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CULTIVATION OF CHLAMYDOMONAS INCERTA IN PALM OIL MILL EFFLUENT: EFFECT OF PHOTOPERIOD AND CARBON DIOXIDE CONCENTRATION IN THE GROWTH

AND BIOMASS PRODUCTIVITY

Shahabaldin Rezania1,2, Mohd Fadhil Md Din1,2,* Mazen Abdo Alqadi2, Shazwin Mat Taib2, Hesam Kamyab3, Shreeshivadasan Chelliapan3,

Arham Abdullah4

(Author names are listed here. The correspondent author must be clearly mentioned in a footnote)1 Centre for Environmental Sustainability and Water Security (IPASA), Research Institute for Environmental Sustainability, Block C07, Level 2, Universiti Teknologi

Malaysia, 81310 Johor Bahru, Malaysia

2 Department of Environmental Engineering, Faculty of Civil Engineering, Universiti Teknologi Malaysia (UTM), 81310, Johor, Malaysia

3 Engineering department, UTM Razak School of Engineering & Advanced, Universiti Teknologi Malaysia, Malaysia

4 Department of Structure and Material, Faculty of Civil Engineering, Universiti Teknologi Malaysia (UTM), 81310, Johor, Malaysia

Abstract. Microalgae are sustainable sources of biomass for fuel, food, and feed as well as pollutant removal from wastewater. The aim of this study was to determine the optimal pH for the growth of chlamydomonas incerta (c.incerta) as well as the effect of different light/dark cycle and CO2 concentration on the biomass productivity when cultured in palm oil mill effluent (POME). In addition, the ability of C. incerta for the pollutant removal from POME was investigated. The performance was determined by measuring the optical density (OD), cell dry weight (CDW), chlorophyll content and the removal rate of COD, TN and TP for 17 days. Based on the results, light/ dark cycle influenced the growth of microalgae while by decreasing CO2 injection, the growth rate and removal efficiency increased. The maximum biomass productivity and specific growth rate for 12/12 L/D cycle were 0.04 g/L.d and 0.118 g/L.d, respectively. In addition, the maximum biomass productivity and specific growth rate for 16/8 cycle were (0.024 g/L.d and 0.093) and for 24/0 L/D (0.043g/L.d and 0.122). The removal for COD was 88%, for TN 97.3, and for TP 99.8%.

Keywords: Microalgae; Chlamydomonas incerta, biomass production, palm oil mill effluent (POME); Light/Dark cycle

1. Introduction

Algae is one of the major photosynthetic organism group beside plants and bacteria which are divided into microalgae and macroalgae based on their size. It has a great role in food and agriculture, and in exploiting microbial activities for producing valuable human products, generating energy, and cleaning up the environment (Hadiyanto and Nur, 2012; Kamyab et al, 2014a; Feng et al, 2011; Nurul-Adela et al, 2016) . Microalgae are microscopic unicellular species, prokaryotic or eukaryotic, photosynthetic microorganisms that are found in both marine and freshwater. In fact, they can be completely exposed to light so that every cell can conduct photosynthesis at full speed and double within several hours (Wiltshire et al, 2000; Sukumaran et al, 2014; Kamyab et al, 2015a). Microalgae growth rate and their simple structure have gained tremendous focus and interest from researchers and industries. The cellular components of Microalgae such as carbohydrate, proteins, and lipids can be used for biotechnological applications like producing biofuel, nutritional products, pharmaceutical, and cosmetic (Cho et al, 2015; Ding et al, 2016). In addition, microalgae are also used in wastewater treatment for pollutant and nutrient removal (Becker,1994). This is because of microalgae growth which is dependent on the nutrient concentration as well as light, temperature, salinity and pH (Sukumaran et al, 2014).

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Malaysian palm oil industry has grown rapidly over the years where Malaysia has become one of the world's largest producer and exporter of palm oil. Malaysia is the second largest producer of palm oil mill in the world that contributes significantly towards the economic growth, rapid development and increasing the Malaysians living standard (Kamyab et al, 2016). In the other hand, it has also contributed to the environmental pollution of huge quantities of by-products from the oil extraction process (Jacob-Lopes et al, 2009;Wahidin et al, 2013; Ying et al, 2014) .

