Assessing the feasibility of microalgae cultivation in agricultural wastewater: The nutrient characteristics

Environmental Technology & Innovation 15 (2019) 100402

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Environmental Technology & Innovation journal homepage: www.elsevier.com/locate/eti

Assessing the feasibility of microalgae cultivation in agricultural wastewater: The nutrient characteristics ∗

Azianabiha A Halip Khalid a,b , , Zahira Yaakob a , Siti Rozaimah Sheikh Abdullah a , Mohd Sobri Takriff c a

Chemical Engineering Programme, Faculty of Engineering & Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia b Faculty of Civil Engineering, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia c Research Center for Sustainable Process Technology, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia

highlights • • • • •

High concentrations of essential nutrients (C, N and P) were found in POME. The nutrients were largely in soluble form thus increase the bioavailability. Ammonium and phosphate constitute the majority of TN and TP respectively. Chlorella sorokiniana from native and commercial source produced highest biomass. POME is a feasible alternative for sustainable biomass production from microalgae.

article

info

Article history: Received 2 November 2018 Received in revised form 18 March 2019 Accepted 2 June 2019 Available online 4 June 2019 Keywords: Microalgae biomass Chlorella sorokiniana Agricultural wastewater Palm oil mill effluent (POME) Nutrient characterization Nutrient solubility highlights

a b s t r a c t This study investigated the feasibility of microalgae cultivation using palm oil mill effluent (POME). The availability of nutrient was assessed as well as the components and solubility of those nutrients in POME. The growth of native and commercial strains was evaluated based on their specific growth rate (µ) and biomass production. Characterization of POME shows high concentration of three essential nutrients; carbon (C), nitrogen (N) and phosphorous (P) at 2364 mg L−1 , 385 mg L−1 and 106 mg L−1 respectively. Crucially, more than 80% of C and N along with 72% of P were in soluble form, thus readily available for microalgae assimilation. Major constitute of N and P were ammonium and phosphate respectively, suitably the preferred form by the microalgae. A native strain, Chlorella sorokiniana produced the highest growth with µ of 0.24 day−1 . Interestingly, Chlorella sorokiniana obtained from commercial source produced comparable result with µ of 0.23 day−1 . High concentration of nutrient in POME has resulted in productive accumulation of biomass with both Chlorella sorokiniana species produced more than 100 mg L−1 day−1 . Outcome from this study indicated that POME is much suitable option for microalgae cultivation for subsequent production of valuable biomass. © 2019 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Chemical Engineering Programme, Faculty of Engineering & Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia. E-mail address: [email protected] (A.A.H. Khalid). https://doi.org/10.1016/j.eti.2019.100402 2352-1864/© 2019 Elsevier B.V. All rights reserved.

