Nutrient Removal Using Algal-Bacterial Mixed Culture

Nutrient Removal Using Algal-Bacterial Mixed Culture

Vaishali Ashok, Amritanshu Shriwastav & Purnendu Bose

Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-014-1229-z

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Author's personal copy Appl Biochem Biotechnol DOI 10.1007/s12010-014-1229-z

Nutrient Removal Using Algal-Bacterial Mixed Culture Vaishali Ashok & Amritanshu Shriwastav & Purnendu Bose

Received: 22 June 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014

Abstract Simultaneous nitrate (N), phosphate (P), and COD removal was investigated in photobioreactors containing both algae and bacteria. The reactors were operated in the semibatch mode with a hydraulic retention time of 2 days. Reactors were operated in two phases, (1) with 33 % biomass recycle and (2) with no biomass recycle. In both phases, more than 90 % of N and P and 80 % of COD present in synthetic wastewaters with initial N and P concentrations of up to 110 and 25 mg/L, respectively, and initial COD of 45 mg/L could be removed. Biomass growth in reactors did not increase with the increase in initial N and P concentration in either phase. However, biomass growth was slightly more in reactors operated with no biomass recycle. In both phases, N and P uptake was greater in reactors with greater initial N and P concentrations. Also in all cases, N and P uptake in the reactors was far in excess of the stoichiometric requirements for the observed biomass growth. This “luxury uptake” of nitrogen and phosphorus by biomass was responsible for excellent nitrogen and phosphorus removal as observed. However, based on the results of this study, no advantage of biomass recycling could be demonstrated. Keywords Algal-bacterial consortia . Chlorella vulgaris . Chlamydomonas reinhardtii . Nitrates . Phosphates removal . Semi-batch reactors

Introduction Greater than 90 % of the BOD of domestic wastewater can be removed by secondary treatment [1]. However, substantial amounts of nutrients, i.e., nitrogen (N) and phosphorus (P), remain in the secondary treated effluent. Discharge of secondary treated effluent into natural water bodies may cause eutrophication [2]. Traditional processes for nutrient removal from wastewater can be time consuming, create toxic by-products, result in leakage of ammonia or nitrates, require large area, costly chemicals, organic substrate, aeration, etc. [1]. The residual BOD and nutrients in secondary treated effluent may be removed in photobioreactors using algal-bacterial mixed culture [3]. Modern photobioreactor configurations provide necessary conditions like high surface area to volume ratio, stable temperature, light availability, mixing, etc. [4] required for this purpose. Many such designs of efficient V. Ashok (*) : A. Shriwastav : P. Bose Department of Civil Engineering, IIT, Kanpur, India e-mail: [email protected]

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photobioreactors are available, e.g., tubular [5, 6], columnar, air-lift, stirred tank [7], bubble column [8, 9], vertical, horizontal, helical, etc. [10]. Current efforts in photobioreactor technology have focused on improving nutrient removal efficiencies with lower HRT provisions, with the eventual goal of applying such photobioreactors for tertiary treatment of wastewater. It has also been observed that a consortium of algae and bacteria is more efficient than algae for treatment of wastewater in photobioreactors [11]. The reason for this is the symbiotic relationship between algae and bacteria, wherein the oxygen released by algal photosynthesis is used by bacteria to mineralize organic nutrients, while carbon dioxide released by bacterial respiration is used for algal photosynthesis [12]. The main objective of the present study was to examine N and P removal by algal-bacterial mixed culture under various conditions and also comment on the possible mechanisms for such removal. Parameters like chlorophyll, polysaccharide, dissolved oxygen, dissolved organic carbon, etc. were also monitored to gain additional knowledge about various parameters that can affect the nutrient removal.