One of the main waste product of Palm oil industry is the palm oil mill effluent (POME). POME is very rich in organic matters and nutrients and slightly acidic with a pH between 4 to 5 (Guo et al, 2015). It has a high chemical oxygen demand (COD) and biochemical oxygen demand (BOD). Due to these high levels of organic matter and nutrient elements (nitrogen and phosphorous), the effluent could cause severe pollution to the environment, typically pollution to water resources (Kin-Chung et al, 2016). Hence, POME has a substantial amount of inorganic nutrients and trace metals that encourage plants and algae growth. Interestingly, it was found that POME can be used as a medium for microalgae cultivation. Microalgae can reduce the POME pollutants by utilizing nutrients, CO2 and organic compounds (Kumar et al, 2010).

One of the important factors for algal growth is pH that can affect the activity of different enzymes. In general, different algal species have various ranges of tolerance to pH. (Kamyab et al, 2015b). Although, uptake of inorganic carbon by phytoplankton during photosynthesis may increase pH (Travieso et al, 2006). Moreover, different microalgae species are affected differently by pH. For instance, Kamyab et al. (2014b) found out that biomass was highest at pH 7.5 (320 ± 29.9 mg biomass L−1 day−1 and and pH 7 (407±5.5) mg biomass L−1 day−1 for T. suecica CS-187 and Chlorella sp, respectively. The growth rate was the highest at pH 7.5 to 8.0 in all species (C. lineatum, H. triquetra, P. minimum). In these species the growth rate decreased when pH exceeded to more than 8 (Travieso et al, 2006).

Some studies reported the efficiency of C. inserta for treatment of POME. For instance, Kamyab et al. [18] obtained 67.35% of COD removal using C. incerta in 250 mg/L of POME concentrations in 28 days. In another study, by using C. inserta, 12.5%, 11.3%, and 70.47% of NO3−, NH3-N, and PO4-P is removed from POME (kamyab et al, 2017). The aim of this study is to propose the newly discovered local tropical microalgae C.incerta as an alternative for other imported specie and to find the potential of this species for biomass productivity and pollutant removal from POME.

2. Material and methods

The experimental work in this study was divided into three phases, which are preparation phase, accumulation phase, and steady phase. Preparation phase involves the collection and analysis of POME, and preparing of Bold’s Basel Medium (BBM). The accumulation phase involves culturing C.incerta in BBM and finding the optimum pH. The Steady phase was the main point of this study that involves the cultivation of C. incerta in POME under different light/dark cycle and CO2 concentrations.

2.1. Microalgae strain and culture medium

The freshwater local tropical microalgae strain, C.incerta, was obtained from previous research work from the environmental laboratory, faculty of civil engineering, UTM. 100 mL sample with optical density of 0.5 nm, was initially used to culture more microalgae stock in

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Bold’s Basel Medium (BBM), which is essential for growing a variety of algal cultures (Hadiyanto and Nur, 2012). The initial pH was in the range of 6.4 to 7.2 and adjusted to the desired pH using HCL and NaOH solutions. After the adjustment of pH, BBM solution was autoclaved at 121°C for 15 min (Sukumaran et al, 2014).

2.2. POME collection

The raw POME sample was collected from facultative ponds at Felda Palm Oil Industries Sdn. Bhd., in Kulai Johor Bahru. The collected sample was stored in plastic containers with proper label and preserved in the cool room at 4 o C to prevent any contamination and to limit the activity of biodegradation process. Large and bulky materials in the raw POME sample were removed before dilution. In order to accurately characterize the POME for analysis, serial dilution was done using distilled water. Prior to sample preparation for microalgae cultivation, POME sample was left over for 2 hours in order to return the POME to room temperature and analyzed for COD, MLSS, MLVSS, TN, and TP.

2.3. Accumulation and optimization phase

The unialgal species was cultured in BBM to provide enough stock for the steady phase. C.incerta culturing experiments were performed in different sizes of conical flasks in order to find the optimal surface area to volume ratio. Each conical flask was felled with 200 ml of BBM and inoculated with 10% (v/v) of pure C.incerta. The culturing was in an environmental chamber equipped with fluorescent lamps (Illumination 3000 lux) at room temperature and continuous light with manual swirling applied once a day. However, different set of pH (6.8, 7, 7.15) were examined to find the optimal pH value suitable for healthy and fast growth of C. incerta. Immediately after incubation, optical density, at single wavelength of 600 nm, of the culture was measured and compared with the measurement taken daily to monitor the microalgae growth rate.