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1. Introduction The concept of wastewater recycling for microalgae cultivation has attracted much attention over the last few decades. Recent studies have demonstrated that microalgae could grow well in different types of wastewater especially from agricultural industry (Hariz et al., 2018). Agricultural sectors such as palm oil industry released huge amount of effluent characterized by high nutrients load that is consumable by microalgae (Ahmad et al., 2016). The use of nutrient-rich wastewater for microalgae cultivation is considered as an ideal scenario due to the potential of simultaneous effluents remediation and biomass production (Ammar et al., 2018). The nutrients will be assimilated into the microalgae cell thus prevented from entering the receiving water bodies. Besides, this culturing approach is a feasible way to reduce the cost of microalgae biomass production by reducing the needs of expensive synthetic growth medium. However, one of the main problems associated with this application is the high amount of suspended solid in the wastewater. It will not only blocks light transmission, but could also result in majority of nutrients retained in the particulate phase rather than in liquid form. The insoluble component of the nutrient could not be directly consumed by microalgae in contrast to dissolved nutrient (Griffiths, 2009). Thus, the extent of readily available nutrient for microalgae assimilation could define the suitability of the wastewater as culture medium. However, wastewater characterization based on the soluble and insoluble nutrient forms has been scarcely reported within the literature. The ability of microalgae to sustain growth in wastewater environment is also species specific (Hwang et al., 2016). Although growth is expected to be lower in harsh environment, but certain species are highly adaptable thus more suited for wastewater cultivation. On the contrary, some microalgae might struggle to cope with high concentration of nutrient. For instant, ammonium (NH4 + ) which is the most common chemical forms of N in wastewater could be potentially toxic to low-tolerance species. Reports from the literature indicated strong inhibition of Chlorella sp. and Scenedesmus sp. growth within NH4 + range from 210–1600 mg L−1 (Ayre et al., 2017; Park et al., 2010) On the other hand, present of trace metals such as Fe, Mg and K are beneficial for microalgae but it can be toxic in high concentration (Daneshvar et al., 2018). Previous researches have demonstrated superior acclimation of native microalgae in wastewater application (Rizza et al., 2017). However some might argue that given excellent adaptability of microalgae, commercial strains that fulfil desirable traits for a particular application can be genetically modified towards desired applications (Kothari et al., 2017). Thus screening of robust microalgae candidate is the primary step in ensuring the success of microalgae cultivation using wastewater effluent. The aim of this study was to evaluate the feasibility of microalgae cultivation using palm oil mill effluent. Available nutrients in palm oil mill effluent (POME) were compared with the standard medium to evaluate the adequacy of POME to supply important nutrient. Then, the forms and solubility of the nutrient components was determined to assess the fraction of nutrient that is immediately available for microalgae growth. The growth ability of native and commercial microalgae was compared based on their specific growth rate (µ) and biomass production to determine the most suitable strain. Outcome of this study can lead to more understanding on nutrient characteristic of POME and the ability of different microalgae to growth and accumulate biomass in this agricultural wastewater. 2. Materials and methods 2.1. Wastewater collection and pre-treatment The POME was obtained from an anaerobic pond and final effluent from Seri Ulu Langat palm oil mill in Sepang, Malaysia. The mill adopted open ponding system which is the most conventional method for treating high organic wastewater. This biological approach relies on bacteria to break down the organic pollutants. POME is initially treated in a series of anaerobic ponds, each about 6 metres deep that excludes O2 . The anaerobic condition aims for significant decomposition of organic matter by anaerobic bacteria. The retention times at this stage normally lasts about 40 days and data showed that 11% and 39% of TN and TP respectively was removed. The next stage in the treatment process is the facultative ponds, with a typical retention time between 20 to 40 days. The pond were constructed about 2 m deep and equipped with mechanical aeration. A marked reduction of 78% and 63% of TN and TP respectively was achieved, with nitrification and denitrification process by aerobic bacteria is thought to be the major N removal path. Then, the POME is further treated in aerobic ponds for 15 to 20 days, in which about 88% and 93% of TN and TP respectively was removed from the initial concentration. Photosynthesis of plankton in aerobic pond leads to an increase of pH thus volatilization of ammonia and phosphorus precipitation could be significant nutrient removal mechanism at this stage. Finally, the POME is transferred to the polishing pond and released as final effluent to the environment. Although these combined ponding systems have generally performed well in the removal of high organic matter content, it is not entirely efficient to treat nutrients. The final effluent still contained about 45 mg L−1 and 21 mg L−1 of TN and TP respectively. Furthermore, long retention period, totalling about 80 to 100 days also added to the disadvantages of this particular treatment method. The collected samples were transferred in 20 L labelled containers and immediately stored in 4 ◦ C cold room to preserve the characteristics. Prior to experimentation, the POME was filtered using filter cloth and further centrifuged to remove any visible solid particle. The supernatant was autoclaved at 121 ◦ C for 20 min and the characterization of POME before and after autoclaving is shown in Table 1. It has been reported in previous study that sterilization of wastewater by autoclaving reduces nutrients and might have an effect on microalgae growth (Ramsundar et al., 2017). However, as

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Table 1 Characteristics of BBM and POME from different ponds, mean (±SD). Parameter

Anaerobic

Autoclaved anaerobic (supernatant)

Final effluent

BBM

pH TSS (mg L−1 ) Turbidity (NTU) COD (mg L−1 ) TN (mg L−1 ) TP (mg L−1 ) Al (mg L−1 ) Ca (mg L−1 ) Cd (mg L−1 ) Cu (mg L−1 ) Fe (mg L−1 ) K (mg L−1 ) Mg (mg L−1 ) Mn (mg L−1 ) Na (mg L−1 ) Ni (mg L−1 ) Zn (mg L−1 )

7.6 ± 0.05 2980.0 ± 268.2 1940 ± 155.2 2364.0 ± 211.8 385.0 ± 25.0 106.0 ± 4.5 0.5 ± 0.03 41.9 ± 3.1 0.8 ± 0.06 0.4 ± 0.03 2.3 ± 0.1 2419.3 ± 237.1 591.0 ± 34.8 2.9 ± 0.1 42.4 ± 2.8 0.3 ± 0.01 0.5 ± 0.39

