Biodegradation of wastewater pollutants by activated sludge coimmobilized with Scenedesmus obliquus

Biodegradación de contaminantes de aguas residuales por lodos activados coinmovilizado con Scenedesmus obliquus

 Alejandro Ruiz–Marin^*, Yunuen Canedo–Lopez, Silvia del C. Campos–Garcia, Mirna Y. Sabido–Perez y José del C. Zavala–Loria

Facultad de Química, Universidad Autónoma del Carmen. 24180. Avenida Concordia Esquina Avenida 56 No. 4. Ciudad del Carmen, Campeche, México. * Autor responsable.(aruiz@pampano.unacar.mx).

Received: April, 2012.
Approved: March, 2013.

Abstract

Wastewater treatment plants produce large quantities of biomass (sludge) that require about one–third of the total investment and plant operation costs for their treatment. With microbial immobilization and coimmobilization with microalgae, it is possible to handle high cell concentration in the column reactor and increase its efficiency. The experimental design consisted in immobilized and coimmobilized S. obliquus with activated sludge in alginate beads cultured under continuous light or photoperiods (12 h/12 h) in artificial wastewater medium to evaluate the growth, nutrients and biochemical oxygen demand (BOD) removal. The results showed that the microalgae S. obliquus immobilized and coimmobilized in cultures under continuous light had higher growth rates (0.624 d^–1 and 0.552 d^–1) than under photoperiods (0.456 d^–1 and 0.312 d^–1). Also, a higher cell density (4X10⁶ cells bead^–1 and 2.9X10⁶ cells bead^–1) was obtained under continuous light than under photoperiods (2.0X10⁶ cells bead^–1 and 1.0X10⁶ cells bead^–1). The removal of nitrogen by S. obliquus, immobilized (92 %) and coimmobilized (60 %) under continuous light, was higher (40 %) than under photoperiods. Also, 43 % of BOD was removed by the coimmobilized system under continuous illumination. The coimmobilization system favored growth of algae and bacteria under continuous light, suggesting a possible exchange of gases (CO₂ and O₂): oxygen produced by microalgae and CO₂ by bacteria.

Key words: activated sludge, coimmobilization, growth, Scenedesmus obliquus, nitrogen and organic matter removal.

Resumen

Las plantas de tratamiento de aguas residuales producen grandes cantidades de biomasa (lodo) que requieren alrededor de un tercio de la inversión total y los costos de operación de la planta para su tratamiento. Con la inmovilización microbiana y la coinmovilización con microalgas, es posible manejar una concentración alta de células en el reactor de columna y aumentar su eficiencia. El diseño experimental consistió en inmovilizar y coinmovilizar S.obliquus con lodos activados en esferas de alginato cultivadas con luz continua o fotoperíodos (12 h/12 h) en un medio artificial de aguas residuales para evaluar el crecimiento, los nutrientes y la eliminación de la demanda bioquímica de oxígeno (DBO). Los resultados mostraron que las microalgas S. obliquus, inmovilizadas y coinmovilizadas en cultivos bajo luz continua, registraron tasas de crecimiento más altas (0.624 d^–1 y 0.552 d^–1) que bajo fotoperíodos (0.456 d^–1 y 0.312 d^–1). Además, se obtuvo una densidad celular mayor (4X10⁶ células esfera^–1 y 2.9X10⁶ células esfera^–1) con luz continua que con el fotoperíodo (2.0X10⁶ células esfera^–1 y 1.0X10⁶ células esfera^–1). La eliminación de nitrógeno por S. obliquus, inmovilizado (92 %) y coinmovilizado (60 %) fue más alto (40 %) con luz continua que con el fotoperíodo. También, 43 % de la DBO se eliminó por el sistema coinmovilizado con iluminación continua. El sistema de coinmovilización favoreció el crecimiento de algas y bacterias con luz continua, lo que sugiere un posible intercambio de gases (CO₂ y O₂): el oxígeno producido por microalgas y CO₂ por bacterias.

INTRODUCTION

The aerobic treatment of domestic wastewater takes place in conventional activated sludge systems. However, this process is mainly focused on removing BOD (biochemical oxygen demand) and COD (chemical oxygen demand), but nitrogenous compounds and phosphate are insufficiently removed (Pipes and Zmuda, 1997; Nagadomi et al., 2000). A high technology treatment process is therefore required to remove such compounds in the activated sludge process as nitrification and denitrification (Bitton, 1994).