2.4. Steady Stage and experimental setup

The steady phase consists of three different light/dark cycles (24/0, 16/8, and 12/12). All cycle have same four set of CO2 injection concentration 0 % (control), 25 %, 50%, and 100%. The cultivation was done in an environmental chamber equipped with white fluorescent lamps (Illumination 3000 lux). The lamps were connected to an electronic automatic switch equipped with timer to apply 24/0, 16/8, 12/12 h light/dark cycle. The standard conditions for cultivation were Temperature 24-28 oC, and according to the photoperiod, fluorescence white light (Phillips) of 21.8 µmol photons m–2s–1 intensity. POME was diluted to a concentration of 250 mg/L COD as this concentration was found to be the optimal value for the microalgae growth (kamyab et al, 2014b). The pH value of the diluted POME was adjusted to the optimal pH value (7.15) as found in the optimization phase.

After setting up the cultivation condition, the stock culture (with optical density of 0.35 nm) was inoculated into each 500 mL Erlenmeyer culture flask to get 10% (v/v) inoculum density. The percentage of CO2 concentration was controlled by valves. All the glass-wares used in the experiment were sterilized by autoclaving at 121°C for 20 mins. Three replications were used for the all cultures and control media. The flowrate of CO2 was controlled to be 600 PSI. The injection was done daily at the same time for all light/dark cycle for 20 minutes. The cell concentration was monitored daily.

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2.5. Analytical Methods

2.5.1 POME Analysis

The characteristic of the raw POME, such as pH, chemical oxygen demand (COD), mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS), total nitrogen (TN), and total phosphorus (TP) were determined according to the Standard Methods for the Examination of Water and Wastewater (APHA) and DR 6000 Spectrophotometer Procedure Manual. In this study, POME characteristic was measured twice, immediately after sampling and at the end of cultivation period. At the last day (day 16) of the cultivation time and during the stationary phase, 50 ml were harvested by centrifugation at 6000 RPM for 5 min. The pellets were kept in the 4 oC cool room for lipid estimation. The supernatant was used for the measurement of COD, TN, and TP in order find the pollutant removal of microalgae. The removal efficiencies of COD, TN and TP were calculated using the following equation (Feng et al, 2011):

Removal Efficiency (% )=P1−P2

P1×100 (1)

Where P1 and P2 are the initial and final pollutants concentration, respectively.

2.5.2 Measurement of microalgae growth

The C.incerta growth was estimated by measuring the OD, CDW, and chlorophyll contents.

2.5.3 Optical Density

A sample of 8 mL was taken every alternative day from each flask for the measurement of OD. OD was measured using hatch DR 6000 at a single wavelength of 600 nm (Kamyab et al, 2015). The sample is returned back to the flask once the measurement is done.

2.5.4 Cell dry weight

The dry weight of microalgae biomass was determined using gravimetric method, and the growth factor was expressed as g/L of dry biomass (Cho et al, 2015). Thirty six filter papers were dried at 105 oC for 90 minutes before they weighted. Then, 1 mL of each microalgae flask was filtered using Whatman glass micro-fibers (Grade GF/C 1.2 mm) under vacuum, and rinsed twice with distilled water and incubated in the oven for one hour (kamyab et al, 2016; Wahidin et al, 2013). After cooling in the desiccator, their final weight were obtained and recorded. This process was repeated until constant dry weight was obtained. The final cell dry weight of each sample was determined as follows:

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Cell dry weight ( gL

)=A1−A2

V (2)

Where A1 and A2 are the empty filter paper and the filter paper with the dry biomass, respectively. Whereas V is the volume of the filtered sample.

2.5.5 Chlorophyll contents

Chlorophyll contents were determined according to Jacob-Lopes et al. (2009). 8 ml of microalgae cells were collected and harvested by centrifugation at 4000 rpm for 4 min and discarded the supernatant for chlorophyll a content. The pellets were put in sonicator for 1 min and top up 5 ml of 90% aqueous of acetone solution. The solution was homogenized and the chlorophyll content was measure using hatch DR 6000 at a single wavelength of 663 and 645 nm. Using these wavelength values, chlorophyll a and chlorophyll b were measured according to the following equations:

Ca=12.7 A663−2.69 A645 (3)

Cb=22.9 A645−4.64 A663 (4)

Ca+b=20.2 A645+8.02 A663 (5)

2.5.6 Kinetic parameter

The specific growth rate (µ) was determined by the following equation [5, 14, 15]:

μ=1t

ln (X2

X1¿)¿ (6)

Where t is the time of cultivation run (days) and X2 and X1 are the biomass concentration at the end and beginning of cultivation, respectively. In addition, the biomass productivity was calculated by the equation (Ying et al, 2014):

Biomass Productivity=X2−X 1

t (7)

3. Result and Discussion

3.1 Optimization phase

The objective of this phase was to determine the optimal cultivation condition in term of pH and surface area to volume ratio. The cultivation was done in different conical flask size with different pH value. Figure 1shows the plot of daily biomass concentration in different pH.