9.0 ± 0.05 Not measured 116 ± 17.7 2105 ± 153.3 313.7 ± 32.0 59.5 ± 9.7 0.31 ± 0.04 36.5 ± 6.8 0.33 ± 0.03 0.24 ± 0.02 0.95 ± 0.07 2371.47 ± 296 530.08 ± 68.9 1.35 ± 0.13 35.11 ± 5.96 0.13 ± 0.02 0.28 ± 0.05

9.2 ± 0.05 300.0 ± 23.4 64.5 ± 5.7 238 ± 22.3 45.0 ± 3.9 21.0 ± 1.4 0.14 ± 0.01 24.87 ± 1.6 0.4 ± 0.01 0.2 ± 0.01 0.8 ± 0.1 1306.4 ± 113.7 319.2 ± 31.6 0.3 ± 0.02 35.5 ± 2.6 0.2 ± 0.02 0.2 ± 0.01

7.0 – – – 41.2 53.4 – 6.8 – 0.4 1.0 105.5 10.5 0.4 77.5 – 2.0

shown in Table 1, even the nutrient concentration decreased after autoclaving, the TN and TP were still higher than those in standard Bold Basal medium (BBM). Thus, it did not markedly affect POME suitability as growth medium, demonstrated by high microalgae growth rate in the obtained results. The pH was adjusted to 7.0 ± 0.05 using 3M HCl before being used as culture medium. 2.2. Microalgae strain and culture condition The native microalgae strains used in this study were Characium sp. UKM1 (hereafter CR), Chlorella sorokiniana UKM3 (CS-N) and Coelastrella sp. UKM 4 (CO) with the NCBI accession number of KJ143753, KP262477 and KP691597 respectively. All strains were obtained from POME sources after series of isolation, purification and screening experiments by the previous researchers. The CR and CS-N was isolated from Sime Darby Palm Oil East Mill (Carrey Island) (2◦ 53′ 85′′ N, 101◦ 26′ 10.8′′ E) by Tamil Selvam et al. (2015), while CO was isolated by Ding (2016) from the effluent of Dominion Square Palm Oil Mill (Gambang) (3◦ 43′ 36.6′′ N, 103◦ 06′ 22.6′′ E). Meanwhile for the commercial strains, three microalgae were selected namely Chlorella sorokiniana SAG211-31 (CS-C), Desmedesmus armatus SAG276.4d (DS) and Chrocystic sp. SAG2512 (CT). The strains were obtained from culture collection of algae at the University of Göttingen, Germany (SAG). The microalgae were cultured in a standard medium (BBM) as a control experiment. The ingredients for BBM was prepared as per described by Ding et al. (2016). The experiments were conducted in batch mode under controlled temperature of 25 ± 2 ◦ C and exposed to 12:12 light/dark cycle at 7000 Lux intensity. The inoculation was performed under sterile condition to prevent any contamination using 20% (v/v) inoculum size. The aeration was provided continuously with 5% (v/v) of CO2 and 95% of atmospheric air. The CO2 used in the study was of purity 99.9% and obtained from high pressure cylinders supplied by Leeden gases, Malaysia. The ratio volume of compressed atmospheric air and CO2 gas was properly measured using flow metre and mixed, prior to entering the culture system. The CO2 supplied was used to maintain the initial pH of the cultures as well as to provide carbon source for the microalgae growth in BBM. 2.3. POME characterization The characteristics of POME and BBM were examined for essential nutrient and trace metal as shown in Table 1 (Mohd, 2017). The nutrient such as COD, TN and TP was measured according to HACH DR 2800 spectrophotometer manual (Ding et al., 2016). A 10 mL of POME sample was taken from the culture and centrifuged at 8000 rpm for 10 min. The supernatant was appropriately diluted prior to the analysis of respective elements (Hariz et al., 2018). The PO4 3− and NH4 + were determined using Ion Chromatography (882 Compact IC plus conductivity detector 1) (Metrohm, Switzerland) using a Metrosep A Supp 5 (150/4.0 mm) column and Metrosep C 4 100/4.0 respectively (Chantara et al., 2019; Halim et al., 2016). For nutrient characterization, the total nutrient was measured from the homogenized samples. Then, the soluble constituent was determined after filtering the sample through 0.45-µm membrane filter (Hülsen et al., 2016). The concentration of metals was analysed using Inductive Coupled Plasma-Optical Emission Spectroscopy (ICPOES) (PerkinElmer, USA). Calibration curve with appropriate dilutions of certified multi-element standard solution was performed to determine the elements concentration. The concentrations of total solids solid (TSS) and turbidity were determined according to the procedures described in APHA 1995 using a 10-ml sample in the 105 ◦ C oven and HACH 2100 AN Turbidimeter respectively.