Material and Methods Algal-Bacterial Culture Water collected from an oxidation pond at IIT Kanpur, India was filtered through 1.2-μm pore size GF/C filter paper, and the concentrated biomass thus obtained was re-suspended in 25 L mineral salt medium (MSM). The MSM is a solution containing the following chemicals [12]: EDTA-Na2 0.5 g/l, H3BO3 0.114 g/l, CaCl2.2H2O 0.12 g/l, MgSO4.7H2O 0.63 g/l, FeSO4.7H2O 0.05 g/l, ZnSO4.7H2O 0.088 g/l, MoO3 0.007 g/l, CuSO4.5H2O 0.158 g/l, Co(NO3)2.6H2O 0.0049 g/l, and MnSO4.H2O 0.012 g/l. Nitrate and phosphate salts were added as nutrients. No additional organic carbon source was added. This suspension was incubated for 7 days with alternate periods of 8-h illumination and 6-h darkness. Illumination was provided using four 14 W CFL (Compact Fluorescent Lamps; Philips India). Five hundred milliliter of incubated suspension was filtered every week and resuspended in fresh MSM. After four such cycles, algal culture without any bacteria was successfully isolated, as verified with light microscopy. Chlamydomonas reinhardtii and Chlorella vulgaris were identified as the algal species in the culture. Aerobic bacteria were collected from domestic wastewater treatment plant (130 MLD) and cultured separately using dextrose as carbon source under aerated conditions. A mixed algal-bacterial stock culture prepared by mixing 20 mL each of algal and bacterial stock cultures in 300-mL MSM containing N and P as nutrients and dextrose as the carbon source. The algal and algal-bacterial stock cultures were maintained in illuminated conditions. All stock cultures renewed every 7 days with fresh medium [13]. Light Chamber The photobioreactors were maintained in a light chamber (Fig. 1) of dimension of 2 m×1 m× 0.9 m; the base plate of the chamber was capable of to and fro’ motion. The 500-mL reagent bottles used a photobioreactors were placed on this base plate to ensure complete mixing. Five CFL bulbs (100 W) were attached to the ceiling of the light chamber. The light intensity provided inside photobioreactors was 0.63 W/L as determined by an actinometrical procedure [14]. The emission spectra of CFL bulbs (data not shown) had a large overlap with the absorption spectra of the algae species used in this study. The illumination inside the chamber was controlled by a timer. The illumination strategy involved provision of alternate


Fig. 1 Pictorial representation of the reactor chamber

16-h light and 8-h dark period. Alternate light and dark periods were adopted to reduce to the photo inhibition and photo toxicity damage to algae arising out of continuous illumination [15]. The chamber was equipped with an exhaust fan to control and equalize the temperature. When required, a 100 W incandescent light bulb was put inside the chamber as a heat source to maintain temperature. Experimental Procedure Synthetic wastewater samples with four different concentrations of nutrients were prepared in the laboratory. These samples were put in the four photobioreactors and each reactor seeded with 20-mL algal-bacterial culture. The final volume in each reactor was 300 mL. The initial characteristics of the synthetic sample in each photobioreactor are given in Table 1. The photobioreactors were then put inside the light chamber and maintained for 36 days in the semi-batch mode, i.e., by removing some amount of reactor contents and adding an equal amount of feed. To prepare the feed, 20 mL of MSM was diluted to 1 l and 507.6 mg/L Na2CO3 and 504 mg/L NaHCO3 was added as alkalinity. Also, desired concentration of KNO3 was added to the feed as the N source, and K2HPO4 and KH2PO4 were added as the P source. Dextrose was added as the organic carbon source. All reactors were operated with the hydraulic retention time (HRT) for 2 days. This involved daily removal of 150 mL of the fully mixed reactor contents and its replacement with an equal volume of the feed. The algal-bacterial biomass removed each day from the reactor was, in some cases, partially recycled back the reactor. In the 50 % recycling case, 75-mL of suspension withdrawn from the reactor was centrifuged and the isolated biomass was put back in the reactor along with the fresh feed. In 33 % recycling case, 50 mL of the withdrawn suspension was centrifuged and the isolated biomass was returned to the reactor. Centrifugation was done in 60-mL vials at the rate of 3000 rpm for 10 min [16]. All reactors were maintained for a period of 36 days in the light chamber. A typical cycle of reactor operation started with the addition of daily feed. The N-P concentrations in the reactors after addition of the daily feed were approximately 70–10, 80–15, 90–20, and 110–25 mg/L, respectively. The cycle ended with the withdrawal of 150 mL of reactor contents after 24 h. For all reactors, 50 % recycling was done for the first 12 days of reactor operation, followed by