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1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 310

0.050.1

0.150.2

0.250.3

0.350.4

0.450.5

pH = 6.5 pH=6.8pH=7 pH7.15

Days

Biom

ass C

once

ntra

tion,

g/L

.d

Biomass Productivity Specific Growth rate 0.0000

0.0050

0.0100

0.0150

0.0200

0.0250

6.50 6.80 7.00 7.15

pH

Figure 1: A: Effect of pH on the growth rate of C.incerta cultured in BBM.

B: Biomass productivity and Specific growth rate of C.incerta cultured in BBM with different pH

Figure 1A shows the optimal pH for the growth of C.incerta species is 7.15. Similarly, Kumar et al. (2010) studied the effect of different pH on Dunaliella salina cultivation and growth. They found that under constant supply of CO2, the biomass concentration increasing steadily at the initial pH of 7.15. According to Figure 2A, the pH 6.8 shows a constant but little increment in growth of C.incerta with time. As shown in Figure 1B, pH 7.15, the biomass concentration was doubled by the end of the cultivation time when the final biomass concentration were 1.14, 1.59, 1.28, and 2.03 (g/L.d) at original initial concentration for pH 6.5, 6.8, 7 and 7.15 respectively. However, the slow growth is not desirable since it increases the cost and time.

3.2 Effect of different light/dark cycle and CO2 concentration on the growth rate of C. incerta3.2.1 Light/Dark Cycle

The growth rate and biomass productivity of C.incerta were investigated under different light/dark cycle, and CO2 concentration. Figures 2, 3 and 4 indicate the effect of these conditions on the growth of C.incerta reacted in term of optical density, cell dry weight, and Chlorophyll content. According to Figure 2, the adaptation phase ended on the 3th day of cultivation in all cycles while the stationary phase has not been reached within the 17 days of cultivation. However, all light/dark cycle showed a positive growth and the rate of growth was different for each cycle and CO2 concentration. At zero CO2 injection, the maximum biomass productivity and specific growth rate for 12/12 L/D cycle were 0.04 g/L.d, 0.118 respectively. Whereas, for 16/8 and 24/0 L/D cycle, the maximum biomass productivity and specific growth rate were (0.024 g/L.d and 0.093) and (0.043g/L.d and 0.122), respectively (Table 1). The optical density of algae grown in POME increased steadily which was expected as POME is rich with nutrient (Figure 3).

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A B


1 3 5 9 11 13 15 170.0000.1000.2000.3000.4000.5000.6000.7000.8000.900

0% CO2 100% CO2 50% CO2

25% CO2

Days

Opti

cal D

ensit

y

1 3 5 9 11 13 15 170.000

0.100

0.200

0.300

0.400

0.500

0.600

0% CO2 100% CO2 50% CO2

25% CO2

Days

Opti

cal D

ensit

y

1 3 5 9 11 13 15 170.0000.1000.2000.3000.4000.5000.6000.7000.8000.900

0% CO2 100% CO2 50% CO2 25% CO2

Days

Opti

cal D

ensit

y

Figure 2: Biomass concentration in term of optical density under different light/dark cycles and CO2 concentrations a: 12/12 light/dark cycle b: 16/8 light/dark cycle c: 24/0 light/dark

cycle

The biomass growth rate for OD and CDW was slightly similar except for the day 11 which showed large increment for CDW. Theoretically, TSS and OD curve should be equal. However, it was showed that the CDW and OD results are slightly different, especially the low concentration of CO2. This could be because microalgae could not assimilate all of CO2 to convert cellular biomass in culture system as amount of carbon fixed during photosynthesis was lost from cells to the surrounding culture medium by passive diffusion. The rate of diffusion could be depended on stress condition such as change in pH value or nutrient depletion.

