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Comparative toxicity effect of organic and inorganic substances in palm oil mill effluent (POME) using native microalgae species
Journal of Water Process Engineering 34 (2020) 101165
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Comparative toxicity effect of organic and inorganic substances in palm oil mill effluent (POME) using native microalgae species
T
Jannatulhawa Jasnid, Shalini Narayanan Arishtd, Nazlina Haiza Mohd Yasinb,*, Peer Mohamed Abdula,d, Sheng-Kai Linc, Chun-Min Liuc, Shu-Yii Wuc, Jamaliah Md Jahima,d, Mohd Sobri Takriffa,d a
Research Centre for Sustainable Process Technology, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia b Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia c Department of Chemical Engineering, Feng Chia University, Taichung 40724, Taiwan d Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia
ARTICLE INFO
ABSTRACT
Keywords: Mix-culture microalgae Toxicity POME EC50 Ecotoxicology
Phycoremediation of palm oil mill effluent (POME) using native microalgae promotes sustainable agricultural wastewater treatment technology. However, POME is characterized by a high amount of organic and inorganic compounds. Therefore, the aim of this paper is to study the toxic effect of selected organic (acetic acid, butyric acid, propionic acid, formic acid, oil and grease) and inorganic (copper) compounds presence in POME on the microalgae population. A mix-culture of three native microalgae species consisted of Coelastrella sp. UKM 4, Chlorella sp. UKM 8 and Scenedesmus sp. UKM 9 were exposed for 120 h to different organic and inorganic substances. Then, microalgae growth response, the changes of pH and the half-maximal effective concentration (EC50) were studied for both short-term (24 h) and long-term (96 h) exposure. The EC50 indicated that acetic, butyric, formic and propionic acids promote microalgae growth at both short and long-term exposure (survival rate > 50 % at all selected concentrations) whereas copper, as well as oil and grease, suppress microalgae growth (survival rate < 50 %) during short-term exposure. The most favorable growth rates of microalgal were in the pH range of 7–9. These findings show that it is feasible to cultivate microalgae in POME with a suitable amount of organic and inorganic compound.
1. Introduction For the past decade, palm oil mill effluent (POME) has been treated in a series of treatment pond that includes anaerobic, aerobic or facultative ponds to reduce the contaminant level in wastewater before being flown to the river. However, these conventional methods are listed as non-sustainable and non-environmental friendly due to the discharge of a large amount of nutrientloaded waste into the water bodies contributing emission of greenhouse gaseous and air pollution [1]. Due to the worsening effect in environmental health, the regulation implemented on industrialized discharge to be more stringent [2]. In conjunction with zero-waste management technologies, every palm oil mill must take part in research and development for better waste management [3,4]. Recently, there has been a great interest from researcher to obtain
algal biomass grown in wastewater to minimize overall production cost as it can simultaneously offer a synergistic effect for wastewater nutrient removal, carbon capture and production of valuable biomass for various commercial applications such as animal feed, biofertilizer and biopharmaceutical products [5]. A recent study conducted by [6] had proven that POME could be utilized as a growing medium for marine diatom Phaeodactylum tricornutum and subsequently produce sulfated exopolysaccharide and nutrient removal. It is also suggested that the optimum concentration of POME for microalgae cultivation is 30 % as the growth rate could be inhibited beyond that concentration [6]. To date, phycoremediation has been used for over 70 years in wastewater treatment as it will further enhance the treatment activities through their efficient photosynthetic metabolism and effectively fix CO2 [7]. However, the amount of organic and inorganic materials in the wastewater sometimes can exceed the optimum acceptable level that
⁎ Corresponding author at: Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. E-mail address: [email protected] (N.H. Mohd Yasin).
https://doi.org/10.1016/j.jwpe.2020.101165 Received 25 September 2019; Received in revised form 23 January 2020; Accepted 24 January 2020 2214-7144/ © 2020 Elsevier Ltd. All rights reserved.
Journal of Water Process Engineering 34 (2020) 101165
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microalgae can adapt. Therefore, the knowledge of toxic effects and concentration of non-inhibiting concentrations of wastewaters is important to retain a high biomass rate for high treatment capacity. As characterized, POME contains a high amount of organic materials and nutrients that make it as a potential low-cost substrate for cultivating microorganism especially microalgae [8]. However as per other wastewaters, POME is also known to contain a certain range of inhibitors and toxicants. Roughly, it consists of 0.6–0.7 % palm oil, 95–96 % water, 4–5 % total solid, and 2–4 % suspended solids [9]. According to [10], the nutrients in POME consist of 2726 (mg/L) of COD, 1270 (mg/L) of TOC, 316.7 (mg/L) of TN and 257.6 (mg/L) of phosphate. Among these components, there are compounds which can inhibit the growth of microalgae by reducing microalgae growth, photosynthetic activity, enzymatic activity and respiration [11]. The cultivation of microalgae in POME is challenging since the dark brown POME inhibit light penetration. However, POME pre-treatment (i.e. chemical, biological and mechanical pre-treatment) can be done prior to microalgae cultivation to permeate light to pass through. Hence, appropriate microalgae selection is important. In this study, mixotrophic microalgae were used. The advantage of using mixotrophic microalgae is it uses both inorganic and organic carbon sources under both photo-autotrophic and heterotrophic process simultaneously [12]. Therefore, if the POME unfavorable, the