Simultaneous removal of inorganic nutrients and organic carbon by

Biotechnology and Bioprocess Engineering 20: 1114-1122 (2015) DOI 10.1007/s12257-015-0421-5

RESEARCH PAPER

Simultaneous Removal of Inorganic Nutrients and Organic Carbon by Symbiotic Co-culture of Chlorella vulgaris and Pseudomonas putida Ghulam Mujtaba, Muhammad Rizwan, and Kisay Lee

Received: 30 June 2015 / Revised: 8 September 2015 / Accepted: 5 October 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract The co-culture system of photosynthetic microalgae Chlorella vulgaris and aerobic heterotrophic bacteria Pseudomonas putida was investigated as a possible combination of symbiotic mixed culture for the simultaneous removal of nutrients (ammonium and phosphate) and organic contaminants. Using synthetic municipal wastewater, the co-culture system exhibited symbiotic enhancement in the removal of nutrients and organic carbon compared to each of axenic cultures. The co-culture system performed successfully in removing both of ammonium and chemical oxygen demand (COD), showing around 80% removal for 4 days. Strategies of nitrogen and phosphorous starvation in C. vulgaris for two days prior to main treatment did not increase the performance of nutrients removal, indicating that the nutrient starvation as a pretreatment is unnecessary. Without alkalinity (as bicarbonate), nutrients and COD were not removed significantly, implying that the existence of alkalinity is essential for symbiotic treatment of both nutrients and organics. Results demonstrated that coculture system composed of C. vulgaris and P. putida can be a potential candidate of mixed culture system for the simultaneous removal of nutrients and organic carbon in wastewater treatment using a single reactor. Keywords: Chlorella vulgaris, Pseudomonas putida, symbiotic co-culture system, nutrients removal, COD removal, wastewater treatment

Ghulam Mujtaba, Muhammad Rizwan, Kisay Lee* Department of Environmental Engineering and Energy, Myongji University, Yongin 449-728, Korea Tel: +82-31-330-6689; Fax: +82-31-336-6336 E-mail: [email protected]

1. Introduction The presence of inorganic nutrients, such as nitrogen and phosphorous, in municipal wastewaters are responsible for eutrophication of natural water bodies. These nutrients should be removed from the wastewaters before they are discharged into the aquatic systems in order to prevent algal blooming. Nutrient removal is a complex and costly process which involves several steps with different conditions. Therefore, developing cost effective and efficient technologies for one-step tertiary treatment of wastewater has a high priority [1]. Biological nutrient removal (BNR) systems have been extensively used to treat wastewater containing nitrogen and phosphorus. Anaerobic-anoxic-oxic (A2O) process is the most commonly used standard method, from which many BNR methods have been derived. A2O-related processes require at least three bioreactors (anaerobic, anoxic, and aerobic) in series with relatively different operating conditions and complicated internal recycle system. Phosphate removal is achieved through anaerobic-anoxic coupling. Nitrification and denitrification takes place in aerobic and anoxic reactors, respectively. Aerobic reactor is responsible for organics removal. Since denitrifiers and phosphate-accumulating microorganisms need organic carbon source to complete the reactions, the availability of biological oxygen demand (BOD) is often a vital limiting factor for the removal of nitrogen and phosphorous [2]. Furthermore, methanol is usually added as a supplement carbon source in conventional BNRs. Microalgae have a high potential to remove inorganic nutrients from the wastewater [3]. The main mechanism of algal nutrient removal from wastewater is uptake of them into the cell biomass [4]. The major advantages of using microalgae for nutrients removal are the possibility of

Simultaneous Removal of Inorganic Nutrients and Organic Carbon by Symbiotic Co-culture …