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A B

C


Table 1: Biomass productivity, specific growth rate, and pollutant removal efficiency under different light/ dark cycle and CO2 concentration

CO2 Concentration

12/12 Light/Dark Cycle 16/8 Light/Dark Cycle 24/0 Light/Dark Cycle

0 25 50 100 0 25 50 100 0 25 50 100

Biomass Productivity

(g/L.d)0.040 0.017 0.017 0.020 0.015 0.017 0.024 0.024 0.014 0.033 0.043 0.042

Specific growth rate 0.118 0.078 0.076 0.080 0.072 0.076 0.093 0.093 0.065 0.103 0.122 0.119

Xi / Xf 7.4 3.8 3.6 3.9 3.4 3.6 4.9 4.8 3 5.8 7.9 7.6

At day 11, the biomass attached to the conical flask was removed manually so it is fully homogenized on day 11. With adequate mixing, nutrients can be evenly distributed in the medium thereby disrupting diffusion barriers at the algal cell surfaces. Moreover, sufficient mixing of the medium can uniformly expose the algal cells to the light source, ensure quick removal of the oxygen produced by the microalgae during photosynthesis and subsequently avoid potential oxidative stress on the algal cells (Kamyab et al, 2015b).

3.2.2 CO2 Concentration

According to Figures 3 and 4, CO2 injection was controlled by valve in proportion ratio of 1:0.5:0.25 of the original CO2 concentration 500 PSI. Different CO2 concentration at different L/D cycle had different biomass productivity and specific growth rate as well as different COD, TN, and TP removal which are shown in Figures 4, 5, 6 and Table 1. The results showed that C.incerta could grow 7.4, 4.9, and 7.9 times more than initial biomass concentration after 17 days for the conditions (12/12 L/D, 0 % CO2), (16/8 L/D, 50% CO2), and (24/0 L/D, 50 % CO2), respectively. This result was in consistent with the obtained results by Ying et al., (2014). In this study, the injection of CO2 reduced the biomass productivity when there was no CO2 injection. This could be due to the reduction of pH in the culture system. The optimum growth and metabolism of various microalgae could be affected by the different pH value. It could affect both gas absorption and nutrient availability.

Based on the Figure 2A, the optimal pH for the growth of C.incerta is 7.15 which was set to be the initial pH for all cultures. The injection of CO2 in water would result in decline of pH due to carbonic acid formation. Therefore, it is assumed that with the injection of CO 2 gas, initial pH value start to decrease resulting in low biomass productivity compared to the biomass growth in the culture system with no CO2 injection. Moreover, the effect of different CO2 concentration in the biomass productivity and specific growth rate was not significant. The injection of CO 2

concentration enhanced the amount of total dissolve inorganic carbon source as bicarbonate in the culture medium. Some researchers found that the lower pH results in the decrease of the extracellular carbonic anhydrase activity (Travieso et al, 2006; Kamyab et al, 2014b).

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1 3 5 9 11 15 170.000.100.200.300.400.500.600.700.800.90

0% CO2 100% CO2 50% CO225% CO2

Days

CDW

, g/L

.d

1 3 5 9 11 15 170.00

0.20

0.40

0.60

0.80

1.00

1.20

0% CO2 100% CO2 50% CO225% CO2

Day

CDW

, g/L

.d

1 3 5 9 11 15 170.000.200.400.600.801.001.201.401.601.80

0% CO2 100% CO2 50% CO2 25% CO2Day

CDW

, g/L

.d

Figure 3: Growth rate and biomass concentration based on cell dry weight under different light/dark cycles and CO2 concentrations. A: 12/12 light/dark cycle B: 16/8 light/dark cycle C:

24/0 light/dark cycle

As shown in Figure 4, chlorophylla concentration could be increased by CO2 injection in all light/dark cycle except for 12/12 L/D cycle where control culture had higher chlorophylla concentration. The highest chlorophyll-a concentration was 14.66 mg/ L at 50 % and 100 % CO2 injection, 24/0 L/D cycle in day 14. In all days, control culture had the lowest chlorophylla content. After the 14th day of cultivation, chlorophyll content started to decline. From Figure 4, it can be concluded that, in term of chlorophylla, the best growth condition of C.incerta was the continuous illumination with high CO2 injection.