Wastewater treatment plants also produce large amounts of sludge which must be separated from wastewater in the secondary clarifier and treatment to comply with Mexican environmental norms. To remove sludge from wastewater, technologies such as aerobic and anaerobic digestion, and centrifugation are used (Metcalf and Eddy, 2003). However, the drawback of these methods is that they consume about one–third of the total cost of investment and operation of the wastewater treatment plant (Martínez and Bustos, 2009). New and more efficient biotechnology for wastewater treatment to remove pollutants and control sludge production are needed. Immobilization of bacteria and microalgae has been studied, and they have several benefits over free cells for the treatment of domestic wastewater including increased conversion rate, lower growth rates, higher cell concentration, unnecessary cell separation, and elimination of washout possibility (Tam et al., 1994; Ruiz–Marin et al., 2010).

Cell immobilization technology are applied for mammal cells cultures (Uludag et al., 2000), for production of hydrogen (Wu et al., 2003) and compounds used commercially in the food industry (Kawaguti et al., 2006). There are other studies with immobilized cells, such as in wastewater treatment to remove nutrients (N and P) with microalgae, phenol and hexavalent chromium (Bandhyopadhyay et al., 2001; Humphries et al., 2005; Ruiz–Marin et al., 2010). However, there are fewer applications in wastewater treatment involving immobilization of mixed–culture systems and coimmobilized cultures.

Cell immobilization is an option for wastewater treatment. This technology provides the possibility of efficiently incorporating symbiotic bacteria (Travieso et al., 1996; De Bashan et al., 2002), with the likely advantage of reducing sludge production (Chena et al., 2002). The interaction between two microorganisms is called coimmobilization and the associations in the same matrix can be positive with higher growth and thus better removal of pollutants. Bashan et al. (2002) report rapid growth and nitrogen and phosphorus removal capacity, as well as an increase in the pigment content of Chlorella vulgaris coimmobilized with Azospirillum brasilense in alginate (growth–promoting bacteria in plants) in cultures with artificial wastewater.

Not all associations tend to be positive, as reported by González et al. (2000) for C. vulgaris coimmobilization with Phyllobacterium myrsinacearum. However, coimmobilization of microorganisms is beneficial, stimulating production of extracellular materials such as alpha–amylase produced by Bacillus subtilis when coimmobilized with Scenedesmus obliquus (Chevalier and de la Noüe, 1988).

There is little information regarding the association of microalgae and activated sludge coimmobilized in alginate beads under autotrophic and heterotrophic pathway. The aim of this study was to determine whether the association between S. obliquus coimmobilized with activated sludge improved growth of both microorganisms and removal of nitrogen and organic material under autotrophic and heterotrophic pathways. In this study, both microorganisms were confined by coimmobilization in small alginate beads, a practical means of using microorganisms for environmental applications.

Routine algal culture and acclimatization

The microalga used was S. obliquus isolated from a hypereutrophic soil. The microalga was obtained from the culture collection of the Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Baja California, Mexico, and cultured in artificial wastewater with the following composition (mg L^–1): NaCl, 7; CaCl₂, 4; MgSO₄.7H₂O, 2; KH₂PO₄, 15 and NH₄Cl, 115. These concentrations were used simulating the mean values of the secondary effluent of a wastewater treatment plant. Trace metals and vitamins were added following guidelines for ''f/2" medium preparation (Guillard and Ryther, 1962). During acclimatization at 25 °C±2 and light intensity of 100 μE m^–2 s ^–1 (1 month), the microalgae were transferred to fresh artificial wastewater every 7 d.

The activated sludge was taken from the aerated reactor of the wastewater treatment plant, washed twice with saline solution (0.85 %) and separated by centrifugation at 2500 rpm. Sludge was cultured in medium as described by Nakashio et al. (1972), prepared with the following concentrations (mg L^–1): D–glucose, 100; KH₂HPO₄, 15; K₂HPO₄, 10; Na₂HPO₄.12H₂O, 30; NH₄ Cl, 17; MgSO₄.7H₂O, 56; CaCl₂, 10 and peptone, 150. During acclimatization at 25±2 °C and 100 μE m^–2 s ^–1 (1 month), the activated sludge was transferred to fresh medium every 24 h.

Bead preparation

After acclimatization, microalgae and activated sludge were grown in three–liter polyethylene terephthalate (PET) photobioreactors (volume of operation 2.5 L). A volume of cell suspension of known concentration in each reactor was harvested for microalgae and activated sludge, as well as for immobilization and coimmobilization.