Author's personal copy Appl Biochem Biotechnol Table 1 Initial characteristics of synthetic wastewater in photobioreactors Parameter

70/10

80/15

90/20

110/25

MSM (mL/L)

20

20

20

20

Na2CO3 (mg/L)

507.6

507.6

507.6

507.6

NaHCO3 (mg/L)

504.04

504.04

504.04

504.04

KNO3 (mg/L) K2HPO4 (mg/L)

101.4 10.82

115.88 16.23

130.37 21.64

159.34 27.05

KH2PO4 (mg/L)

5.056

7.584

10.116

12.64

Temperature (°C)

11.5

11.5

11.5

11.5

pH

9.57

9.59

9.62

9.62

Dissolved oxygen (DO) (mg O2/L)

4.6

4.6

4.6

4.6

COD Added (mg O2/L)

45

45

45

45

Biomass (mg/L)

31.7

25.2

27.2

36.1

Total alkalinity (mg/l as CaCO3) Chlorophyll a (mg/L)

150 2.8

150 2.3

150 2.2

150 2.7

Polysaccharides (mg/L)

61

61

61

61

Nitrate (mg/L)

71.8

78.4

79.98

111.5

Nitrite (mg/L)

0.1

0.0

0.1

0.1

Phosphate (mg/L)

9.6

13.5

16.83

19.32

Dissolved organic carbon (DOC) (mg/L)

29.7

46.1

26.2

33.9

reactor operation at 33 % recycling for the next 12 days and reactor operation with no recycle for the final 12 days. The objective of biomass recycle was to enhance the initial biomass concentration at the start of a reactor operation cycle, such that the extent of biomass growth over the operation cycle is enhanced. Analytical Methods Temperature was measured using a clinical thermometer once a day between 10 am to 2 pm, i.e., during the dark period of reactor operation. pH was measured using pH meter (Toshniwal, India, Model CL-54). DO was measured as described in Shriwastav et al. (2010) [17] in 1-mL volume samples. Alkalinity was also determined as per standard methods (method no. 2320) [18]. Chlorophyll a was measured according to the procedures described by Sartory and Grobbelaar (1984) [19] and Porra et al. (1989) [20]. Suspended biomass concentration was measured gravimetrically. For this purpose, 0.22-μm Teflon filter (Millipore, USA) was preconditioned by heating at 105° for 2 h and cooling in a desiccator for 24 h. Next, 5 mL of the suspension was passed through the filter. The filter was then put in an oven for drying at 105 °C for 24 h and finally weighed. Following parameters were measured in filtered samples: nitrate and phosphate concentration was analyzed in ion chromatography (model—882 Compact IC Plus, Metrohm) using a standard method specified by instrument manufacturer. Sample volume used was 20 μL. COD was measured using the closed reflux method (standard method no. 5220) [18]. Total, inorganic, and organic carbon concentrations (TC, IC, and OC) were analyzed using TOC analyzer (TOC-V CPN, Shimadzu, Japan). Polysaccharides were measured as per procedure described by Dubois et al. (1956) [21].