recycling the assimilated nitrogen and phosphorus into algal biomass as a fertilizer, which enables us to reduce sludge handling problem, and the generation of oxygenated effluent, as a result of photosynthesis, into the water body. In addition, the process has no requirement of organic carbon for nutrient removal if autotrophic metabolism of algae is utilized [5]. Therefore, microalgal process can be employed as an alternative tertiary treatment system for the removal of nutrients from wastewater [6]. The co-culture system comprising of microalgae and bacteria is drawing a huge attention recently as an alternative biosystem for the treatment of municipal wastewater as well as industrial wastewaters [7-9], because heterotrophic metabolism of aerobic bacteria and algal capabilities of nutrient assimilation and photosynthetic oxygenation can be mutually symbiotic if the growth and maintenance of two different microorganisms are compatible. Municipal wastewater contains both organic carbon and nutrients that can be assimilated into biomass of microalgae and bacteria. Aerobic heterotrophic bacteria are responsible for oxidative degradation of organics (or removal of BOD) and producing CO2 in conventional biological treatment systems (such as activated sludge process). Photoautotrophic microalgae assimilate nitrogen and phosphorous as their biomass constituents and also consume CO2 or its dissolved forms (such as bicarbonate) as a carbon source. Through photosynthetic carbon fixation, inorganic carbons are converted into carbohydrates or lipids, and accompanied O2 generation is utilized by aerobic bacteria as an electron acceptor [3,10]. Therefore, if aerobic heterotrophic bacteria and autotrophic microalgae are carefully chosen and co-cultured successfully, organics and nutrients can be removed simultaneously in a single reactor system. Green microalgae Chlorella sp. have fast growth rate and short reproduction time [11]. Chlorella sp. have exhibited a good potential in wastewater treatment because they can tolerate the rigorous environmental conditions found in municipal wastewater and some other industrial wastewaters, and efficiently assimilate nitrogen and phosphorus from the wastewaters [12]. Pseudomonas sp., heterotrophic aerobic bacteria which are popularly found in activated sludge process, have a good BOD removal capability and are known to stimulate the growth of Chlorella [13]. The performance of nutrient removal is generally influenced by nutrient availability, wastewater characteristics and activity of involved biomass. It has been reported that the performance of nutrient uptake can be enhanced by controlling nutrient availability, such as the existence of nutrient starvation stress prior to main treatment step [14,15]. Among wastewater characteristics, alkalinity is one of the factors that affect pH and inorganic carbon source for microalgae, which in turn influences biomass

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activity and nutrient uptake [16,17]. The motivation and purpose of using microalgaebacteria co-culture system in this study was to develop a simplified wastewater treatment process through removing nutrients and organics simultaneously in a single reactor. In this study, as a first time, co-culture system of Chlorella vulgaris and Pseudomonas putida was used for the removal of nitrogen, phosphorous, and organic carbon in one reactor. The efficiency of removing nitrogen and phosphorous by Chlorella and COD (or BOD) by Pseudomonas may be mutually enhanced by symbiotically mechanisms. A comparison was made among axenic C. vulgaris, axenic P. putida, and co-culture to eliminate the nutrients and COD. The effects of nutrient starvation and the existence of alkalinity were investigated in order to enhance the removal efficiency. The stability of the co-culture system was also examined through a semi-continuous treatment of wastewater.

2. Materials and Methods 2.1. Microorganisms The freshwater green alga Chlorella vulgaris AG30007 was obtained from Korea Collection for Type Culture (KCTC) of Korea Biological Resource Center (Daejeon, Korea). C. vulgaris was cultivated routinely using the BG-11 medium [18] at 25°C with continuous illumination of 50 µmol/m2/sec using fluorescent lamps. A bubble- column photobioreactor was used [19] in order to achieve faster growth rate by supplying 5% (v/v) CO2 and light intensity of 100 µmol/m2/sec before wastewater treatment. The gram-negative aerobic bacterium Pseudomonas putida ATCC 17514 was used in this study and cultivated routinely in LB-broth at 30°C prior to the use in wastewater treatment. 2.2. Synthetic wastewater Synthetic wastewater, similar to general municipal wastewater composition, was prepared as (mg/L): 190 NH4Cl, 40 K2HPO4, 15 KH2PO4, 275 NaHCO3, and 450 glucose, so that initial values were total nitrogen 50, total phosphorous 10, alkalinity 200, inorganic carbon 50, and COD 490. Prepared wastewater medium was autoclaved at 121°C for sterilization. The initial pH of synthetic wastewater was 8.8. 2.3. Experimental setup After the individual growth of microorganisms, each of microalgal and bacterial cells was centrifuged (3,000 rpm for 5 min) and washed with deionized water three times to remove residual nutrients. For the co-culture system, C. vulgaris and P. putida were mixed together, in which their initial cell densities were 2 × 106 and 1 × 105 cells/mL,

