Abstract
The use of freely suspended cells of microalgae culture to treat wastewater is of current global interest because of their effective photosynthetic uptake of pollutants, carbon dioxide sequestration, and biomass production for desirable high value-products. Biomass immobilization is a promising option to overcome the harvesting problem that is encountered when using free-cells upon completion of the wastewater treatment process. In this study, Nannochloropsis sp. cells were immobilized in sodium alginate beads to eliminate the harvesting limitation. The microalgal beads were further cultivated in treated palm oil mill effluent (TPOME) for removal of chemical oxygen demand (COD). The effect of POME concentration on COD removal and microalgal cells growth was investigated, respectively. It was found that the maximum biomass concentration of 1.23 g/L and COD removal of 55% from 10% POME were achieved after 9 days. An increment of POME concentration did not cause any improvement to the treatment efficiency due to the inhibitory effect of high initial COD of POME on the biomass concentration and was further responsible for low COD removal. The immobilized cells showed a systematic growth, demonstrating that the beads are biocompatible as immobilization carrier. In conclusion, the immobilized microalgal cells could be a viable alternative technology system for POME treatment as well as biomass production.
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Phycoremediation of Treated Palm Oil Mill Effluent (TPOME) using Nannochloropsis sp. Cells Immobilized in the Biological Sodium Alginate Beads: Effect of POME Concentration
Quin Emparan, Razif Harun,* and Yew Sing Jye
The use of freely suspended cells of microalgae culture to treat wastewater is of current global interest because of their effective photosynthetic uptake of pollutants, carbon dioxide sequestration, and biomass production for desirable high value-products. Biomass immobilization is a promising option to overcome the harvesting problem that is encountered when using free-cells upon completion of the wastewater treatment process. In this study, Nannochloropsis sp. cells were immobilized in sodium alginate beads to eliminate the harvesting limitation. The microalgal beads were further cultivated in treated palm oil mill effluent (TPOME) for removal of chemical oxygen demand (COD). The effect of POME concentration on COD removal and microalgal cells growth was investigated, respectively. It was found that the maximum biomass concentration of 1.23 g/L and COD removal of 55% from 10% POME were achieved after 9 days. An increment of POME concentration did not cause any improvement to the treatment efficiency due to the inhibitory effect of high initial COD of POME on the biomass concentration and was further responsible for low COD removal. The immobilized cells showed a systematic growth, demonstrating that the beads are biocompatible as immobilization carrier. In conclusion, the immobilized microalgal cells could be a viable alternative technology system for POME treatment as well as biomass production.
Keywords: Wastewater pollution and treatment; Microalgae; Biomass growth; COD; Removal efficiency; Sustainability
Contact information: Department of Chemical & Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang, Malaysia; *Corresponding author: mh_razif@upm.edu.my
INTRODUCTION
Malaysia is the world’s largest producer of palm oil, which is one of the world’s leading agricultural commodities. There are more than 400 mills throughout the country. Most of the mills have generated around 800 tonnes/day of palm oil mill effluent (POME) resulting from the palm oil processing (Halim et al. 2016). POME has a high concentration of organic matter identified as chemical oxygen demand (COD) and biochemical oxygen demand (BOD3) which are up to 51000 and 25000 mg/L, respectively (Zainal et al. 2017). The accidental discharge of untreated or treated POME into the environment could result in the eutrophication of water bodies and accelerate the dwindling of freshwater resources at a high rate. Eventually this can result in low levels of dissolved oxygen, a high mortality rate of zooplankton, depletion of aquatic life, and murkiness of water systems. Conventional technologies for POME treatment are sometimes still not capable of reducing the level of pollutants sufficiently to meet the discharge standards set by the department of environment (DOE) (Kamyab et al. 2014). Therefore, a highly efficient, cheap, and environmentally friendly approach is required for the POME treatment.
Microalgae are microscopic photosynthetic organisms that can be found in marine ecosystems and freshwater. They consume organic matter and nutrients including nitrogen and phosphorus present in the wastewater for their growth (Chinnasamy et al. 2010). According to Emparan et al. (2019), phycoremediation is defined as the use of algae to remove or transform pollutants, including nutrients and toxic chemicals from wastewater, and removal of CO2 from waste air accompanied by biomass production. Microalgae in the form of suspended free-cells of culture has been used for phycoremediation of wastewater including POME for removal of BOD, COD, and nutrients (Rajkumar and Takriff 2015). Pollutants present in the wastewater can be assimilated by the algae and can accumulate in the biomass, since the algal cell walls are porous which allow free passage of molecules and ions in aqueous solution (Lage et al. 2018). Recent studies have shown that the biomass of suspended free-cells from microalgae culture of the treated POME can be used to produce high-value products such as biodiesel (Selmani et al. 2013) and feedstock in aquaculture industry (Selvam et al. 2015).