Results Preliminary Observations All reactors were operated in the semi-batch mode with 50 % biomass recycling for the first 12 days. During this period, the biomass concentration in all four reactors increased from ∼30 and stabilized at ∼80 mg/L. The data reported in this paper pertain to the reactor operation for the next 24 days. During the first 12 days of this period, the reactors were operated with 33 % biomass recycling, while for the final 12 days, the reactors were operated with no biomass recycling. Nitrate, Phosphate, and COD Removal The N, P, and COD removal data from the four reactors are summarized in Table 2. During reactor operation at 33 % biomass recycle, average N removal in all four reactors was greater than 97 %. P removal was also greater than 97 % in three reactors. However, in the reactor with influent P concentration of ∼25 mg/L, P removal was 92 %. N removal was also greater than 97 % in all rectors during operation with no biomass recycle. P removal was also greater than 97 % in two reactors during this phase of operation. However, P removal declined to 95 and 92 % in reactors with influent P concentrations of ∼20 and ∼25 mg/L, respectively. COD removal was nearly 80 % or greater in all reactors at all phases of reactor operation. These results indicate the excellent N and P removal; along with simultaneous COD removal was possible in photobioreactors operated with algal-bacterial mixed culture. N and P in the dissolved phase was incorporated in the algal and bacterial biomass, while the COD removed was partially incorporated into bacterial biomass and partially mineralized. Daily withdrawal of biomass from the photobioreactors enabled removal of nutrients and COD from the reactor on a sustained basis. Quality of Treated Effluent The quality of treated effluent as measured in the four reactors has been summarized in Table 3. In general, the treated effluent from all reactors have low N, P, and COD concentrations, which is consistent with the excellent removals of these substances as reported earlier. The effluent polysaccharide and organic carbon concentrations are consistent with the reported COD concentrations in all cases. The effluent alkalinity and inorganic carbon concentrations were also consistent with each other. Over a reactor operation cycle, there is a net removal of inorganic carbon and hence decline in alkalinity in all the reactors. This is consistent with the growth of algal biomass in the reactors. The decline in alkalinity resulted in pH increase. Hence, pH of the effluent from the reactors was always ∼10. Temperature of the effluent was ∼10 °C. However, this relatively low temperature is unlikely to adversely affect the treated effluent quality. Maxwell et al., in 1994 [22] had conducted experiments with C. vulgaris species in photo-bioreactors with temperatures as low as 5 °C. In addition, C. reinhardtii have been reported to grow optimally at temperatures in wide environmental ranges of 15–37 °C [23]. During operation at 33 % biomass recycle condition, the initial biomass concentration in all reactors was ∼80 mg/L. The biomass concentration at the end of a cycle and hence in the effluent was ∼100 mg/L in all cases. During operation under no biomass recycle condition, the corresponding values were ∼60 and ∼90 mg/L, respectively. Surprisingly, both the initial and final biomass concentrations in the reactors did not vary appreciably with the initial nutrient

%COD removal

97.7±2.4

97.4±0.9

97.8±1.3

80/15

90/20

110/25

92.8±7.6

97.2±3.0

100±0 86.0±9.1

80.0±11.7

88.1±10.6

82.0±9.8

97.0±1.9

97.4±2.2

97.2±1.7

99.6±0.6

%N removal

%P removal

99.7±0.8

%N removal

98.0±1.3

70/10

No recycle

33 % recycle

Table 2 Photobioreactor performance

%P removal

93.5±6.4

95.7±5.8

97.3±2.9

98.6±3.3

%COD removal

84.2±6.1

78.3±8.9

82.2±6.5

86.2±8.9


mg/L

mg/L

Dissolved oxygen

Chlorophyll

10.1±0.3

mg/L

Alkalinity (as CaCO3)