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C

BA


5 10 14 180.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0% CO2 100% CO2 50% CO225% CO2

Days

Chl A

5 10 14 170.001.002.003.004.005.006.007.008.009.00

0% CO2 100% CO2 50% CO2 25% CO2Days

Chl A

5 10 14 170.002.004.006.008.00

10.0012.0014.0016.00

0% CO2 100% CO2 50% CO2 25% CO2

Days

Chl A

Figure 4: Growth rate and biomass concentration in term of Chlorophyll A content under different light/dark cycles and CO2 concentrations. A: 12/12 light/dark cycle B: 16/8 light/dark

cycle C: 24/0 light/dark cycle

3.3 Efficiency of Microalgae Chlamydomonas on the Removal of Pollutants

Figure 5A, shows the effect different L/D cycle in based on CDW. Based on the results, that biomass reached maximum concentration when the culture was under continuous light 24/0 and injected with 100% CO2. Therefore, the favorable L/D cycle for higher biomass productivity was 24/0 followed by 12/12, and 16/8 as shown in Figure 5B. One of the objectives of this study was to assess the efficiency of C.incerta in reducing COD, TN, and TP. The initial COD, TN, and TP in the diluted POME sample prior to the cultivation were 250, 37 and 52.8 mg/L respectively. According to Figure 5B, C.incerta was effective in reducing the pollutants in POME. It was observed that; (i) CO2 concentration injection has a direct proportion with the COD, TN, and TP removal efficiency where, the higher concentration of CO2 injection resulted in the higher removal efficiency; (ii) different light/dark cycle had no significant effect on the removal of pollutant as they have close removal efficiencies.

10

C

BA


1 3 5 9 11 13 15 170.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

12/0 % CO2 16/0 % CO2 24/0 % CO2 12/25 % CO2 16/25 % CO2 24/25 % CO2 12/50 % CO2

16/50 % CO2 24/50 % CO2 12/100 % CO2 16/100 % CO2 24/100 % CO2

Days

CDW

, g/L

12/0 % CO2

12/100 % CO2

12/50% CO2

12/25% CO2

16/ 0 % CO2

16/ 100 % CO2

16/50% CO2

16/25% CO2

24/0 % CO2

24/100 % CO2

24/50% CO2

24/25% CO2

0.0

20.0

40.0

60.0

80.0

100.0

120.0

Rem

oval

Effi

cienc

y (%

)

Figure 5: A: Effect of Light/Dark cycle and CO2 [Light/ CO2 Concentration] on the biomass concentration B: Removal of COD, TN, and TP in different L/D cycle and CO2 concentration.

The highest COD removal was achieved at 12/12 L/D cycle with 100% CO2 concentration with removal efficiency of 88%. Whereas the highest TN, and TP removal efficiency was at 16/8 and 24/0 L/D cycle with 50% CO2 concentration with the removal percentage of 97.3 and 99.8 mg/L, respectively. The Lowest COD, TN, and TP removal efficiency were found to be 52, 43.2, and 65 mg/L respectively and was at 24/0, 16/8, and 24/0 light/dark cycle with 0% CO2 concentration, respectively. These results were close to the results obtained by Kamyab [20]. In another study, Kamyab [18] found out in POME medium with same initial COD (250 mg/L COD), the highest removal efficiency of COD by C.incerta was 67.35 %. Moreover, these finding were also supported by the finding of (Kamyab et al, 2015b) and (Travieso et al, 2006). Kamyab et al., (2014b) found the cultivation of mixed micro-macro algae in a 250 mg/L COD POME resulted in 71.16 % reduction in COD while this study had higher removal efficiency. The removal occurred

11

B

A


because microalgae utilize the organic matter and nutrient in the wastewater for their photosynthesis.

3. Conclusion

In this study, the maximum biomass productivity and specific growth rate for 12/12 L/D cycle were 0.04 g/L.d, 0.118 respectively which was achieved at zero CO2 injection. Whereas for 16/8 and 24/0 L/D cycle, the maximum biomass productivity and specific growth rate were (0.024 g/L.d and 0.093) and (0.043g/L.d and 0.122), respectively. Moreover, the highest COD removal was achieved at 12/12 L/D cycle with 100% CO2 concentration with removal efficiency of 88%. While the highest TN, and TP removal efficiency was at 16/8 and 24/0 L/D cycle with 50% CO2 concentration with the removal efficiency of 97.3 and 99.8 mg/L, respectively. As the conclusion, the abundance growth of C.incerta in POME provides dual benefit such as cost reduction for wastewater treatment and by-product production as a low cost method that can create impact on the environment.

Acknowledgment

The authors would like to acknowledge the government research grant Tier1 (Q.J130000.2517.10H25) as well as FRGS grant (R.J130000.7809.4F472) from Ministry of Higher Education, Malaysia.

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