However, recovery of suspended free-cells from microalgae biomass using the treated wastewater including POME is one of the challenges during a phycoremediation process. The suspended free-cells culture is the condition of microalgae living cells that move independently within the bottles containing medium under a condition to ensure uniform cells distribution (Katarzyna et al. 2015; Emparan et al. 2019). Therefore, the application of immobilized microalgal cells is a good approach to overcome the harvesting problem. The immobilized cells are living microalgal cells that are prevented from flowing freely away from their original location to all parts of the medium (Katarzyna et al. 2015; Emparan et al. 2019). In comparison to the other immobilization methods (adsorption, covalent binding, cross-linking, encapsulation), entrapment of microalgal cells in natural and synthetic gel polymers presents advantages of higher nutrients/products diffusion rates, more environmentally friendly character, and greater stability (Das and Adholeya 2015; Eroglu et al. 2015).
Natural polymers such as alginate are the most commonly used materials to immobilize the microalgal cells before the phycoremediation of wastewater. The advantages of using natural polymers as immobilizing carriers are their non-toxic, transparent, permeable, hydrophilic, nutrient enriched, environmentally friendly, and bio-compatible nature; they also have high product diffusion rates and produce less hazardous waste upon completion of the process (Shi et al. 2007; Zhang et al. 2008; Moreno-Garrido 2008; Eroglu et al. 2015; Sumithrabhai et al. 2016; Emparan et al. 2019). Since the immobilized beads have a larger size than suspended free-cells, a simpler method by sieving/netting can be employed to harvest the beads from water without requiring high energy input (Lam and Lee 2012) compared to suspended free-cells. Solvents are used to dissolve the beads and then oven-dried (where this process is also done in suspended free-cells). Also, the immobilized microalgal cells exhibited higher removal efficiency of pollutants from wastewater as compared to the suspended free-cells of microalgal culture (Zeng et al. 2013a).
Therefore, this study investigated the potential of microalgae, Nannochloropsis sp. cells immobilized in sodium alginate beads for phycoremediation of treated POME. So far, there have been no such studies carried out for removing pollutants from POME using immobilized Nannochloropsis sp. cells. In fact, the main objective of this study is to treat the POME without any chemical usage and apply the biological method via microalgae sodium alginate beads.
EXPERIMENTAL
Algal Cultivation
The species of microalgae used in this study was Nannochloropsis sp., which was supplied by the CSIRO Microalgae Research Centre (Tasmania, Australia). The microalgal cell was cultivated in F/2 culture media under the axenic condition at the algal research laboratory (Department of Chemical and Environmental Engineering, Universiti Putra, Malaysia). The suspended free-cells of microalgae culture was maintained by routine sub-culturing to prolong their life and to expand the cells number as stock media. The microalgal cells were harvested prior to the immobilization procedure.
Preparation of Sodium Alginate-Immobilized Microalgae
Sodium alginate beads with immobilized microalgal cells were obtained from sodium alginate-microalgae suspension. Approximately 4% (w/v) of sodium alginate solution was prepared and autoclaved at 121 °C and 15 psi for 15 min. Then, the suspended free-cells in microalgae culture were collected from the stock medium. They were then mixed with the 4% of sodium alginate solution at a ratio of 1:1 (v/v) to produce a 2% suspension of sodium alginate-microalgae. The mixture was extruded through a sterile disposable 5 mL syringe and a 25 gauge hypodermic needle into 0.3 M calcium chloride (CaC12). About 520 microalgae beads were produced from 40 mL of microalgae sodium alginate solution for sample A1 as shown in Table 1. All experiments were performed in duplicates. Therefore, the total amount of microalgae beads used in this batch studies were about 3120. The microalgae beads were left in CaC12 solution overnight for hardening stage. After that, the beads were washed with sterile distilled water. Strict aseptic precautions were adopted throughout the immobilization procedure.