–

mg/L

Inorganic carbon

mg/L °C

mg/L

Organic carbon

Biomass Temperature

mg/L

Polysaccharide

pH

95.8±10.2

mg/L

COD

7.6±1.5

7.6±5.6

109.0±17.3 10.2±1.2

143.1±14.6

11.6±5.7

15.2±3.9

8.1±4.4

1.5±1.0 0.03±0.07

mg/l mg/L

70/10

Unit

Nitrate Phosphate

33 % recycle

Parameter

Table 3 Treated effluent quality

7.1±1.3

8.2±4.6

103.4±10.0 10.2±1.2

10.1±0.3

87.5±13.7

139.9±12.8

12.1±4.9

11.7±6.4

6.4±4.4

5.0±7.2 0.0±0.0

80/15

6.6±2.4

7.1±3.8

96.5±25.5 10.2±1.2

10.1±0.2

91.6±10.2

140.5±8.0

11.5±6.2

14.2±2.5

9.9±5.3

4.8±5.9 0.2±0.2

90/20

7.0±3.1

7.4±5.7

103.2±36.7 10.2±1.2

10.1±0.3

91.7±12.9

142.2±10.7

5.3±6.6

17.6±3.9

6.3±4.1

7.0±11.1 1.6±1.6

110/25

6.3±0.9

15.0±3.0

102.6±12.7 12.8±0.4

10.3±0.2

95.8±10.2

135.2±8.9

11.9±8.3

17.1±3.6

6.2±4.0

0.2±0.4 0.1+0.3

70/10

No recycle 80/15

6.3±1.3

14.4±2.7

93.4±14.2 12.8±0.4

10.3±0.1

91.7±12.9

127.9±5.2

11.8±8.4

17.0±3.2

8.0±2.9

2.5±1.4 0.4±0.4

6.1±1.7

13.0±0.9

97.2±12.4 12.8±0.4

10.4±0.2

87.5±13.7

128.0±11.2

7.4±7.1

16.1±2.7

9.8±4.0

2.3±1.9 0.8±1.1

90/20

110/25

4.8±0.7

14.8±2.9

85.1±14.4 12.8±0.4

10.2±0.2

91.7±12.9

140.7±7.1

7.6±9.2

18.5±5.7

7.1±2.7

10.4±17.2 1.5±1.5

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Appl Biochem Biotechnol

53.6

Synthetic

Swine slurry

Ammonia (O) nitrogen

Ammonium-nitrates (A,B)

36 4.9

Secondary settled

Sludge liquor and municipal WW 906 28 Synthetic 90–110 10–25

Ammonium-nitrates (A) phosphates

Ammonium-nitrogen (A) phosphates

25–84 88–94 97–100 92–100

>99 98

88.3 64.8

59

70–90

94–100

2 days

5–7 days

1 day

8 days

10 days

7 days

8h

10 days

∼25 ∼10 91–99.9

20 days

>99

The alphabets given in the parenthesis denote nutrients removed by (A) algae, (B) bacteria, and (O) other species

Nitrates (A,B) phosphates

18.9 4.9

Total kjeldahl (A,B) nitrogen phosphate Second clarifier

117 17

Nitrate (A)

656

Phosphates

Municipal

410 200

Synthetic

Ammonium-nitrates (A) phosphates

53

Synthetic

Total (A,O) nitrogen

Mohammed et al., 2008 [28]

Aslan et al., 2006 [27]

Bich et al., 1999 [26]

Bottles

Tubular

Tubular

Stirred tank

Flasks

Present study

Akerstrom et al., 2014 [31]

Termini et al., 2011 [16]

Su et al., 2011 [30]

Wang et al., 2010 [29]

Tubular biofilm Godos et al., 2009 [5]

Membrane

Flasks

Square

Initial conc. (mg/L) Percent removed (%) Time taken Type of reactor Reference

Type of waste water

Species removed

Table 4 A comparison of present study with other similar studies


Unit

mg

mg

mg (as N)

mg (as N)

mg (as P)

mg (as P)