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Biotechnology and Bioprocess Engineering 20: 1114-1122 (2015)

respectively. Experiments were carried out in 500 mL Erlenmeyer flasks with 400 mL working volume. All the cultures with axenic C. vulgaris, axenic P. putida, and coculture were incubated under normal microalgal growth conditions (such as 25°C and 50 µmol/m2/sec) in a shaking incubator without external aeration and CO2 supply. 2.4. Analytical methods Samples were filtered out first through a 0.45 µm filter paper for the measurements of NH4-N, NO3-N, NO2-N, PO4-P, and COD. All the analyses were made according to the Standard Methods [20], in which ammonium concentration was determined based upon the salicylate method, phosphate measurement based upon the molybdovanadate method, and analysis of COD based upon the K2Cr2O7 oxidation using the commercial kits (Hach, Loveland, CO, USA). Microalgal cell concentrations were determined by Neubauer haemocytometer counting. Bacterial concentrations were estimated as colony forming unit (cfu) on nutrient agar plates.

3. Results and Discussion 3.1. Nutrients and COD removal Removal of nutrients and COD from the synthetic wastewater was carried out in three culture systems: axenic C. vulgaris, axenic P. putida, and co-culture (composed of C. vulgaris and P. putida). Fig. 1A shows the comparison of ammonium removal in three culture systems. Results show that axenic P. putida did not contribute much in removing nitrogen. A substantial amount of nitrogen was removed in axenic C. vulgaris culture (about 75% of removal in 8 days) compared to axenic P. putida. Co-culture system exhibited a better performance of nitrogen removal, showing 75% removal in 5 days and 80% in 8 days. It has been reported that no significant quantity of nitrogen was removed by other heterotrophic bacteria [21]. And the extent of nitrogen removal may be increased by employing co-culturing of algae with appropriately selectedbacterium compared with the culture of algae alone. Known examples are Bacillus licheniformis and Azospirillum brasilense with C. vulgaris [21,22]. In our study, 80% of nitrogen reduction was accomplished in 8 day treatment with an initial concentration of 40 mg N/L. C. vulgaris is known to be very effective in removing nitrogen as < 22 mg N/L [5]. Ammonia can be normally converted to nitrite or nitrate in aerobic treatment system through bacterial nitrification and then the resulting nitrate is removed as nitrogen gas through reductive denitrification in anoxic reactor. Therefore, the contents of nitrate or nitrite should have been increased at least temporarily in aerobic conditions as a consequence

Fig. 1. Changes in (A) ammonium, (B) phosphate, and (C) COD by axenic C. vulgaris, axenic P. putida, and co-culture of C. vulgaris and P. putida.

of ammonia removal, if a certain level of nitrification exists. However, the levels of nitrate and nitrite steadily decreased from their initial values in all three cases of our experiments (data not shown), which indicated that the removal of ammonium in axenic algae reactor and in coculture reactor was not by the conventional nitrification-


denitrification mechanisms. It can be concluded that algal assimilative uptake was the only mechanism of nitrogen removal in this study and that nitrogen was consumed by C. vulgaris regardless of the possible forms of inorganic nitrogen (NH4+, NO3-, or NO2-). The ammonium in aqueous solution is possible to be removed by stripping if a strong aeration is applied at high pH [10]. In our system, aeration was not strong enough to promote ammonia stripping, as only flask-shaking was used. Furthermore, the pH values of culture decreased throughout the cultivation time, and the pH of co-culture system dropped below 7 after 1.5 days (Fig. 2B). Therefore, the loss of ammonia due to stripping effect was negligible and the current nitrogen removal was achieved only by algal uptake in our experimental systems. In Fig. 1A, the reduction of NH4-N in Chlorella culture was 30 mg as N/L for 8 days. The increase of Chlorella biomass during the same period was approximately 0.5 × 106 cells/mL (Fig. 2A), which corresponded to ~ 500 mg/L as dry cell weight. The fraction of N in microalgae biomass is approximately 6.6% since its molecular formula is CO0.48 H1.83N0.11P0.01 [23]. Therefore, this mass balance indicates that the removed N from the medium was mostly converted to Chlorella biomass and the fractions of stripping loss of ammonia or conversion of N to bacterial biomass were negligible. Fig. 1B shows the removal of phosphorous by three culture systems. Axenic P. putida could not remove phosphorous, and the level of phosphorous was even increased during the treatment. Comparatively, axenic C. vulgaris and coculture performed well in phosphorous removal. Coculture system was removing a little more phosphorous than that of axenic C. vulgaris in earlier stage of treatment. However, phosphorous removal performance of axenic microalgal culture became better after 3 days. It seemed that the presence of P. putida played a role in increasing phosphorous level during the treatment. At the end of treatment (after 8 days), axenic C. vulgaris and co-culture systems removed 70 and 60% of phosphorous, respectively. Although this increment of phosphate in P. putida culture was insignificant as only 1 ~ 2 mg/L of P, somehow phosphate level did increase after certain period of culture (Fig. 1B). Experiments were conducted more than three times and the same results were observed repeatedly. It may be happened that some bacterial species uptake phosphate in aerobic environment, while the stored phosphates can be released from the bacteria into wastewater medium usually under anaerobic environment [24-26]. In this study, since external aeration was not provided except microalgal photosynthesis, dissolved oxygen level dropped near to zero (Fig. 2B) and thus it seemed that P. putida released phosphate to the medium. Because P. putida released P in