Collection of POME
The POME used in this study was obtained from Sime Darby Palm Oil Plantation Sdn. Bhd., Labu, Negeri Sembilan (Malaysia). The effluent was obtained after sterilization, extraction, and purification. The raw POME was further treated through a series of ponding systems including an acidification pond, followed by an anaerobic pond, aerobic pond, and final pond. The POME sample was collected from the final pond/algae pond and stored in 20 L plastic containers with proper labeling. The sample was stored in the fridge at 4 °C to prevent contamination and limit the biodegradation process by bacterial activity. The POME was initially filtered using a filter paper to remove bacteria and suspended in sludge prior to microalgae cultivation experiment.
Treatment of POME Using Microalgal Cells Immobilized in Sodium Alginate Beads
Effect of POME concentration on microalgal cells growth and COD removal efficiency
The phycoremediation studies were performed in a batch culture using Duran bottles at the algal research laboratory, Department of Chemical & Environmental Engineering, Universiti Putra Malaysia. Three different concentrations of POME (10%, 25%, and 100 % POME) were prepared in 500 mL bottles as shown in Table 1. The selection of the ranges were based on Ding et al. (2016). In this study, the ratio of microalgae beads to each POME sample was fixed at 1:5 (v/v). In this respect, about 520 microalgae beads were put into each 200 mL POME sample. The fluorescence light energy and aeration using a magnetic stirrer at 100 rpm were continuously supplied to all samples. The bottles were stuffed with cotton plugs to filter and allow movement of air into and out of the bottles thus preventing the entry of fungal spores or bacteria. Furthermore, the cotton plugs are cheap and can be re-autoclaved and recycled for use in the next experimental work. All the experiments were carried out at room temperature. The well-mixed microalgae beads suspension were collected and the microalgal cell growth was measured every 3 days and up to 9 days of the treatment period. The COD of each POME sample on Day 0 and Day 9 were also measured using the following Eq. 1,
COD (%) = [(Co – Ci)/Co] × 100% (1)
where Co (mg/L) and Ci (mg/L) are the mean values of COD concentrations at initial time to (day) and time ti (day), respectively.
Algal Analysis
Morphological examination using a microscope
This method is the fast and easy way to examine the condition of immobilized microalgal cells in the sodium alginate beads. Five microalgae beads were collected and dissolved using 0.1 M sodium citrate (Na3C6H5O7) solution. Approximately 1.0 mL of the dissolved immobilized-microalgae beads was added to one drop of oil red stain. The mixture was vortexed for about 1.0 min and centrifuged at 5 RCF for about 5 min. The supernatant was removed, and about 1.0 mL of 85% propylene glycol was added to wash the pellet. The mixture was again vortexed and centrifuged at 5 RCF for about 5 min and the supernatant was removed again. The procedure was repeated using 50% propylene glycol. After the supernatant was removed, distilled water was added and vortexed to homogenize the pellet. Each sample was then ready to be examined under the JVC Color Video Camera microscope (Leica PTE Ltd, Singapore) with a magnification of 40x and 100x.
Scanning electron microscope (SEM)
The morphological structure of microalgal cells, sodium alginate microalgae beads, and blank sodium alginate beads were investigated using Jeol JSM 6400 SEM (Hitachi, Tokyo, Japan). Prior to microscopic observation, each sample was processed through several processes, including fixation, washing, and dehydration. After that, each sample was ready to be viewed under the SEM.
Optical density (OD) and biomass concentration
The OD of microalgal cells was measured using a GENESYS™ 10S UV-Vis spectrophotometer at an optimum wavelength of 600 nm (OD600). A calibration curve was prepared by plotting OD against biomass concentration (in dry weight, g/L). Prior to analysis, five microalgae beads were dissolved using 2 mL of 0.1 M Na3C6H5O7 solution and placed into a clean UV-cuvette to measure the absorbance value. For suspended free-cell microalgae culture (standard graph), 2 mL of the sample was used. Distilled water was used as a blank sample. The OD was recorded and biomass concentration was obtained from the calibration curve.