microalgae mode switch to the heterotrophic mode and vice versa. Aquatic microalgae are often used in inhibition test than other types of aquatic organism because of its extremely sensitive to the wide range of pollutants [13]. Current ecotoxicology test using mix-culture microalgae as biotest is still limited to monoculture. Mix-culture is better in ecotoxicology as it is more realistic to mimic their real nature conditions [14]. It can be severely affected by environmental changes and therefore, can provide meaningful indicators of ecological change over short time scales [15]. The toxicity inhibition level can be expressed in EC50, the half maximal effective concentration of the toxicant that inhibits the 50 % of the algal growth [16]. Calculation of half maximal effective concentration (EC50) for biomass (cellular density) corresponding to the exponential phase approximation of EC50 for growth rate allows us to estimate complete toxic effects on growth parameters. An organic compound like acetic acid, butyric acid, propionic acid and formic acid are commonly found as POME by-products. It is listed as a volatile fatty acid (VFA) that can negatively impact the environment [17]. However, it also can become a source of carbon for some microalgae [18]. Several studies had also utilized POME as carbon for lipid production and pollutants removal, carbon and nitrogen sources for food-grade yeast biomass production, carbon source for supporting ammonia-oxidizing bacteria (AOB) in ammonia removal, a substrate for bacteria to produce poly-β-hydroxyalkanoates, biohydrogen and biomethane [19–24]. Excessive amounts of oil and grease in POME can hinder sedimentation, which causes losses of biomass and overall affecting a reduction in the efficiency of treatment stations [25]. Hence, an effective handling system of POME, such as through phycoremediation is crucial and has been a great challenge for oil palm industries. This work aims to evaluate the toxic effect of selected organic
(acetic acid, butyric acid, formic acid, propionic acid, copper and oil & grease) and inorganic (copper) compounds which are major constituents in POME towards microalgae population. POME may contain a lot of nutrients. Therefore, this research utilized BBM as growing media to see the sole effect of the selected toxicants on mix-culture microalgae and the concentration range were selected based on the range presence in POME as a standard reference. The test duration and the possible adaptation of the microalgae to the test material and their effect towards toxicants on physicochemical properties, including pH during microalgae growth were investigated. These results reveal the proposed potential toxic concentration of organic and inorganic materials that microalgae can tolerate in POME for wastewater treatment purpose. 2. Materials and methods 2.1. Microalgae and media preparation Three inoculum mix-culture species of native microalgae Coelastrealla sp. UKM4 (KP691597), Chlorella sp. UKM8 (KT452082) and Scenedesmus sp. UKM9 (KU170547) which was previously isolated were prepared in a sterile condition in two-liter Schott bottle using the sterile Bold Basal Medium (BBM). All chemical component of test medium used were dissolved in ultrapure Milli-Ro water (Kemflo AICRO-Q Water Filters, USA) and adjusted to pH 7 using 5 M of NaOH and HCl. The BBM medium, consisted of following components (g/l) [26]: (g/l): CaCl2·2H2O (25), K2HPO4 (75), MgSO4.7H2O (75), H3BO3 (11.4), NaNO3 (250), NaCl (25), EDTA.Na2 (50), KH2PO4 (173.8), FeSO4.7H2O (4.98), H2SO4 (1), ZnSO4.7H2O (8.82), MnCl2.4H2O (1.44), MoO3 (0.71), CuSO4.5H2O (1.572), Co(NO3)2.6H2O (0.49). The incubation conditions were maintained at 25 °C with a continuous air supply at the rate of 1 L/min and continuous illumination at 3000 lx. The activated inoculum was kept in an active condition during the preparation of microalgae to achieve OD 1.0 prior to inoculation. The test microorganism flasks are shaken twice a day and placed in the culturing apparatus. 2.2. Standard Chemical toxicants The concentration range of chemical toxicants in which the effects on the growth of microalgae are likely to occur may be determined based on the results from range-finding tests. For the final test, six concentrations were tested based on the arrangement in a geometric series. The concentration series should preferably cover the range causing 0–90 % inhibition of algal and bacterial growth. Table 1 exhibits the characteristics of POME and test concentration range used in this experiment. 2.3. Incubation Flasks of BBM containing toxicants (Test 1–6) (Table 1) were inoculated with a mixed culture of microalgae. The algal morphology were checked regularly using Olympus BX51 microscope (Olympus, Center Valley, PA, USA) to ensure no sign of contamination. Thirty per
Table 1 Characteristics of POME and test concentration range used in the experiment. Component
Acetic acid (AA) Butyric acid (BA) Formic acid (FA) Propionic acid (PA) Copper Oil & grease (O&G)
Characteristics of organic and inorganic substance in POME [27,28,29]
Test concentration range (mg/L) in Bold Basal Media
Mean (mg/L)
Range (mg/L)
1
2
3
4
5
6
3540 8.15 1000 29.36 0.85 7213
3030-4050 16-290 300-1700 58-720 0.80–0.90 5614–8812
3000 66 300 50 0.4 4500
3200 116 600 200 0.6 5500
3400 216 900 350 0.8 6500
3600 266 1200 500 1.0 7500
3800 316 1500 650 1.2 8500
4100 366 1800 800 1.4 9500
2
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cent (30 ml) from the stock culture was inoculated into 250 mL conical flask and were top up with BBM until 100 mL. The toxicants were added into the culture with their respective concentrations. The cultures were then incubated in the incubator for 120 h at 25 °C with 12 h/12 h illumination (3000 lx) by white fluorescent lamps (Philips) without aeration. The salinity was maintained at 0 ppt and were monitored using a refractometer. The flasks with mix culture of microalgaewere shaken twice a day and placed in the culturing apparatus. The initial biomass was standardized at 100 mg/L, equivalent to 8.6 × 107 cfu/ mL. All the experiments were conducted in triplicates.