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2.4. Microalgae growth measurement in BBM and POME The microalgae growth in BBM was quantified by gravimetric method. A 10 mL of sample culture was filtered through pre-weighted dried filter paper (47 µm GF/C Whatman) and oven-dried at 100 ◦ C for 24 h. Then, the filter paper with biomass residue was placed in a desiccator for at least 30 min and weighted again. The dry cell weight (DCW ) was calculated using Eq. (1): (

)

/

DCW gL−1 =(a−b)×1000 sample v olume(L)

(1)

where; a = weight of filter paper and biomass residue; b = weight of filter paper For growth analysis in POME, 20% of distilled water was added to improve the light penetration in the medium and growth was monitored for 20 days. The biomass was harvested and centrifuged at 8000 rpm for 10 min. Then the pellet was washed twice using distilled water to remove the remaining solid from POME before subsequent determination of DCW as per describe in Eq. (1). 2.5. Determination of the growth parameters The specific growth rate µ was obtained using Eq. (2):

µ = ln x2 −ln x1/∆T

(2)

where; x1 is the initial biomass dry weight, x2 is final biomass dry weight and T is the cultivation time in days. The cell division time, D′ and doubling time td were calculated based on Eqs. (3) and (4) respectively: D′ = td =

µ ln 2 1

(3)

(4) D′ Then, the final biomass accumulated (Y ) and biomass productivity (P) was measured once it reached stationary phase using Eqs. (5) and (6) respectively: Y gL−1 = (x2 − x1 )

(

(

P gL

)

−1

)

= (x2 − x1 )/(Ti − To )

(5) (6)

where; x1 and x2 is initial and final biomass dry weight respectively; Ti and To = Time (day) at x2 and x1 respectively. 2.6. Analysis of biomass nitrogen composition The elemental analysis of the biomass was conducted using CHNS Elemental Analyser (Flash EA 1112, Thermo Electron) to measure the nitrogen weight percentage in the microalgae. 2.7. Statistical analysis The statistical analyses of nutrient concentration in the effluents and microalgae growth kinetic were determined using the paired t-test and one-way analysis of variance (ANOVA) respectively. Data were processed using SPSS 21 software for Windows (SPSS Inc., Chicago). For all statistical tests, the significance level was set at 0.05. The experimental values represent the means of the triplicates along with the standard deviations and error bars in figures. 3. Result and discussions 3.1. Characterization of POME Table 1 shows the physicochemical characteristic of POME and BBM used in this study. The initial pH for anaerobic POME was 7.6 but higher pH of 9.2 was determined for final effluent. The pH is one of the important factor affecting microalgae growths since it influences cell metabolism and nutrient availability (Chavan et al., 2014). Generally, pH range between 7 and 9 is considered as the optimum for most microalgae but certain species could tolerate extreme pH levels (Ghosh et al., 2017). The total suspended solid (TSS) and turbidity were 2980 mg L−1 and 1940 NTU respectively in anaerobic POME. These noticeably high values might resulted in reduced light penetration into the medium and subsequently inhibit the growth of microalgae. Thus dilution of anaerobic POME may be required to improve its suitability for microalgae growth. In contrast, the light availability should increase significantly in final effluent due to much lower TSS and turbidity with 300 mg L−1 and 64.5 NTU respectively. The main nutrients required for production of microalgae biomass are C, N and P. These are the essential recipe in BBM solution, with addition of few trace elements as supplement. Importantly, analysis revealed that most of the nutrients in

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Table 2 Percentage of soluble nutrient in POME, mean (±SD).