Wastewater Analysis
Chemical oxygen demand (COD)
In this study, the COD measurement was carried out according to the standard method 5220C (Closed Reflux, Titrimetric) (APHA 2017). Initially, about 2.5 mL of POME sample was added into a COD vial. Another test vial filled with 2.5 mL of distilled water was prepared as a blank sample. After that, each sample was mixed with 1.5 mL of potassium dichromate (K2Cr2O7) (9.05 mol/L), followed by 3.5 mL of sulfuric acid (H2SO4) (18.01 mol/L). The vials were mixed well and placed in the COD reactor at 150 °C for 2 h. After 2 h, all the vials were allowed to cool to reach room temperature. Each sample was then placed into a conical flask and added with 3 drops of ferroin indicator. Then, each sample was titrated with 0.10 M ferrous ammonium sulfate (FAS) solution until the color change from pale blue-green into reddish-brown. The volume of FAS used was recorded. The COD of the each was calculated based on the following Eq. 2,
(2)
where A is the volume of FAS (mL) used in blank sample and B is the volume of FAS (mL) in POME sample.
Turbidity
The turbidity of each sample was determined based on HACH Method 10047 (Attenuated Radiation Method) using a DR/4000 spectrophotometer. Firstly, the spectrophotometer was turn on and Hach Program: 3750 at 860 nm was selected. After that, about 10 mL of distilled water was put into a vial as blank. Then, about 10 mL of the sample was put into other vials. Each vial was put into the spectrophotometer, and the turbidity reading (FAU) was measured and recorded.
Hydrogen ion concentration
The pH of the sample was determined based on Model 3505 user guide using pH meter (Brand JENWAY).
Statistical Analysis
All the experiments were carried out in duplicate. The analysis of variance (ANOVA) SPSS version 20 was used to determine the statistical significance among treatments at p < 0.05.
Table 1. Composition of POME and Sodium Alginate Microalgal Cells Solution
RESULTS AND DISCUSSION
Immobilization of Microalgal Cells in the Sodium Alginate Beads
Immobilization of the microalgal cells using sodium alginate was carried out to enhance the harvesting and efficiency of POME treatment. As mentioned in the methodology section, the microalgal cell culture was mixed with the sodium alginate solution at a ratio of 1:1 (v/v) and extruded using a syringe to form a spherical shape of the beads in CaC12 solution. The microalgal beads were then left overnight for the hardening stage. The procedure was repeated without the addition of microalgal cells culture with blank sodium alginate bead samples.
The images of blank and sodium alginate microalgal cell beads are shown in Fig. 1. According to Fig. 1, it was observed that the blank sodium alginate beads were transparent in color. After mixing with microalgal cell culture, the color of beads changed into light green. The color of the cultivated microalgal beads changed to darker green after the POME treatment for 9 days, hence demonstrating that microalgal cells were successfully grown inside the beads.
Fig. 1. Image of (A) blank sodium alginate beads; (B) immobilized sodium alginate microalgal cell beads before treatment; and (C) immobilized sodium alginate microalgal cell beads after 10% POME treatment.
Morphological examination using a microscope
The analysis was carried out using a microscope to determine the condition of microalgal cells in the beads after the immobilization process. The oil red stain is a natural lipophilic dye with known spectral properties. As shown in Fig. 2, the microalgal cells exhibited green and red color where its lipid had been dyed with the red stain. The structure and appearance of microalgal cells did not show any changes before and after immobilization into the beads. This showed that the immobilization will not affect the morphological structure and metabolic activity of microalgae. Eventually, microalgae can survive and grow in the beads.
Scanning electron microscope (SEM)
The morphological structure of immobilized cells, blank sodium alginate beads, and sodium alginate microalgal cell beads were examined using a scanning electron microscope (SEM). The outer and inner surfaces of blank sodium alginate beads are shown in Fig. 3. The outer surface of blank sodium alginate bead was smooth. The blank sodium alginate beads were then cut into half to observe the cross-section of the inner surface and some pores with size 300 nm to 500 nm were observed. The images of immobilized microalgal cell beads are shown in Fig. 4. From the outer surface, it was observed that there were insoluble particulates attached to microalgal cells. The particulates might be the insoluble sodium alginate powder. From the cross-sectional surface, the microalgal cells were bonded with each other and entrapped by the sodium alginate. The pores were used to exchange materials such as CO2, oxygen, nutrient sources, and metabolites inside and outside the capsules (Zeng et al. 2013b).
Fig. 2. Nannochloropsis sp. cells (A) without beads; (B) without beads; (C) with sodium alginate microalgal cell beads; and (D) with sodium alginate microalgal cell beads
Fig. 3. Blank sodium alginate bead: (A) outer surface; and (B) inner surface pore