Y= aX + c ( a is in+ ve or
3. Result and discussion 3.1. Algae growth response curve to toxic substances The toxic effect of organic (acetic acid, butyric acid, formic acid, propionic acid) and inorganic (copper and oil & grease) on algal growth were examined by measurement of microalgae biomass concentration throughout cultivation. As described by [36], non-competitive inhibition indicates that transformation can occur at every concentration level of the inhibiting compound. The inhibition was the dependent variable, while the concentrations of organic and inorganic substances were the independent variables. Conflicting the other orders of reaction, a zero-order reaction has a rate that is independent of the concentration of the substances. As such, increasing or decreasing the concentration of the reacting algal species will not speed up or slow down the reaction rate as they have the same level of inhibition towards the algal cell. The initial microalgae cell density in this experiments were kept constant at 100 mg/L, as shown in Figs. 1 and 2. From the results, it was found that organic matters used in this experiment has a stimulant effect on microalgae growth at different concentrations. AA was found as the best growth stimulant. Among tested concentration of AA (Fig. 1a), the highest biomass was observed during the exposure to the highest concentration of AA, 4100 mg/L. The lowest biomass of microalgae at AA more than 3.0 × 100 mg/L was observed at concentration of 3400 mg/L, which is still higher than the others which their lowest biomass nearly to zero. According to [37], AA is one of the most common carbon sources for many microbial species, including microalgae. Metabolically, microalgae assimilate AA into coenzyme A to form Acetyl-CoA for carbon skeletons of ATP and NADH. Meanwhile, butyric acid, formic acid and propionic acid show a similar trend of response towards microalgae growth by showing a stimulant effect at lower concentrations and inhibition effect at the higher concentrations. This might be because most of the single compound of fatty acids provide an additional carbon source for the growth of microalgae. As explained by [38,39], AA could be toxic for many microorganisms at high concentrations with the exception to Chlamydomonas mundana which proliferated in the presence of acetate. For this reason, AA is frequently used to buffer the pH value in bioreactors for microalgae culture. PA, on the other hands, was suggested to provide oxaloacetate to the cells and therefore, can improve the cellular growth [40]. Furthermore, the mixotrophic nature of mix-culture microalgae used in this experiment can heterotrophically utilize organic carbon sources to increase their biomass production. The decline in microalgae biomass at the end of the experiment can be explained due to a depletion of nutrients, overheating, pH disturbance, or contaminations. An inorganic matter like copper (Fig. 1e) used in this experiment shows a negative impact on microalgae growth. It can be seen that the additional copper in the growth media proportionally inhibits microalgae growth at any concentrations. Thus, copper concentration should be kept low if the wastewater to be used as a media for microalgae culture. As for oil & grease (Fig. 1f), it may cause a reduction in microalgae growth as the amount increased. However, it does not contain any harmful effect on microalgae growth. The declining growth phase in oil & grease can be observed by the reduction of the cell division rate due to limiting factors, such as nutrients and aeration supply. The oil masked the surface of the media preventing the gas exchange and light penetration, making the anaerobic condition for the microalgae. The mixotroph algae in this experiment do not sustain anaerobic condition.
2.4.1. Growth culture The growth of algae in test substances was determined by measurement of the optical density of a culture with a spectrophotometer at 685 nm. Meanwhile, the biomass was measured by analysis of dry weight by using standard profile of OD at 685 nm versus microalgae biomass concentration (mg/L). The microalgal biomass was obtained daily from 15 mL of sample and centrifuged at 2862×g for 10 min. Prior to determine the dry cell weight (DCW), the cell concentration were measured using the Varian Cary 50 spectrophotometer with UV/ VIS detector (Varian Cary, Agilent, Santa Clara, CA, USA). Then, the samples were filtered using 0.45-μm micro fiberglass filters (GF/C). The filtered biomass was dried at 105 °C in a drying oven for 24 h, and the weight was recorded. The standard curve of optical density versus dry weight were constructed. 2.4.2. Toxicants growth curve analysis Scatter plot with linear line was applied to optimize key factors affecting biomass concentration of microalgae. Scatter line plot was used to optimize the level of toxicants (mg/L) and days (120 h) on biomass concentration. The response variable is biomass concentration (x 100 mg/L). The inhibition by a toxic compound can be described with the following model for non-competitive inhibition on the assumption that the inhibition is zero-order:
Si . c Si . c + Si
(1)
Where, Si is concentration of inhibiting compound (mg/1); Si.c is inhibition constant for which kobs.tox at 1 kmax (mg/l); kmax is maximum 2 (0′ order) biomass concentration rate (mg/l/h) and kobs.tox is observed biomass concentration rate, when a toxicant is present (mg/l/h) [30]. The statistical software SigmaPlot14, (Version 14.0, San Jose, CA, USA) is used for regression and graphical analysis of the experimental data. The quality of fit of the quadratic model is expressed by the coefficient of determination, R2, and its statistical significance is checked by the F-test. 2.4.3. Half-maximal effective concentration (EC50) Different concentrations of toxicants were used to culture mixed microalgae. The biomass concentration of each culture in the triplicate samples was calculated by comparison with the biomass concentration in the control culture (BBM) in the absence of toxicants. The average specific biomass concentration rate for a specific period was calculated as the survival rate (%). Average of specific biomass concentration was obtained from Eq. (2) [31] over concentration (mg/L) [32]. Then, it was plotted using Sigma Plot Software 14.0 (Systat Software Inc., USA) and half-maximal toxicity was calculated using linear regression line Equation (3) [33–35] for each substance.
% cell survival=
Bsample
Bblank
Bcontrol
Bblank
× 100
(3)
Where, Y is 50 % from the overall maximum inhibition percentage (y-axis); X is EC50 concentration value in the mg/L concentration at which 50 % inhibition of the toxicants achieved; values a is positive or negative steep slope and c is the intercept on y-axis are generated by the SigmaPlot linear fit curve operation.
2.4. Measurement and analytical test procedure determination
K obs . tox = kmax +
ve depending on the data)
(2)
Where, Bsample is biomass concentration of specific toxicant concentration; Bblank is biomass concentration of blank and Bcontrol is biomass concentration of control. 3
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Fig. 1. Experimental data of the growth kinetics for algae at selected toxicants, Acetic acid, a); Butyric acid, b); Formic acid, c); Propionic acid, d); Copper, e) and Oil & Grease, f) at different days, and fittings to Eq. (1).