For immobilization, cells were harvested by centrifugation at 3500 rpm for 10 min. The activated sludge and microalgae cells were resuspended independently in 50 mL distilled water. The algal and activated sludge suspension was then mixed with a 4 % sodium–alginate solution in 1:1 volume ratio to obtain a mixture of 2 % algae–alginate suspension (Tam and Wong, 2000). The mixture was transferred to a 50 mL burette and drops were formed when titrated into a calcium chloride solution (2 %). This method produced approximately 6500 uniform algal beads of approximately 2.5 mm in diameter with an initial algal cell number of 300X10³ cells bead ^–1, and bacteria number was 200 CFU 100 bead^–1 for every 100 mL of the alginate mixture. Beads were kept 4 h at 25±2 °C in the CaCl₂ solution for hardening, then rinsed with sterile saline solution (0.85 % NaCl) and subsequently with distilled water. A concentration of 2.6 beads mL^–1 of wastewater (equivalent to 1:25 bead:wastewater v/v) and an approximate volume of each bead 0.01538 mL was obtained after the immobilization.

A similar procedure was used for coimmobilization, with the difference that the concentrate of activated sludge and microalgae was mixed to form a 50 mL volume and then mixed with 50 mL of alginate (25 mL algal culture+25 mL bacteria culture +50 mL alginate). This procedure allowed retaining the same concentration of cells in all experiments.

Experimental setup and procedure

Microalgae and activated sludge were immobilized and coimmobilized and then placed in polyethylene terephthalate (PET) reactors with culture medium (2.5 L) with composition (mg L^–1):7NaCl, 20 CaCl₂, 2MgSO₄.7H₂O, 20KH₂PO₄, 120 NH₄Cl, 2 K₂HPO₄, 4 Na₂HPO₄.7H₂O, 10 D–glucose and 150 peptone.

All reactors with immobilized and coimmobilized cells were used under aerobic conditions. Dissolved oxygen concentration (OD) at 2.3 mg L^–1was maintained by supplying air at a rate of 10 mL min^–1. The supplied air also allowed the mixture within the reactors to maintain the beads distributed homogeneously. Water samples (150 mL) were collected from each reactor every 12 h for 96 h of treatment and analyzed for N–NH₄⁺ and biochemical oxygen demand (BOD₅) following the technical guidelines of Standard Methods for the Examination of Water and Wastewater (APHA, AWWA, WEF, 1998).

After dissolving one bead in 5 mL of 0.25 M of Na₂HPO₄.7H₂O solution (pH 7.0) in triplicate, the number of algal cells in the beads was counted using the Neubauer chamber (hemocytometer). To quantify bacteria, the Most Probable Number (CFU bead^–1) technique was applied, with a series of five tubes for analysis, using both probabilistic and confirmative tests (APHA, AWWA, WEF, 1998). The specific growth rate was calculated with the equation:

Where Xm and Xo are the concentrations of biomass at the end and the beginning of a batch run, respectively, and t is the duration of the run.

Statistical analysis

The effects of immobilization and coimmobilization under two culture conditions of continuous light and cycles of light/darkness (12h/12h) on growth and on nitrogen and organic material (BOD) removal were analyzed by covariance (ANCOVA; p≤0.05) with Statistical Software (StatSoft Inc., Tulsa, OK, USA). The Tukey test (p≤0.05) was applied when results showed significant differences.

RESULTS AND DISCUSSION

Algal growth and bacteria

The specific growth rates (μ) for immobilized and coimmobilized S. obliquus cells showed significant differences (ANCOVA, p≤0.032). A higher algal rate (μ) in immobilized culture (LCM: 0.624 d^–1) was obtained in reactors with continuous illumination with respect to coimmobilized culture (LCC: 0.552 d^–1).

The Tukey test showed that, under conditions of photoperiods μ of S. obliquus in LDM and LDC, there were no significant differences (p>0.053), but, because of the effect of the photoperiod, μ was lower (0.456 d^–1 and 0.312 d^–1) than under continuous illumination (Table 1). However, μ for all treatments was higher than those reported by Jiménez–Pérez et al. (2004) and Ruiz–Marín et al. (2010) for immobilized S. obliquus (0.264 d^–1 and 0.110 d^–1) under continuous illumination.