Parameter

Initial biomass

Biomass increase

Initial dissolved N

Dissolved N uptake

Initial dissolved P

Dissolved P uptake

Table 5 Mass balance

0.5±0.1

0.5±0.0

4.9+0.1

5.0±0.6

8.4±3.9

24.3±2.6

70/10

33 % recycle

0.6±0.0

0.6±0.0

6.0±0.5

6.3±0.6

7.8±3.4

23.7±3.9

80/15

0.6±0.0

0.6±0.0

5.9±0.4

6.2±0.5

8.0±5.3

26.1±3.5

90/20

0.6±0.1

0.7±0.0

7.3±0.7

7.7±0.7

7.9±9.0

24±2.8

110/25

0.9±0.0

0.9±0.0

4.8±0.2

4.9±0.5

10.3±3.7

20.5±1.7

70/10

No recycle 80/15

1.4±0.0

1.4±0.1

5.9±0.1

6.1±0.3

10.4±4.4

17.7±0.9

90/20

1.7±0.1

1.8±0.3

5.8±0.2

6.0±0.5

9.9±2.5

19.3±2.9

2.2±0.2

2.4±0.4

7.0±1.1

7.7±0.9

10.8±3.6

14.7±2.7

110/25

Author's personal copy

Appl Biochem Biotechnol


concentrations in the reactors. The chlorophyll a concentration in the effluent was greater when reactors were operated at 33 % biomass recycle. This is consistent with the higher biomass concentration in the effluent under these conditions. Dissolved oxygen concentrations were higher during reactor operations in no recycle condition. Considering that dissolved oxygen production is related to algal growth, higher algal biomass (total biomass production is ∼ 30 mg/L/cycle) production per cycle under no recycle conditions as compared to the 33 % biomass recycle condition (total biomass production is ∼20 mg/L/cycle) is thought to be the reason for this observation.

Discussion Literature review shows that several other studies have reported removal of N and P from a variety of wastewaters using algae or algal-bacterial mixed cultures. Some of these studies have been summarized in Table 4. Though in most studies, the time required for substantial N and P removals was several days, one study (Termini et. al., 2011) [16] does report nearly complete N and P removal by algae in tubular reactors within a day. However, the influent N and P concentrations in this study were quite low. The unique feature of the present study was that it demonstrates almost complete N and P removal from wastewater containing relatively high concentration of these nutrients. This removal was possible using an algal-bacterial culture in a photobioreactor of retention time of 2 days. Amount of biomass growth and corresponding N and P uptake in the reactors has been summarized in Table 5. During reactor operation in the 33 % biomass recycle condition, the average increase in biomass in the reactor was ∼8 mg/cycle. This increase was approximately same in all four reactors, i.e., was not influenced by differences in influent nutrient concentrations between reactors. Corresponding N and P uptake by biomass was between 5.0–7.0 mg (as N)/cycle and 0.5–0.7 mg (as P)/cycle, respectively. N and P uptake was however seen to increase with the increase in the initial N and P concentration in the reactors. This apparent disconnect between biomass growth and nutrient uptake is instructive. Further, the amount of N and P uptake as reported above is far in excess of the stoichiometric N and P requirement for the corresponding growth of biomass. The above observations indicate that the N and P incorporation into biomass as reported in this study can mostly be attributed to the “luxury uptake” of these nutrients by both algal and bacterial biomass. It has been reported that the N and P uptake by algae can be used for two purposes in the algal cell, (1) storage for future metabolic activities, and (2) for immediate biomass growth [24, 25]. Ambrose et al. 2006 [24] and Chapra et al. 2007 [25] have also reported that algae can uptake maximum 0.72 mg N/mg algae/day and 0.05–0.2 mg P/mg algae/day. Photobioreactor operation under no recycle conditions also produced results which lead to a similar conclusion. Biomass recycle was adopted to enhance the initial biomass concentration at the start of a reactor operation cycle. Results presented in Table 5 show that this objective was fulfilled and the initial biomass concentrations during photobioreactor operation at 33 % biomass recycle condition was higher than during reactor operation at no recycle condition. However, the expectation that biomass recycle would increase the extent of biomass growth in reactors was belied. Results presented in Table 5 indicate that the net biomass growth was actually higher in the reactors with no recycle. Considering that all other conditions were the same, this was probably due to higher biomass concentration in the reactors during operation with 33 % biomass recycle. This resulted in diminished light availability as a result of mutual “selfshading” of algal cells. Moreover, results presented in Table 2 clearly show that percent removals of N and P were similar in both modes of reactor operation. Thus, based on the results of this study, no advantage of biomass recycle was evident.