Overall, the organic matters have both positive and negative impact on the microalgae growth at a certain range of concentration by which AA becomes the most favorable towards the microalgae growth. In contrast, copper has a negative effect on the microalgae growth at any concentrations.
3.2. Effects of pH towards microalgae growth activity The effect of pH on microalgae growth was initially investigated in this study. The pH reading for O&G are not shown on this figure as pH meter is sensitive to high oil content. The pH was a crucial factor 4
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influencing metabolic effect in microalgae cells. pH 7 was used as the initial point for all analysis as at the neutral pH of most freshwater habitats (pH 6–8), proton acidity is the most important component while mineral or organic acidity are minor contributors [41]. Two pHdependent conceptual models were proposed for test organisms based on observed responses to variable pH values (Fig. 2a to 2e). These include Model-I: decreasing toxicity with increasing pH (positive correlation), Model-II: increasing toxicity with decreasing pH (negative correlation) [42]. The effect of pH on microalgae growth as shown in Fig. 2a to 2e plays a crucial role as the pH decreases at 120 h exposure, the survival rate decreases. The growth rates for all five organic and inorganic matter exhibited a similar pattern over the pH range (around pH 3.0–10.0) with the optimum pH values for growth obtained between pH 7.0 and 9.0. By comparing Fig. 1 and Fig. 2, algae growth rates decreased when the pH values drop below pH 5. In general, pH values of 7–9 might be most conducive to increased algae production and minimizing invading organisms [43]. The pH of acetic acid, formic acid, propionic acid and copper drop below neutral pH after 24 h exposure except for butyric acid. Butyric acid showed a slightly increase in pH after 24 h exposure and decrease its pH after 48 h exposure. The same pattern can be observed for acetic acid, formic acid and propionic acid after 72 h, which the pH was increasing and started to fall back after 96 h exposure. However, butyric acid and copper continuoes to increase its pH until 120 h of exposure. The mix-culture microalgae used in this experiment seems to withstand low pH condition after manage to regain back their neutral pH after 72 h. However, at higher concentration of butyric acid (316 and 366 mg/ L) propionic acid (650 and 800 mg/L) and copper (1.2 and 1.4 mg/L), the pH maintains in acidic condition (< pH 5) after 72 h. According to [44], the nature of the freshwater microalgae are robust microorganism, and it has a wide range of pH adaptation. They can increase tolerance to acidic and alkaline pH as an adaptive response to widely fluctuating pH levels that occur commonly in many productive freshwater environments [45]. The homeostasis maintained by proton efflux and influx mechanism of microalgae is based on the cell to the environment communication [44,46]. Microalgae can regulate their internal pH through proton exchange, so whatever the extracellular pH may be, the cytoplasmic pH always remains near neutral [44]. Besides, microalgae cell wall and production of extracellular polysaccharide to neutralize basic conditions can protect microalgae cell from pH damage [44,47]. In this study, the reduction of pH values does affect the survival rate of mixed microalgae culture. The previous report has shown that butyric acid at concentrations of 1–5 mM was found to be highly toxic corresponded to the pH values around 4–5. However, it was relatively harmless at pH 6. On the other hand, propionic acid was highly toxic within pH range around 4–6 [48]. The results obtained from this study support previous findings that the complex forms of the organic acids are more toxic. This is due to the drop of pH values which corresponded to the low pKa value in the media. However, some microalgae favor acidic condition, which they may utilize this condition for better growth and survival. These observations suggest that pH assessment over growth rates possess resistance and enhanced growth rate.
substances have negative slope graph except for acetic acid, which has a positive slope. The negative linear fit line shows that increasing substance concentration reduces survival rate whereas positive fit line shows vice versa. From Fig. 3, the EC50 indicated that AA, BA, FA and PA promote microalgae growth at both short and long-term exposure (survival rate > 50 % at all selected concentrations) whereas, copper and oil grease suppress microalgae growth (survival rate < 50 %) during shortterm exposure. This might be due to the sensitivity differences in uptake rates across the plasma membrane and internal binding mechanisms of microalgae towards toxicants [49]. AA seems to have a high survival rate (> 100 %) at short-term exposure and being highest (113.83 % survival rate) at a concentration of 4100 mg/L (P < 0.05) during 96 h exposure as it is proven to be more easily consumed by several microorganisms by previous research. As stated by [50], acetate is preferred during the early exponential phase for microalgae growth and their presence as an organic compound supplies nutrient assimilation for microalgae carbon skeleton. The metabolic oxidation for AA and BA are very much the same except for butyrate uptake by microalgae is much slower and can reduce the microalgae growth when both are present. It was found that at much higher concentrations (above 500 ppm), the y-butyric acids were found to be highly toxic, whereas the acetic acids have an inhibitory effect on growth [51]. This explains why the survival rate in AA are much higher than BA during long-term exposure. Fifty percent effective dose (the ecological dose resulting in a 50 % decrease in activity), EC50 values in BA, FA, and PA significantly decreases (P < 0.05) from 24 hours–96 hours, 896.68 mg/L to 498.84 mg/L, 6108.79 mg/L to 2412.28 mg/L and 1798.34 mg/L to 828.55 mg/L respectively. Conversely, the EC50 value for AA Table 2, Cu and O &G significantly increases (P < 0.05) from 91.93 mg/L to 1947.56 mg/ L, 0.1766 mg/L to 0.7485 mg/L and 6402.14 mg/L to 8901.83 