Cell density of immobilized S. obliquus (LCM) was higher in reactors with continuous light and a maximum concentration of 4x10⁶ cells bead^–1 compared to reactors with coimmobilized cells (LCC) of 2.9x10⁶ cells bead^–1. Under photoperiod, algal cell concentration was lower for both systems, with 2.0x10⁶ cells bead^–1 and 1.0x10⁶ cells bead^–1 (Figure 1A), suggesting that the microalga has greater capacity for growth when exposed to continuous light. Although growth was low in those reactors with photoperiod, it appeared to have been uninterrupted; therefore, other factors may have contributed to increase cell density.

The increase in the number of bacteria (activated sludge) in immobilized and coimmobilized cultures showed significant differences between treatments (ANCOVA; p=0.395). The Tukey test confirmed that bacterial growth in treatment LCC was significantly different (p=0.019) with respect to other treatments, suggesting that bacteria coimmobilized with S. obliquus under continuous light (Figure 1B) had a higher cell growth (400 CFU bead^–1 to 9100 CFU bead^–1) than treatments LCB, LDB and LDC (400 CFU bead^–1 to 8000 CFU bead^–1).

According to specific growth rates (Table 1), the number of bacteria rapidly increased with respect to S. obliquus, suggesting that during the exponential phase oxygen content was sufficient to sustain bacterial growth. Coimmobilization under continuous light was beneficial for the bacteria since the number of colonies increased. There are similar results for S. obliquus coimmobilized with B. subtilis with an increase in the number of bacteria by a factor of about 500 and the number of algae only by five during the experiment (48 h) (Chevalier and de la Noüe, 1988).

In the present study oxygen was an important factor for bacterial growth in coimmobilized systems. That is, the cells located in the center of the bead may have major limitations in terms of substrate and oxygen, implying greater growth on the surface (Hill and Khan, 2008). However, coimmobilized bacteria did not exhibit these limitations. It is likely that oxygen produced by the microalgae was distributed within the bead and maintained the levels necessary to sustain growth and degradation of organic matter.

Although microalga can change its metabolism from autotrophic to heterotrophic and can utilize inorganic and organic carbon (Mandal and Mallick, 2011), results from the present study indicated that this was not possible. To achieve this, the alga needs time to adapt to such conditions in order to change its metabolism to heterotrophic.

Results from this study show that the coimmobilization system favored growth of both organisms under continuous light, suggesting a possible exchange of CO₂ and O₂, as reported by Chevalier and de la Noüe (1988) for Scenedesmus sp. coimmobilized with B. subtilis.

Consumption of nitrogen and organic matter

Most of the studies about using microalgae for wastewater treatment are based on the use of monoculture to remove specific nutrients, mainly nitrogen and phosphate; only a few studies report using mixed algal cultures for wastewater treatment (Gantar et al., 1991). One area of interest is to study immobilized systems for nutrient removal since these systems incorporate two microorganisms in the same matrix (coimmobilization). This technology can increase in biomass and improve nutrient removal in wastewater. González and Bashan (2000) report significant growth and higher pigment content in C. vulgaris when coimmobilized with A. brasilense. In addition, coimmobilization achieved greater removal of ammonia (100 %) than immobilized monoculture cells (35 %). Chevalier and de la Noüe (1988) coimmobilized the microalga S. obliquus with the bacterium B. subtilis, and conclude that the coimmobilization system favored bacterial and algal growth and high production of alpha–amylase.

Wastewater contains a variety of nutrients and a single strain cannot simultaneously remove all of them. In activated sludge and pond stabilization processes, a complex mixture of microorganisms is found, and their proportions vary with composition of wastewater and treatment stage. The association of these populations of bacteria and photosynthetic organisms occurs to some extent with the exchange of nutrients and gases (oxygen and CO₂); therefore, it is possible to select a good combination of organisms for simultaneous removal of nutrients from the wastewater. However, because not all associations are positive, it is important to assess the relationship between the two microorganisms in coimmobilized systems (González et al., 2000).

In the present study, nitrogen removal showed significant differences among treatments (ANCOVA, p=0.002). Half of the nitrogen was removed in treatments LDM, LCB and LDB but there were no significant differences (Tukey, p=0.32). The most efficient removal (from 30 mg L^–1 to 2.8 mg L^–1; 92 %) was obtained with immobilized S. obliquus (LCM) after 100 h of treatment (Figure 2A). Also, reactors with coimmobilized cells (LCC) eliminated more nitrogen (60 %) and continuous light (from 22.9 mg L^–1 to 9.0 mg L^–1) compared with that eliminated in reactors (32.4 %) under photoperiods (LDC) (from 22.0 mg L^–1 to 14.8 mg L^–1) after 100 h of treatment (Figure 2B).

