Claude Talbot for many stimulating dis-cussions and for technical assistance. Support fromAlpha Biotech Inc. in the form of a scholarship toValentino M. Kaya is gratefully acknowledged.

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