Total nutrient Soluble nutrient Soluble (%)

Total nitrogen (mg L−1 )

Total phosphorus (mg L−1 )

COD (mg L−1 )

Anaerobic

Final effluent

Anaerobic

Final effluent

Anaerobic

Final effluent

385.0 ± 25.0 315.0 ± 30.9 81.8%

45.0 ± 3.9 40.6 ± 4.3 90.2%

106.0 ± 4.5 76.0 ± 5.9 71.7%

21.0 ± 1.4 16.5 ± 0.9 78.6%

2364.0 ± 211.8 1950 ± 121.9 82.5%

238 ± 22.3 214 ± 30.5 90.0%

BBM were sufficiently available in POME, thus showed its high potential as substitution to synthetic medium. The COD, TN and TP in anaerobic POME were found at 2364 mg L−1 , 385 mg L−1 and 106 mg L−1 respectively. These values exceeded the concentration found in BBM that has about 41.2 mg L−1 and 53.4 mg L−1 of TN and TP respectively. However, the TP of 21.0 mg L−1 in final effluent was lower than the concentration in BBM. Thus to prevent P-limited culture, addition of phosphorous might be needed to ensure proper microalgae growth in the final effluent. Further POME analysis shows the necessary trace elements to enhance microalgae growth such as potassium (K), iron (Fe), magnesium (Mg) and zinc (Zn) were also found to be adequate match to BBM. Beside C, macronutrients like N and P are the most important for microalgae growth and cell division. N is used mainly for the synthesis of amino-acids and proteins while P is needed in producing ATP and energy transfer (Dahmani et al., 2016). In anaerobic POME and final effluent, the concentration of N and P were significantly lower than their respective C concentration. The result was consistent with previous study that identified N and P as the key limiting nutrients for microalgae growth in wastewater (Cai et al., 2013). For that reason, the subsequent analysis was focused on these two elements to further assess the suitability of POME as growth medium. 3.2. Analysis of soluble nutrient in POME Nutrient in wastewater can be present in both solid and soluble forms (Van Moorleghem et al., 2013). This information is useful to anticipate the ability of microalgae to assimilate nutrients from the medium. Although insoluble nutrient can be slowly converted into an available form by natural processes, but soluble form has higher bioavailability to microalgae (Stark, 2000). Rapid nutrient uptake could be expected in dissolved form hence increased growth of the microalgae (Griffiths, 2009). The distribution of TN, TP and COD in the solid and soluble forms of the POME effluent is shown in Table 2. Majority of the TN and COD in the effluent was in dissolved form, with less than 20% in solid phase. A slightly lower percentage of 71.7% and 78.6% of TP in anaerobic and final effluent respectively were in dissolved form; which likely due to the precipitation of phosphate. P is more susceptible to pH changes and tend to react quickly with calcium and magnesium to form less soluble compounds at pH above 8 (Ferreira et al., 2017). In wastewater, nutrient concentration was commonly reported as the total concentration. However, it is worth noting that part of the total nutrient may be associated with particulate matter which is not immediately available to the microalgae. In this study, a significant amount of nutrient was present in soluble form thus signify POME high potential for microalgae cultivation. Nevertheless, separation of the dissolved nutrient compound through filtration is only a vague indication on the bioavailability. Unlike NH4 + and PO4 3− , some soluble organic are complex thus reduce their availability for uptake (Gerke, 2010). Furthermore, the bioavailability is also depending on several environmental factor such as pH, redox condition and occurrence of chelators (Boström et al., 1988). 3.3. Nutrient component in POME Further analysis was carried out to determine the distribution of nutrient components in TN and TP. Studies indicated that microalgae are known to prefer certain form of nutrient over others. For instance, NH4 + is regarded as favoured nitrogen source for microalgae due to lesser energy involve during assimilation and for the same reason, PO4 3− uptake is faster than other phosphorous species (Komolafe et al., 2014). Characterization of POME revealed that majority of TN and TP in the POME effluent were present in the form of NH4 + and PO4 3− respectively. As shown in Table 3, 97.4% of 315 mg L−1 TN in anaerobic POME was made up by NH4 + while PO4 3− constituted about 86.6% of 76.0 mg L−1 TP. A similar pattern was observed for final effluent where both NH4 + and PO4 3− constituted about 88.2% and 85.5% of TN and TP respectively. After the treatment using pond system at the mill, the concentration of PO4 3− in the final effluent was significantly reduced from 65.8 mg L−1 to 14.1 mg L−1 . The net PO4 3− removal in open pond system is generally the sum of microorganism absorption (such as bacteria, protozoa and fungi) (Krishnaswamy et al., 2009), uptake by phytoplankton (Sforza et al., 2018) as well as phosphorus precipitation (Ferreira et al., 2017). The latter is commonly brought about by the increased pH level, due to photosynthetic phytoplankton growth on the pond surface (Delgadillo-Mirquez et al., 2016). This phenomenon can be observed in final effluent, where pH reached 9.2 thus support further reduction of PO4 3− through precipitation. On this matter, the low level of phosphorous might induced P-limited condition hence decreased of microalgae growth and biomass accumulation (Sforza et al., 2018). On the other hand, higher concentration of N and P at 306.7 mg L−1 and 65.8 mg L−1 respectively found in anaerobic