mg/L, respectively through 24 hours–96 hours incubation (Fig. 3). During the 24 h and 96 h exposure, majority all of the organic matters survival rate above 50 % whereas majority survival rate for O&G and Cu fall below 50 % (Table 2),(P < 0.05). Microalgae survive better in organic matter because they serve as carbon sources to support microalgae growth [18] while inorganic matter like copper always suppresses their growth by messing their metabolic pathway. Growth inhibition in microalgae is mainly related to free ions and its sensitivity varies among microalgae [52]. As explained by [53], the effect of Cu towards microalgae depends on the composition of culture medium, experimental protocol and species used. Therefore, direct comparison of sensitivity among microalgae due to copper exposure is difficult but can be minimized at least by using a similar experimental protocol. Cu often causes the generation of reactive species (RS) which cause algal growth and photosynthetic inhibition [54]. It is proven that the increasing level of RS can lead to severe cellular injury or death to cells [55] [52,55] reported that copper in ionic form is able to inhibit the synthesis of D-aminolevulinic acid and protochlorophyllide reductase, peroxidative breakdown of pigments and membrane lipids by reactive oxygen species at higher concentration thus causes chlorophyll-α reduction. From the results obtained, algae have high toxicity tolerance due to its microorganism characteristics, such as has a thicker cell wall being eukaryotic cell. Some microalgae can tolerate at a very high concentration of AA. However, BA also promotes the growth of microalgae at a very much lower concentration as compared to AA. Therefore, it is somewhat to say that AA and BA could be used as a microalgae enhancer. It is suggested that if the microalgae are to be cultured in wastewater containing both AA and BA, the algal biomass can be manipulated either by increasing the initial mix culture biomass or by manipulating the initial acetate: butyrate ratio [51]. Based on the comparison of previous studies on toxicity level [56,57], and shows that using mixed culture as test microorganism has better toxicity fortitude towards toxicants chemical substance as compared to using single
3.3. Half maximal effective concentration (EC50) Microalgae are particularly suitable for toxicity testing because they possess the majority of the same biochemical pathways as higher organisms. Half maximal toxicity concentration assessment on test organism are expressed in EC50 value for 24 h and 96 h. The 24 h incubation could be used to measure short term toxicity, while long-term toxicity test incubation can be tested using 96 h incubation period [35]. From the results achieved in this study, microalgae showed different survival rate and responses towards the organic and inorganic compounds used in this experiment. Fig. 3 linear line graph shows that all 5
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Fig. 2. Concentrations (mg/L) of the toxicants as function of initial pH over time (hr).
culture. Mixed cultures consist of community dynamics where the competitive advantage of more tolerant ones resulted in a change in community composition and survival rate.
4. Conclusion The analysis of the data on toxic effects of organic and inorganic toxicants showed that BA, FA and PA promote the growth of microalgae at a lower level, whereas microalgae are more tolerant towards AA at a 6
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Fig. 3. Toxicity of selected toxicants to a mixed algae population: (a) 24 h; (b) 96 h of exposure at 50 % survival rate. Data are the average of results from triplicate parallel experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
higher level. Oil and grease can be harmful to microalgae growth at high concentration, but copper on the other hand, suppress microalgae growth even at low concentration. Because of the dynamicity of microalgae, it was hard to determine the actual value of the inhibition test. This protocols also tend to have large variability in test result concerning the variation of physicochemical and biological parameters.
This study shows that generally, no single toxicity test can be characterized as being more sensitive than the others. In assessing the toxicity of pollutants to the microorganism, attention must therefore be focused on the specific physicochemical abiotic factors of the recipient environment, which may mediate or potentiate the toxicity of the contaminant.
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Table 2 Algae survival rate of acetic, butyric, formic, propionic acid, oil and grease and copper at 24 and 96 h. Toxicants (mg/L)
AA
BA
FA
PA
Cu
Oil & Grease
[6]
Survival Rate (%)
3000 3200 3400 3600 3800 4100 66 116 216 266 316 366 300 600 900 1200 1500 1800 50 200 350 500 650 800 0.1 0.3 3.0 6.0 9.0 12.0 4500 5500 6500 7500 8500 9500
24 h
96 h
100.03 101.21 104.34 103.95 102.35 106.15 97.99 99.43 108.83 101.72 81.67 77.80 99.97 99.41 100.02 96.83 96.82 83.00 109.76 111.21 106.52 93.05 89.20 87.77 47.43 36.71 32.87 28.85 27.70 18.98 43.28 66.30 44.48 46.36 52.10 41.45
84.53 80.82 81.71 84.84 90.59 113.83 107.56 117.42 127.41 127.97 65.60 51.79 77.90 80.47 80.46 76.74 72.62 48.27 103.53 112.38 108.77 72.89 55.47 54.78 73.46 59.44 44.27 38.92 18.53 2.92 97.06 87.50 79.11 63.66 55.85 43.16
[7] [8]
[9] [10]
[11] [12]
[13]
[14]
[15]
[16] [17]
[18]
Acknowledgement
[19]
The author want to thanks Universiti Kebangsaan Malaysia-Yayasan Sime Darby (UKM-YSD) Chair for Sustainable Development: Zero Waste Technology for the scholarship of J. Jasni during this study. We would like to thank Universiti Kebangsaan Malaysia for funding this work through Geran Galakan Penyelidik Muda (GGPM-2017-060) and Geran Universiti Penyelidikan (GUP-2018-092). We also thank the Ministry of Science and Technology of Taiwan for funding (grant numbers: MOST 105-2622-E-035-005- CC2; MOST 106-2632-E-035-001-; MOST 1062221-E-035-077-). Thanks, are also due to the Precision Instrument Support Center of Feng Chia University and Makmal Berpusat i-CRIM of UKM for providing the fabrication and measurement facilities.