Conclusions The objective of the present study was to examine the extent of N and P removal by algalbacterial mixed culture in photobioreactors operated under various conditions and also to elucidate the mechanism for N and P uptake. The study resulted in the following conclusions:

&

&

& &

Synthetic wastewater samples with initial N and P concentrations of up to 110 and 25 mg/ L, respectively, and initial COD of 45 mg/L were treated in photobioreactors containing algal-bacterial mixed cultures. More than 90 % of N and P and 80 % of COD present in the synthetic wastewater could be removed when operating the reactors at a hydraulic retention time of 2 days. Biomass growth in all four reactors operating under 33 % biomass recycle condition was roughly equal. However, the N and P uptake was greater in reactors with greater initial N and P concentrations. Same observation was made during the operation of the reactors under no recycle condition. In all cases, N and P uptake in the reactors was far in excess of the stoichiometric requirements for immediate biomass growth. This “luxury uptake” of N and P by biomass was the main reason for excellent N and P removal observed during this study. Contrary to expectations, biomass growth was more in reactors operated under no recycle condition. This was probably due to diminished light availability as a result of mutual “self-shading” of algal cells when the reactors were operated under 33 % biomass recycle condition. As such, based on the results of this study, no advantage of biomass recycling during reactor operation was evident.

References 1. Arceivala, S. J., & Asolekar, S. R. (2007). 3rd Edition, 2007. Tata McGraw-Hill Publishing Company: Limited. Wastewater Treatment for Pollution Control and Reuse. 2. Olguin, E. J. (2003). Phycoremediation: key issues for cost-effective nutrient removal processes. Biotechnol Adv, 22, 81–91. 3. Campbell M. N. (2008). Biodiesel: Algae as a renewable source for liquid fuel. Guelph Engineering Journal, (1)2-7, 2008 4. Vasumathi, K. K., Premalatha, M., & Subramanian, P. (2012). Parameters influencing the design of photobioreactor for the growth of microalgae. Renew Sust Energ Rev, 16, 5442–5450. 5. Godos, I., Gonzalez, C., Becares, E., Encina, P. A. G., & Munoz, R. (2009). Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor. Appl Microbiol Biotechnol, 82, 187–194. 6. Salces, B. M., Gonzalez, M. C. G., & Fernandez, C. G. (2010). Performance comparison of two photobioreactors configurations (open and closed to the atmosphere) treating anaerobically degraded swine slurry. Bioresour Technol, 101, 5144–5149. 7. Mallick, N. (2002). Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals, 15, 377–390. 8. Carvalho, A. P., & Malcata, F. X. (2001). Transfer of carbon dioxide within cultures of microalgae: plain bubbling versus hollow-fiber modules. Biotechnol Prog, 17, 265–272. 9. Ozbek, B., & Gayik, S. (2001). The studies on the oxygen mass transfer coefficient in a bioreactor. Process Biochem, 36, 729–741. 10. Singh, R. N., & Sharma, S. (2012). Development of suitable photobioreactor for algae production—a review. Renew Sust Energ Rev, 16, 2347–2353. 11. Bratbak, G., & Thingstad, T. F. (1985). Phytoplankton-bacteria interactions: an apparent paradox? Analysis of a model system with both; competition and commensalism. Mar Ecol Prog Ser, 25, 23–30. 12. Munoz, R., Jacinto, M., Guieysse, B., & Mattiasson, B. (2005). Combined carbon and nitrogen removal from acetonitrile using algal-bacterial bioreactors. Appl Microbiol Biotechnol, 67, 699–707.