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Table 3 Fraction of NH4 + and PO4 3− from TN and TP respectively in POME, mean (±SD). Anaerobic Final effluent

TN (mg L−1 )

NH4 + (mg L−1 )

TP (mg L−1 )

PO4 3− (mg L−1 )

315.0 ± 30.9 40.6 ± 4.3

306.7 ± 19.9 35.8 ± 3.0

76.0 ± 5.9 16.5 ± 0.9

65.8 ± 6.1 14.1 ± 1.0

Fig. 1. Growth profile of microalgae species cultured in Bold Basal Medium (BBM) [Characium sp. UKM1 (CR), Chlorella sorokiniana UKM3 (CS-N), Coelastrella sp. UKM4 (CO), Chlorella sorokiniana SAG211-31 (CS-C), Desmedesmus armatus SAG276.4d (DS), Chrocystic sp. SAG251-2 (CT)].

POME could support prolong growth. As a result, higher biomass is accumulated thus more appealing from biomass commercialization perspective. However, according to Sharma et al. (2016), the elevated level of NH4 + in wastewater might cause detrimental effect to the microalgae. Even though higher nutrient concentration is important to increase biomass production, but excessive level can becomes toxic and retards their growth (Tan et al., 2016). Therefore, growth analysis was performed to assess the tolerance of different microalgae species in anaerobic POME. 3.4. Comparison of microalgae growth in BBM and potential of POME as culture medium 3.4.1. Microalgae growth in BBM Fig. 1 show the growth curve for six microalgae cultivated in BBM. There was no lag phase observed from all species except for minimal lag showed by CT. Synthetic medium such as BBM contains known composition of chemical that could stimulate the growth for microalgae. The complete nutrient composition in BBM has contributed to rapid growth of the microalgae. Evidently, the microalgae growth started instantly without the need of physiological acclimation. In addition, the nature of synthetic medium that is clear from any suspended solid provides plenty of light for photosynthesis activity. It can be observed that all species entered stationary phase after 9 days of exponential growth. However, an exception is showed by CR that went into stationary phase in shorter period of 5 days after steepest exponential phase seen amongst other species. The growth was further assessed by determining the kinetic parameter as shown in Table 4. The highest specific growth rate, µ of 0.5 day−1 was achieved by CR. This was almost fourfold over the lowest µ of 0.13 day−1 recorded by CT. Interestingly, both C. sorokiniana strains; a locally isolated CS-N and commercial strains, CS-C produced near identical result even though they were originated from different geographical location. This shows that the same species isolated from different sources will develop the same growth characteristic when cultured in standard medium under controlled condition. The microalgae cell divisions per day and generation time were also used as indicators for growth comparison between species. High division rate naturally show the cells are dividing rapidly. In contrast, a large doubling time corresponds to a low specific growth rate. The doubling time, or generation time, td was found to be the least for CR with 33 h (1.4 day) that represent 1.7 times faster than both CS-N and CS-C on 57 h (2.39 day) and 59 h (2.45 day) respectively. Notably, the maximum biomass production was also in accordance to the growth rate of respective microalgae. Highest biomass yield of 0.315 mg L−1 was accumulated by CR with estimated 36 mg L−1 daily productivity. However, despite comparable specific growth rate between CS-N and CS-C, lower biomass was produced by CS-C. This was due to a lower initial biomass density during inoculation, where 25 mg L−1 was used for CS-C as opposed to 40 g L−1 inoculum size used during CS-N inoculation.

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Table 4 Microalgae growth kinetics in BBM and anaerobic POME, mean (±SD). Microalgae strains

BBM CR CS-N CS-C CO DS CT

Division, D′ (day−1 )

Growth rate, µ (day−1 )

0.50 0.30 0.28 0.18 0.16 0.13

POME

± ± ± ± ± ±

0.04 0.02 0.02 0.01 0.01 0.01

0.17 0.24 0.23 0.17 0.12 0.09

± ± ± ± ± ±

BBM 0.01 0.02 0.02 0.01 0.01 0.01

0.72 0.42 0.41 0.27 0.23 0.20

Generation time, td (day) POME

± ± ± ± ± ±

0.06 0.03 0.02 0.02 0.02 0.02

0.24 0.35 0.33 0.25 0.17 0.13

± ± ± ± ± ±

BBM 0.01 0.03 0.03 0.01 0.01 0.01

1.40 2.39 2.45 3.75 4.40 5.13

Productivity (mg L−1 day−1 ) POME

± ± ± ± ± ±

0.12 0.14 0.14 0.30 0.37 0.46

4.19 2.85 2.99 4.06 5.74 7.65

± ± ± ± ± ±

0.24 0.21 0.27 0.28 0.46 0.73

Biomass, Y (mg L−1 )