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Fig. 2. Changes in (A) cell population and (B) corresponding DO and (C) pH in C. vulgaris, P. putida and co-culture reactors.

anaerobic environment, the co-culture showed higher P level than the case of axenic C. vulgaris, after 4 days (Fig. 1B) in which DO level was completely consumed. Nurdogan and Oswald (1995) pointed out that abiotic phosphorous removal can take place mainly in the form of orthophosphate precipitation at high pH such as 9 ~ 11 [27]. The pH range in our system was sufficiently low as mentioned earlier (Fig. 2B) and thus it can be concluded

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that the major sole mechanism for phosphorous removal was microalgal uptake. The performances of COD removal by three culture systems are compared in Fig. 1C. The COD removal rate was highest in the co-culture reactor, which displayed 80% removal in 3 days. In axenic P. putida culture, compared to axenic C. vulgaris culture, COD removal rate was faster at early period. However, the final level of COD removal was relatively higher in C. vulgaris. The mechanism of COD removal by P. putida would be oxidative degradation through heterotrophic metabolism for energy and carbon usages, which is the general mechanism of aerobic biological treatment. The reason for COD reduction by C. vulgaris would be because of mixotrophic metabolism that took place in the presence of light and organic carbon. Microalgae normally follow photoautotrophic metabolism to acquire energy and carbon in the presence of light and inorganic carbon like CO2. However, some microalgal species have the ability to switch trophy metabolism from autotrophic to heterotrophic or mixotrophic, depending upon the environmental conditions [28,29]. Heterotrophic metabolism usually turns on in the dark when organic carbons are available, while mixotrophic metabolism can be activated in the light and in the presence of both organic and inorganic carbons. Chlorella vulgaris has been known to change its trophy metabolism (including mixotrophic behavior) depending upon the availability of carbon sources and environmental conditions for growth [30-32]. Fig. 1C indicates that the present C. vulgaris strain has a good ability of metabolizing organic carbons (mainly glucose in this study) and its trophy category is mixotrophic since both glucose and bicarbonate existed and irradiation was also supplied. Conclusively, the results in Fig. 1 showed that the coculture system of C. vulgaris and P. putida exhibited better performance in both nutrients and COD removal than each of axenic cultures. This symbiotic enhancement of treatment performance can be explained by mutual and reciprocal benefits between these two species. Microalgae generate oxygen which is utilized as an electron acceptor by the bacteria. Meanwhile, CO2 is produced by bacteria through aerobic oxidation and is consumed by microalgae as a carbon source. The mutual symbiotic behaviors are possible either by the elaborated selection of compatible species or by utilizing bacterial consortium like activated sludge [33].