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Comparative toxicity effect of organic and inorganic substances in palm oil mill effluent (POME) using native microalgae species
T
Jannatulhawa Jasnid, Shalini Narayanan Arishtd, Nazlina Haiza Mohd Yasinb,*, Peer Mohamed Abdula,d, Sheng-Kai Linc, Chun-Min Liuc, Shu-Yii Wuc, Jamaliah Md Jahima,d, Mohd Sobri Takriffa,d a
Research Centre for Sustainable Process Technology, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia b Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia c Department of Chemical Engineering, Feng Chia University, Taichung 40724, Taiwan d Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia
ARTICLE INFO
ABSTRACT
Keywords: Mix-culture microalgae Toxicity POME EC50 Ecotoxicology
Phycoremediation of palm oil mill effluent (POME) using native microalgae promotes sustainable agricultural wastewater treatment technology. However, POME is characterized by a high amount of organic and inorganic compounds. Therefore, the aim of this paper is to study the toxic effect of selected organic (acetic acid, butyric acid, propionic acid, formic acid, oil and grease) and inorganic (copper) compounds presence in POME on the microalgae population. A mix-culture of three native microalgae species consisted of Coelastrella sp. UKM 4, Chlorella sp. UKM 8 and Scenedesmus sp. UKM 9 were exposed for 120 h to different organic and inorganic substances. Then, microalgae growth response, the changes of pH and the half-maximal effective concentration (EC50) were studied for both short-term (24 h) and long-term (96 h) exposure. The EC50 indicated that acetic, butyric, formic and propionic acids promote microalgae growth at both short and long-term exposure (survival rate > 50 % at all selected concentrations) whereas copper, as well as oil and grease, suppress microalgae growth (survival rate < 50 %) during short-term exposure. The most favorable growth rates of microalgal were in the pH range of 7–9. These findings show that it is feasible to cultivate microalgae in POME with a suitable amount of organic and inorganic compound.
1. Introduction For the past decade, palm oil mill effluent (POME) has been treated in a series of treatment pond that includes anaerobic, aerobic or facultative ponds to reduce the contaminant level in wastewater before being flown to the river. However, these conventional methods are listed as non-sustainable and non-environmental friendly due to the discharge of a large amount of nutrientloaded waste into the water bodies contributing emission of greenhouse gaseous and air pollution [1]. Due to the worsening effect in environmental health, the regulation implemented on industrialized discharge to be more stringent [2]. In conjunction with zero-waste management technologies, every palm oil mill must take part in research and development for better waste management [3,4]. Recently, there has been a great interest from researcher to obtain
algal biomass grown in wastewater to minimize overall production cost as it can simultaneously offer a synergistic effect for wastewater nutrient removal, carbon capture and production of valuable biomass for various commercial applications such as animal feed, biofertilizer and biopharmaceutical products [5]. A recent study conducted by [6] had proven that POME could be utilized as a growing medium for marine diatom Phaeodactylum tricornutum and subsequently produce sulfated exopolysaccharide and nutrient removal. It is also suggested that the optimum concentration of POME for microalgae cultivation is 30 % as the growth rate could be inhibited beyond that concentration [6]. To date, phycoremediation has been used for over 70 years in wastewater treatment as it will further enhance the treatment activities through their efficient photosynthetic metabolism and effectively fix CO2 [7]. However, the amount of organic and inorganic materials in the wastewater sometimes can exceed the optimum acceptable level that
⁎ Corresponding author at: Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. E-mail address: [email protected] (N.H. Mohd Yasin).
https://doi.org/10.1016/j.jwpe.2020.101165 Received 25 September 2019; Received in revised form 23 January 2020; Accepted 24 January 2020 2214-7144/ © 2020 Elsevier Ltd. All rights reserved.
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microalgae can adapt. Therefore, the knowledge of toxic effects and concentration of non-inhibiting concentrations of wastewaters is important to retain a high biomass rate for high treatment capacity. As characterized, POME contains a high amount of organic materials and nutrients that make it as a potential low-cost substrate for cultivating microorganism especially microalgae [8]. However as per other wastewaters, POME is also known to contain a certain range of inhibitors and toxicants. Roughly, it consists of 0.6–0.7 % palm oil, 95–96 % water, 4–5 % total solid, and 2–4 % suspended solids [9]. According to [10], the nutrients in POME consist of 2726 (mg/L) of COD, 1270 (mg/L) of TOC, 316.7 (mg/L) of TN and 257.6 (mg/L) of phosphate. Among these components, there are compounds which can inhibit the growth of microalgae by reducing microalgae growth, photosynthetic activity, enzymatic activity and respiration [11]. The cultivation of microalgae in POME is challenging since the dark brown POME inhibit light penetration. However, POME pre-treatment (i.e. chemical, biological and mechanical pre-treatment) can be done prior to microalgae cultivation to permeate light to pass through. Hence, appropriate microalgae selection is important. In this study, mixotrophic microalgae were used. The advantage of using mixotrophic microalgae is it uses both inorganic and organic carbon sources under both photo-autotrophic and heterotrophic process simultaneously [12]. Therefore, if the POME unfavorable, the microalgae mode switch to the heterotrophic mode and vice versa. Aquatic microalgae are often used in inhibition test than other types of