BBM

POME

BBM

36 ± 2.84 21 ± 1.32 13 ± 0.68 19 ± 1.61 16 ± 1.26 9 ± 0.73

60 ± 3.12 107 ± 8.56 115 ± 10.00 61 ± 3.96 53 ± 3.98 20 ± 1.96

315 271 173 243 218 126

POME

± ± ± ± ± ±

28.35 19.24 9.86 22.11 17.66 10.71

1430 ± 87.2 2400 ± 194.4 2500 ± 215.0 1450 ± 88.4 1322 ± 103.1 174 ± 16.53

Fig. 2. Growth profile of microalgae species cultured in palm oil mill effluent (POME) [Characium sp. UKM1 (CR), Chlorella sorokiniana UKM3 (CS-N), Coelastrella sp. UKM4 (CO), Chlorella sorokiniana SAG211-31 (CS-C), Desmedesmus armatus SAG276.4d (DS), Chrocystic sp. SAG251-2 (CT)].

3.4.2. Microalgae growth in anaerobic POME Fig. 2 shows the growth of microalgae in anaerobic POME for 20 days of cultivation period. Expectedly, there was prolonged lag phase seen for all strains as the microalgae require time to acclimatize in the new surroundings. The natural characteristic of POME that contains extremely high suspended solid has blocked the light penetration thus impeded the microalgae growth. However, it can be seen from the graph that both CS-N and CS-C was clear distinguished from the rest. This result is in agreement with previous research that demonstrated extreme ability of Chlorella sp. to adapt to wastewater environment (Hu et al., 2016). Chlorella sp. is well known to be mixotrophic microalgae which mean they can simultaneously consume inorganic carbon in the present of light (photoautotrophic) and organic carbon in the dark (heterotrophic) (Subramaniyam et al., 2016). A study by Ding et al. (2016) indicates that acetate which commonly found in POME has been used as carbon source by microalgae in the dark section of the culture medium. In all probability, this has contributed to remarkable adaptability of the C. sorokiniana in dark coloured POME. Another native strains, CO was fairly adapted after initial 8 days of lag phase and followed by exponential growth that lasted for 6 days. Meanwhile, CR needed considerable long time of about 10 days before it started to growth in POME, a stark contrast compare to its high growth rate in BBM. As for the other two commercial strains, DS shows comparable growth as per CR, while CT seems to be struggling to adapt in hostile POME conditions throughout the experiment period. Table 4 shows the specific growth rate for all species in POME and it was noticeably lower than the results obtained in BBM. This could mainly due to the limited light penetration that significantly reduced the ability of microalgae to conduct photosynthesis. The CS-N and CS-C shows the highest growth with µ of 0.24 day−1 and 0.23 day−1 respectively with both averaging of 0.34 day−1 division rate. As predicted, extended doubling time was attained for cultivation in POME as compared to BBM with the shortest was 68 h (2.85 day) for CS-N followed by 72 h (2.99 day) by CS-C. The rest of the strains required much longer period with minimum of 97 h (4.06 day) up to 184 h (7.65 day) thus confirmed the Chlorella sp. superiority for application in wastewater effluent (Mohammadi et al., 2018). In general, native strains showed better growth rate as compared to commercial strains. CR and CO recorded higher growth rate in comparison to DS and CT. Result is consistent with previous research that highlighted better tolerance of stress by native strains upon cultivation in local habitat (Jazzar et al., 2015). Interestingly, despite the limited light, all microalgae produced more biomass in POME than in BBM with the exception of CT. This could be attributed to microalgae growing in POME was able to adopts mixotrophic growth strategy, as opposed

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Table 5 Comparison of growth kinetic for microalgae in different wastewater sources. Wastewater type

Microalgae

Specific growth, (µ day−1 )

Productivity, (mg L−1 day−1 )

References

Piggery wastewater Textile wastewater Domestic wastewater Dairy wastewater Aquaculture wastewater Palm oil mill effluent (POME)