that of co-culture system. In the co-culture reactor, cell population of C. vulgaris was decreased at first day, and then increased and reached the similar (slightly better) level of growth rate compared to axenic culture. The temporary decrease of microalgal population in the coculture system implies that they need an adaptation period in the new medium and co-culture environment. Fig. 2A also shows the growth of P. putida in axenic culture and in co-culture system. P. putida in the co-culture system showed a quicker adaptation till its active growth than axenic culture in first 4 days, but, it was decreased to lower level than that in axenic culture at later period. However, the order of magnitude of cell population was kept constant, indicating that the variation of cell population was not significant as shown in Fig. 2. Although both microorganisms did not promote the growth of the other member significantly in co-culture system, it is obvious that each microorganism did not suppress the growth of other member in the current coculture system, showing cell populations of both species increased from their initial concentrations and maintained a steady value after a certain period. It was assumed that molecular oxygen produced by C. vulgaris provided an aeration effect which was beneficial to P. putida. Fig. 2B shows the dissolved oxygen (DO) values for all the culture systems. A quick increase in DO (exceed 8.0 from initial 2.5 mg/L), as a result of photosynthesis, was observed in axenic C. vulgaris reactor. This change clearly corresponded to the gradual increase in population of C. vulgaris (Fig. 2A). DO value in axenic P. putida reactor decreased to zero quickly because no sufficient aeration was provided and thus DO was completely consumed by the aerobic heterotrophic metabolism of P. putida. In the co-culture reactor, DO level was remained almost constant at a little lowered value from the initial one, indicating that some fraction of the oxygen produced by C. vulgaris was consumed by P. putida. Variations in pH by axenic C. vulgaris, axenic P. putida, and co-culture are shown in Fig. 2C. The pH was decreased under all tested culture systems. The extent of pH drop was largest in the co-culture system and least in axenic C. vulgaris culture. Similar tendency was also observed in the case of co-culture with C. vulgaris and B. licheniformis [21]. The pH drop in the axenic culture of P. putida is because organic acids are formed in the middle of oxidative degradation of organics by aerobic heterotrophs. The reason for pH drop in Chlorella-involved cultures is probably due to the consumption of ammonium ions [34].

3.2. Changes in cell population Changes in cell population of each axenic cultures and co-culture system are shown in Fig. 2. Cell density of C. vulgaris increased in both systems from their initial concentrations as shown in Fig. 2A. The cell population of C. vulgaris in axenic culture was maintained higher than

3.3. Effect of nutrient starvation Because it is known that having certain period of nutrient starvation stress before exposing cells to treatment process


may helpful for enhancing nutrient removal performance in some microalgae species [14,15,35], the effectiveness of nutrient starvation in our co-culture system was investigated as a pretreatment. Microalgal cells were starved to nitrogen and phosphorous prior to nutrients removal experiment; fully grown cells were incubated for 2 days in BG-11 medium deleted of nitrogen and phosphorous. After that, cells were washed three times with 0.85% sterile NaCl solution. Starved microalgae were inoculated into both axenic and co-culture systems. Fig. 3 shows the performances of starved and non-starved C. vulgaris in axenic and co-culture systems in order to remove nitrogen and phosphorous. Although non-starved C. vulgaris in both systems removed more nitrogen than starved ones as shown in Fig. 3A, the differences observed were not significant. It indicates that the starvation pretreatment was unnecessary in C. vulgaris and the present co-culture system with P. putida. Similarly, the efficiency of phosphorous removal could not increase in starved cells either (Fig. 3B). Comparatively,

Fig. 3. Effect of nutrient starvation on the removal of (A) ammonium and (B) phosphate by axenic C. vulgaris and co-culture.

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more phosphorous was removed by non-starved C. vulgaris in both axenic and co-culture systems. Although nutrient demand would be high to starved cells, however, nutrient starvation probably caused the loss of overall cellular activity and thus would responsible for the lowered removal of nitrogen and phosphorous. 3.4. Effect of alkalinity The existence of bicarbonate in wastewater may influence the cell growth and its nutrient removal performance [16,17]. First, the effect of absence and presence (200 ppm as HCO3-) of alkalinity was compared on the performance of nutrients removal by three culture systems. Nitrogen was not removed in any case by axenic culture of P. putida (Fig. 4A). Without alkalinity, C. vulgaris in neither axenic nor co-culture system removed nitrogen. When alkalinity was present, C. vulgaris in both systems removed nitrogen significantly. Highest removal of nitrogen was obtained in the co-culture system with alkalinity. Similar tendency was also observed in phosphorous removal (Fig. 4B). Phosphorous was not removed by any system if alkalinity was not present. In both axenic and co-culture systems, C. vulgaris significantly removed phosphorous in the presence of alkalinity. These results indicate that alkalinity is an important factor for enabling C. vulgaris to uptake nitrogen and phosphorous. COD levels were significantly reduced by all tested systems if alkalinity was present (Fig. 4C), indicating that alkalinity also plays an important role in bacterial activity of organics oxidation. Some extents of COD were also reduced from the wastewater without alkalinity. However, the extents of COD reduction were relatively small when alkalinity was absent. Highest COD removal was achieved by the co-culture system when alkalinity existed. Next, the effects of variations in alkalinity concentrations (0 ~ 250 ppm) were investigated on nitrogen and COD removal by the co-culture system. Fig. 5A shows that nitrogen was successfully removed under the range of 50 ~ 250 ppm alkalinity and 100 ~ 150 ppm seemed optimal. Similar results were found in COD removal too, as shown in Fig. 5B, and the highest removal was achieved at 50 ~ 100 ppm alkalinity. These results demonstrated that a certain amount of alkalinity is necessary in order to remove nutrients and COD simultaneously in the current co-culture system. 3.5. Semi-continuous treatment In order to investigate the stability of the algal-bacterial coculture system in treatment process, four cycles of semicontinuous treatment were run. After one cycle of treatment, cells were harvested from the spent medium (treated wastewater) by centrifugation and re-suspended in fresh