aquatic organism because of its extremely sensitive to the wide range of pollutants [13]. Current ecotoxicology test using mix-culture microalgae as biotest is still limited to monoculture. Mix-culture is better in ecotoxicology as it is more realistic to mimic their real nature conditions [14]. It can be severely affected by environmental changes and therefore, can provide meaningful indicators of ecological change over short time scales [15]. The toxicity inhibition level can be expressed in EC50, the half maximal effective concentration of the toxicant that inhibits the 50 % of the algal growth [16]. Calculation of half maximal effective concentration (EC50) for biomass (cellular density) corresponding to the exponential phase approximation of EC50 for growth rate allows us to estimate complete toxic effects on growth parameters. An organic compound like acetic acid, butyric acid, propionic acid and formic acid are commonly found as POME by-products. It is listed as a volatile fatty acid (VFA) that can negatively impact the environment [17]. However, it also can become a source of carbon for some microalgae [18]. Several studies had also utilized POME as carbon for lipid production and pollutants removal, carbon and nitrogen sources for food-grade yeast biomass production, carbon source for supporting ammonia-oxidizing bacteria (AOB) in ammonia removal, a substrate for bacteria to produce poly-β-hydroxyalkanoates, biohydrogen and biomethane [19–24]. Excessive amounts of oil and grease in POME can hinder sedimentation, which causes losses of biomass and overall affecting a reduction in the efficiency of treatment stations [25]. Hence, an effective handling system of POME, such as through phycoremediation is crucial and has been a great challenge for oil palm industries. This work aims to evaluate the toxic effect of selected organic
(acetic acid, butyric acid, formic acid, propionic acid, copper and oil & grease) and inorganic (copper) compounds which are major constituents in POME towards microalgae population. POME may contain a lot of nutrients. Therefore, this research utilized BBM as growing media to see the sole effect of the selected toxicants on mix-culture microalgae and the concentration range were selected based on the range presence in POME as a standard reference. The test duration and the possible adaptation of the microalgae to the test material and their effect towards toxicants on physicochemical properties, including pH during microalgae growth were investigated. These results reveal the proposed potential toxic concentration of organic and inorganic materials that microalgae can tolerate in POME for wastewater treatment purpose. 2. Materials and methods 2.1. Microalgae and media preparation Three inoculum mix-culture species of native microalgae Coelastrealla sp. UKM4 (KP691597), Chlorella sp. UKM8 (KT452082) and Scenedesmus sp. UKM9 (KU170547) which was previously isolated were prepared in a sterile condition in two-liter Schott bottle using the sterile Bold Basal Medium (BBM). All chemical component of test medium used were dissolved in ultrapure Milli-Ro water (Kemflo AICRO-Q Water Filters, USA) and adjusted to pH 7 using 5 M of NaOH and HCl. The BBM medium, consisted of following components (g/l) [26]: (g/l): CaCl2·2H2O (25), K2HPO4 (75), MgSO4.7H2O (75), H3BO3 (11.4), NaNO3 (250), NaCl (25), EDTA.Na2 (50), KH2PO4 (173.8), FeSO4.7H2O (4.98), H2SO4 (1), ZnSO4.7H2O (8.82), MnCl2.4H2O (1.44), MoO3 (0.71), CuSO4.5H2O (1.572), Co(NO3)2.6H2O (0.49). The incubation conditions were maintained at 25 °C with a continuous air supply at the rate of 1 L/min and continuous illumination at 3000 lx. The activated inoculum was kept in an active condition during the preparation of microalgae to achieve OD 1.0 prior to inoculation. The test microorganism flasks are shaken twice a day and placed in the culturing apparatus. 2.2. Standard Chemical toxicants The concentration range of chemical toxicants in which the effects on the growth of microalgae are likely to occur may be determined based on the results from range-finding tests. For the final test, six concentrations were tested based on the arrangement in a geometric series. The concentration series should preferably cover the range causing 0–90 % inhibition of algal and bacterial growth. Table 1 exhibits the characteristics of POME and test concentration range used in this experiment. 2.3. Incubation Flasks of BBM containing toxicants (Test 1–6) (Table 1) were inoculated with a mixed culture of microalgae. The algal morphology were checked regularly using Olympus BX51 microscope (Olympus, Center Valley, PA, USA) to ensure no sign of contamination. Thirty per
Table 1 Characteristics of POME and test concentration range used in the experiment. Component
Acetic acid (AA) Butyric acid (BA) Formic acid (FA) Propionic acid (PA) Copper Oil & grease (O&G)
Characteristics of organic and inorganic substance in POME [27,28,29]
Test concentration range (mg/L) in Bold Basal Media
Mean (mg/L)
Range (mg/L)
1
2
3
4
5
6
3540 8.15 1000 29.36 0.85 7213
3030-4050 16-290 300-1700 58-720 0.80–0.90 5614–8812
3000 66 300 50 0.4 4500
3200 116 600 200 0.6 5500
3400 216 900 350 0.8 6500
3600 266 1200 500 1.0 7500
3800 316 1500 650 1.2 8500
4100 366 1800 800 1.4 9500
2
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cent (30 ml) from the stock culture was inoculated into 250 mL conical flask and were top up with BBM until 100 mL. The toxicants were added into the culture with their respective concentrations. The cultures were then incubated in the incubator for 120 h at 25 °C with 12 h/12 h illumination (3000 lx) by white fluorescent lamps (Philips) without aeration. The salinity was maintained at 0 ppt and were monitored using a refractometer. The flasks with mix culture of microalgaewere shaken twice a day and placed in the culturing apparatus. The initial biomass was standardized at 100 mg/L, equivalent to 8.6 × 107 cfu/ mL. All the experiments were conducted in triplicates.