C. zofingiensis C. pyrenoidosa C.vulgaris Chlorella sp. Chlorella sp. C. sorokiniana

0.32 0.20 0.21 0.32 0.29 0.24

273.30 8.10 128.20 260.00 459.00 107.50

Zhu et al. (2013) Pathak et al. (2015) Cabanelas et al. (2013) Lu et al. (2015) Kuo et al. (2016) This study

to photoautotrophic growth in BBM. In BBM, the injected CO2 was the only inorganic carbon source for the microalgae (Frumento et al., 2016). Thus in the absent of light during the light/dark cycle, the assimilation of inorganic carbon was inhibited hence reduced the biomass production. On the other hand, the presence of higher nutrient concentration in POME has a direct influence on biomass accumulation (Hu et al., 2016). CS-C attained highest biomass productivity with 115 mg L−1 per day with final yield accumulated was about 2.5 g L−1 . According to He et al. (2016), maximum biomass increased with higher initial concentration of nutrients in the medium. The concentration of TN in POME used in this study was 385 mg L−1 which was nearly ten times higher than BBM. Similarly, the concentration of TP in POME was 106 mg L−1 which doubled the concentration found in BBM. Nevertheless, previous studies have reported excessive amount of NH4 + can be toxic to certain species of microalgae (Muñoz et al., 2005). Based on the result, the high ammonium concentration in POME did not inhibit the growth of microalgae species used in the present study. Therefore it can be concluded that POME shows excellent potential as nutrients source for cultivating different species of microalgae without any notable negative impact. Table 5 summarize the data from previous studies on the growth rate and biomass production of Chlorella sp. in different type of wastewater sources. Generally, the growth rate in POME was comparable to other type of wastewater with µ averaging between 0.20 day−1 (Pathak et al., 2015) to 0.32 day−1 (Lu et al., 2015; Zhu et al., 2013). On the contrary, there were large variations in biomass productivity, most notably between aquaculture (Kuo et al., 2016) and textile wastewater (Pathak et al., 2015). The former produced the highest rate of 459 mg L−1 day−1 while only 8.1 mg L−1 day−1 was produced by the latter. This huge different was definitely influenced by initial nutrient concentration in the experiments. Textile industry is predominantly polluted with dye instead of nutrient, thus cell division rate and biomass accumulation is reduced. Furthermore, the adaptability of each species is varies in different wastewater with specific characteristic (Mondal et al., 2017). On the other hand, the results from piggery (Zhu et al., 2013) and domestic sources (Cabanelas et al., 2013) showed biomass productivity of 273 mg L−1 day−1 and 260 mg L−1 day−1 respectively. Even though these results were higher than POME, it was achieved using much diluted sample coupled with continuous lighting to the cultures. Therefore, the finding of the present study shows the competency of POME to serves as alternative medium for microalgae growth among other source of wastewater. Using appropriate technology, the biomass produced could be utilized for wide range of applications such as biogas, health supplements and animal feed (Gatamaneni and Lefsrud, 2018). Among this great diversity on the potential products, direct application of the biomass as fertilizer has attracted much interest due to their high N and P content (Coppens et al., 2016). This is especially true for the microalgae cultivated in wastewater where assimilation of nutrient into biomass is the major uptake mechanisms. In this study, elemental analysis of CS-N biomass revealed that the percentage of internal N increased from 6.2% to 8.7% when cultured in BBM and POME respectively. Therefore, this nutrient-rich biomass residual could be subsequently harvested and reapplied as fertilizer. Albeit the growing interest and impressive growth of microalgae in POME, the application is still on its infancy. More research must be carried out to improve the growth of microalgae in POME. Growth rate can be accelerated by extensive study on the adaptability of specific microalgae strains in different set of experimental and culture conditions. It is also crucial to explore the most practical and feasible approach to improve light penetration in the culture. As light intensity increase, greater rate of photosynthesis could be attained thus maximize the system performance. Thorough understanding on these matters would definitely increase the success of microalgae growth in POME for the purpose of any specified application. 4. Conclusions The characteristic of POME obtained from this study signify POME suitability as microalgae growth medium. The concentration of C, N and P were found higher than those in BBM. The trace element essential to microalgae such as Fe, Mg and K were also sufficiently present. Importantly, most of the nutrient was in soluble form, thus highly bioavailable to microalgae. The best growth rate in POME were achieved by native strains, however Chlorella sorokiniana from commercial source shows excellent adaptability in POME. The results highlighted the feasibility of POME as sustainable medium for microalgae biomass production. However, comprehensive studies to enhance the growth rate of the microalgae using this wastewater-recycling approach remain essential in the future.

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