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Fig. 5. Effect of different alkalinity concentrations on (A) ammonium and (B) COD removal in the co-culture system.

Fig. 4. Effect of alkalinity on the removal of (A) ammonium, (B) phosphate, and (C) COD by microalgal, bacterial, and co-culture systems.

synthetic wastewater. Fig. 6 shows the removal of nutrients and COD in the semi-continuous treatment. In Fig. 6A, as expected from previous results, no significant removal of nitrogen was achieved in the treatment with axenic P. putida in all the cycles. C. vulgaris was capable to remove nitrogen successfully up to four cycles in both axenic and co-culture systems.

Fig. 6B shows that axenic P. putida did not reduce the concentrations of phosphorous till last cycle (similar performance as shown for nitrogen removal). C. vulgaris in both systems removed a substantial amount of phosphorous in comparison with the performance of P. putida. However, the removal efficiency was gradually reduced after first cycle in both axenic culture and co-culture systems. Overall, C. vulgaris in axenic culture exhibited better performance of phosphorous removal than co-culture system. Other studies mentioned that the relatively lower phosphorous removal efficiency in algal-bacterial consortium is probably due to the fact that nitrogen or other essential micronutrient can be limiting [36]. The optimal ratio for maximum nitrogen and phosphorus uptake by algal-bacterial culture is N:P = 30:1 [37]. However, the ratio was 4:1 in our study. Fig. 6C shows the COD removal. In each cycle, significant amounts of COD were reduced by all culture systems. However, comparatively, co-culture exhibited the best performance. In the co-culture system, both C. vulgaris and P. putida would have made their contribution to the removal of COD as discussed earlier. In other studies, the


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maintenance of the co-culture community and their removal performance in repeated-batch mode is necessary for the basis of later study on continuous treatment. The use of P. putida and C. vulgaris was proved to be a successful combination of consortium in synthetic wastewater. Ammonium, phosphate and glucose were removed simultaneously without loss of both microorganisms. Our ultimate goal is to develop an improved and simplified treatment system for the treatment of real wastewater. In real wastewater, it is expected that the characteristics of N and P removal would be similar because C. vulgaris are able to uptake ammonia, nitrate and some organic nitrogen. However, it is highly possible that COD removal behavior would be different from the present study, because COD constituents in real wastewater are not glucose and some of them are non-biodegradable. In order to increase COD removal performance in real wastewater, development of a co-culture system with C. vulgaris and activated sludge (hopefully containing P. putida) would be necessary.

4. Conclusions The co-culture of C. vulgaris and P. putida was proved to be effective to remove both nutrients and COD from synthetic wastewater in a single reactor system. Nutrient assimilation accompanied by photosynthetic oxygenation of microalgae and oxidative degradation of organics accompanied by CO2 generation of aerobic heterotrophic bacteria are mutually beneficial. Strategies of nutrient starvation as a pretreatment had no significant role in increasing performance. Alkalinity was considered to be an important factor for maintaining nutrient removal activity. The simultaneous removal of inorganic nutrients and organic carbon by the co-culture system of C. vulgaris and P. putida in the semi-continuous operation was also quite stable for at least four cycles of treatment.

Acknowledgement

Fig. 6. Semi-continuous operation of (A) ammonium, (B) phosphate, and (C) COD removal by axenic C. vulgaris ( ●), axenic P. putida (▼), and their co-culture ( ■ ).

COD removal efficiencies with only algae and only activated sludge were much lower than those of the cultures containing both, representing the importance of cooperation between algae and bacteria [38]. The treatment mode in Fig. 6 is a sort of repeated-batch process or sequential batch reactor process. The successful

This research was supported by a grant from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Korea.

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