Author's personal copy Appl Biochem Biotechnol 13. Shekhar, M. (2011). Microfiltration of algal suspensions. IIT Kanpur India: Master’s dissertation. 14. Zamfirescu, C., Naterer, G. F., & Dincer, I. (2012). Actinometry measurements of photon flux to supramolecular complexes for photo-catalytic hydrogen generation. 19th World Dihydrogen Energy Conference 2012, 3–7 June, Toronto, Canada 15. Ogbonna, J. C., & Tanaka, H. (2000). Light requirement and photosynthetic cell cultivation—development of processes for efficient light utilization in photobioreactors. J Appl Phycol, 12(3–5), 207–218. 16. Termini, I. D., Prassone, A., Cattaneo, C., & Rovatti, M. (2011). On the nitrogen and phosphorous removal in algal photobioreactors. Ecol Eng, 37, 976–980. 17. Shriwastav, A., Sudarsan, G., Bose, P., & Tare, V. (2010). Modification of Winkler’s method for determination of dissolved oxygen concentration in small sample volumes. Anal Methods, 10, 1618–1622. 18. Eaton, A. D., Clesceri, L. S., & Greenberg, A. E. (Eds.) Standard Methods for the Examination of water and waste water. 19th ed., American Public Health Association, Washington DC, APHA, AWWA and WEF. 19. Sartory, D. P., & Grobbelaar, J. U. (1984). Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis. Hydrobiologia, 114, 177–187. 20. Porra, R. J., Thompson, W. A., & Kriedemann, P. E. (1989). Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta, 975, 384–394. 21. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem, 28, 350–356. 22. Maxwell, D. P., Falk, S., Trick, C. G., & Huner, N. P. A. (1994). Growth at low temperature mimics highlight acclimation in chlorella vulgaris. Plant Physiol, 105, 535–543. 23. Vitova, M., Bisova, K., Hlavova, M., Kawano, S., Zachleder, V., & Cizkova, M. (2011). Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by temperature. Planta, 234, 599–608. 24. Ambrose, R. B., Martin, J. L., & Wool, T. A. (2006). WASP7 Benthic algae—model theory and user’s guide. USA: Environmental Protection Agency. 25. Chapra, S. C., Pelletier, G. J. & Tao, H. (2007). QUAL2K: A modeling framework for simulating river and stream water quality, Version 2.07. Environmental Protection Agency, USA. 26. Bich, N. N., Yaziz, M. I., & Bakti, N. A. K. (1999). Combination of chlorella vulgaris and eichhornia crassipes for wastewater nitrogen removal. Water Res, 33(10), 2357–2362. 27. Aslan, S., & Kapdan, I. K. (2006). Batch kinetics of nitrogen and phosphorous removal from synthetic wastewater by algae. Ecol Eng, 28, 64–70. 28. Mohammed, T. A., Birima, A. H., Noor, M. J. M. M., Muyibi, S. A., & Idris, A. (2008). Evaluation of using membrane bioreactor for treating municipal wastewater at different operating conditions. Desalination, 221, 502–510. 29. Wang, L., Min, M., Li, Y., Chen, P., Chen, Y., Liu, Y., Wang, Y., & Ruan, R. (2010). Cultication of green algae chlorella sp. In different wastewaters from municipal wastewater treatment plant. Appl Biochem Biotechnol, 162, 1174–1186. 30. Su, Y., Mennerich, A., & Urban, B. (2011). Municipal wastewater treatment and biomass accumulation with a wastewater-born and settleable algal-bacterial culture. Water Res, 45, 3351–3358. 31. Akerstrom, A. M., Mortensen, L. M., Rusten, B., & Gislerod, H. R. (2014). Biomass production and nutrient removal by chlorella sp. as affected by sludge liquor concentration. J Environ Manag, 144, 118–124.