More nitrogen was removed with continuous light treatments, similar to results reported by Chevalier and de la Noüe (1985) for S. obliquus immobilized in carrageenan (100 % NH₄⁺ removed). Moreover, Travieso et al. (1996), though with other species of immobilized microalga (C. vulgaris), report an 80 % removal in urban wastewater, and Lau et al. (1997) 95 % of NH₄⁺ removed by alginate–immobilized C. vulgaris cultivated in artificial wastewater.

It is likely that the relatively low nitrogen removal in LCC reactors compared to immobilized cells can be attributed to the contribution of nitrogen as a result of organic matter degradation. This can be observed (Figure 2B) after an increase in nitrogen during the first 48 h and subsequent decline. The relationship between nitrogen input and consumption by both microorganisms occurs in coimmobilized systems under continuous light (Figure 2B); whereas with photoperiods, a decline of algal activity occurs with a subsequent drop in the consumption of nitrogen. This suggests that the heterotrophic activity in the dark phase provides nitrogen to the culture medium, which is then consumed by the microalgae, a phenomenon that occurs in systems of wastewater treatment and pond stabilization (Munoz and Guieysse, 2006).

The consumption of organic matter (BOD) supports the fact that immobilized bacteria can consume this substrate and provide nitrogen to the medium (Figure 3). The removal of 22 % BOD (from 98 mg L^–1 to 76.7 mg L^–1) and 25 % (from 98 to 73.0 mg L^–1) for LCB and LDB, obtained in the immobilized–bacteria system was lower than that observed in the coimmobilized LCC (43 %) (from 97.9 mg L^–1 to 54.2 mg L^–1). Mandal and Mallick (2011) suggest that free cultures of S. obliquus can use organic matter in mixotrophic conditions, as Chlorella sorokiniana and Chlorella vulgaris (Lee etal., 1996; Ogbonna et al., 2000, Liang et al., 2009). Similarly, Shih–Hsin et al. (2010) conclude that in free culture of S. obliquus under light/dark cycles it is possible to achieve high cell growth, compared with growth under continuous illumination reported by de Morais and Costa (2007) and Mandal and Mallick (2009). The authors conclude that the microalga S. obliquus is capable of growing in urban wastewater and can be cultivated in the open in different seasons.

The high BOD removal (from 98 to 54 mg L^–1; 45 %) in coimmobilized cell reactors under light continuous suggests a positive association with possible exchange of oxygen produced by the microalgae through their photosynthetic activity and contribution of CO₂–gas by bacteria (Chevalier and the Noüe, 1988). Therefore, in terms of economy and efficiency, the treatment of wastewater by microalgae and activated sludge in coimmobilized systems is an option for simultaneous removal of nitrogen and organic matter with a single treatment. Furthermore, the supply of oxygen in aerobic digestion reactors can be reduced because microalgae produce oxygen, thereby maintain the heterotrophic activity of bacteria.

CONCLUSIONS

Bacteria and microalgae were not affected in their growth capacity because of the immobilization and coimmobilization in sodium alginate. The cycles of light/darkness reduce the growth capacity of S.obliquus, as well as nitrogen and BOD removal. No organic carbon was used in the immobilized and coimmobilized systems; thus, microalgae showed no ability to use organic carbon in cultures with continuous illumination and photoperiods.

The positive association between the two microorganisms (coimmobilization) simultaneously removed significant amounts of nitrogen and BOD in reactors with continuous illumination. An exchange of CO₂ may occur due to the activated sludge in the respiration processes with the subsequent supply of nitrogen during the degradation of organic matter, while the microalgae makes use of this nutrient and supplies photosynthetic O₂ within the bead and to the culture medium.

Wastewater treatment by microalgae and activated sludge in coimmobilized systems can be an option for the simultaneous removal of nitrogen and organic matter in a single stage of treatment, saving in the supply of oxygen for reactors of aerobic digestion. Further study is required to determine the compatibility of organisms in a single matrix and to explore the possibility of achieving simultaneous nutrient removal in wastewater, using the urban effluents of treatment plants to grow microalgae and obtain high value products through autotrophic and heterotrophic pathways.

ACKNOWLEDGEMENTS

The authors wish to express our gratitude to UNACAR, Mexico, for funding this study.

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