Y= aX + c ( a is in+ ve or
3. Result and discussion 3.1. Algae growth response curve to toxic substances The toxic effect of organic (acetic acid, butyric acid, formic acid, propionic acid) and inorganic (copper and oil & grease) on algal growth were examined by measurement of microalgae biomass concentration throughout cultivation. As described by [36], non-competitive inhibition indicates that transformation can occur at every concentration level of the inhibiting compound. The inhibition was the dependent variable, while the concentrations of organic and inorganic substances were the independent variables. Conflicting the other orders of reaction, a zero-order reaction has a rate that is independent of the concentration of the substances. As such, increasing or decreasing the concentration of the reacting algal species will not speed up or slow down the reaction rate as they have the same level of inhibition towards the algal cell. The initial microalgae cell density in this experiments were kept constant at 100 mg/L, as shown in Figs. 1 and 2. From the results, it was found that organic matters used in this experiment has a stimulant effect on microalgae growth at different concentrations. AA was found as the best growth stimulant. Among tested concentration of AA (Fig. 1a), the highest biomass was observed during the exposure to the highest concentration of AA, 4100 mg/L. The lowest biomass of microalgae at AA more than 3.0 × 100 mg/L was observed at concentration of 3400 mg/L, which is still higher than the others which their lowest biomass nearly to zero. According to [37], AA is one of the most common carbon sources for many microbial species, including microalgae. Metabolically, microalgae assimilate AA into coenzyme A to form Acetyl-CoA for carbon skeletons of ATP and NADH. Meanwhile, butyric acid, formic acid and propionic acid show a similar trend of response towards microalgae growth by showing a stimulant effect at lower concentrations and inhibition effect at the higher concentrations. This might be because most of the single compound of fatty acids provide an additional carbon source for the growth of microalgae. As explained by [38,39], AA could be toxic for many microorganisms at high concentrations with the exception to Chlamydomonas mundana which proliferated in the presence of acetate. For this reason, AA is frequently used to buffer the pH value in bioreactors for microalgae culture. PA, on the other hands, was suggested to provide oxaloacetate to the cells and therefore, can improve the cellular growth [40]. Furthermore, the mixotrophic nature of mix-culture microalgae used in this experiment can heterotrophically utilize organic carbon sources to increase their biomass production. The decline in microalgae biomass at the end of the experiment can be explained due to a depletion of nutrients, overheating, pH disturbance, or contaminations. An inorganic matter like copper (Fig. 1e) used in this experiment shows a negative impact on microalgae growth. It can be seen that the additional copper in the growth media proportionally inhibits microalgae growth at any concentrations. Thus, copper concentration should be kept low if the wastewater to be used as a media for microalgae culture. As for oil & grease (Fig. 1f), it may cause a reduction in microalgae growth as the amount increased. However, it does not contain any harmful effect on microalgae growth. The declining growth phase in oil & grease can be observed by the reduction of the cell division rate due to limiting factors, such as nutrients and aeration supply. The oil masked the surface of the media preventing the gas exchange and light penetration, making the anaerobic condition for the microalgae. The mixotroph algae in this experiment do not sustain anaerobic condition.
2.4.1. Growth culture The growth of algae in test substances was determined by measurement of the optical density of a culture with a spectrophotometer at 685 nm. Meanwhile, the biomass was measured by analysis of dry weight by using standard profile of OD at 685 nm versus microalgae biomass concentration (mg/L). The microalgal biomass was obtained daily from 15 mL of sample and centrifuged at 2862×g for 10 min. Prior to determine the dry cell weight (DCW), the cell concentration were measured using the Varian Cary 50 spectrophotometer with UV/ VIS detector (Varian Cary, Agilent, Santa Clara, CA, USA). Then, the samples were filtered using 0.45-μm micro fiberglass filters (GF/C). The filtered biomass was dried at 105 °C in a drying oven for 24 h, and the weight was recorded. The standard curve of optical density versus dry weight were constructed. 2.4.2. Toxicants growth curve analysis Scatter plot with linear line was applied to optimize key factors affecting biomass concentration of microalgae. Scatter line plot was used to optimize the level of toxicants (mg/L) and days (120 h) on biomass concentration. The response variable is biomass concentration (x 100 mg/L). The inhibition by a toxic compound can be described with the following model for non-competitive inhibition on the assumption that the inhibition is zero-order:
Si . c Si . c + Si
(1)
Where, Si is concentration of inhibiting compound (mg/1); Si.c is inhibition constant for which kobs.tox at 1 kmax (mg/l); kmax is maximum 2 (0′ order) biomass concentration rate (mg/l/h) and kobs.tox is observed biomass concentration rate, when a toxicant is present (mg/l/h) [30]. The statistical software SigmaPlot14, (Version 14.0, San Jose, CA, USA) is used for regression and graphical analysis of the experimental data. The quality of fit of the quadratic model is expressed by the coefficient of determination, R2, and its statistical significance is checked by the F-test. 2.4.3. Half-maximal effective concentration (EC50) Different concentrations of toxicants were used to culture mixed microalgae. The biomass concentration of each culture in the triplicate samples was calculated by comparison with the biomass concentration in the control culture (BBM) in the absence of toxicants. The average specific biomass concentration rate for a specific period was calculated as the survival rate (%). Average of specific biomass concentration was obtained from Eq. (2) [31] over concentration (mg/L) [32]. Then, it was plotted using Sigma Plot Software 14.0 (Systat Software Inc., USA) and half-maximal toxicity was calculated using linear regression line Equation (3) [33–35] for each substance.
% cell survival=
Bsample
Bblank
Bcontrol
Bblank
× 100
(3)
Where, Y is 50 % from the overall maximum inhibition percentage (y-axis); X is EC50 concentration value in the mg/L concentration at which 50 % inhibition of the toxicants achieved; values a is positive or negative steep slope and c is the intercept on y-axis are generated by the SigmaPlot linear fit curve operation.
2.4. Measurement and analytical test procedure determination
K obs . tox = kmax +
ve depending on the data)
(2)
Where, Bsample is biomass concentration of specific toxicant concentration; Bblank is biomass concentration of blank and Bcontrol is biomass concentration of control. 3
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Fig. 1. Experimental data of the growth kinetics for algae at selected toxicants, Acetic acid, a); Butyric acid, b); Formic acid, c); Propionic acid, d); Copper, e) and Oil & Grease, f) at different days, and fittings to Eq. (1).
Overall, the organic matters have both positive and negative impact on the microalgae growth at a certain range of concentration by which AA becomes the most favorable towards the microalgae growth. In contrast, copper has a negative effect on the microalgae growth at any concentrations.
3.2. Effects of pH towards microalgae growth activity The effect of pH on microalgae growth was initially investigated in this study. The pH reading for O&G are not shown on this figure as pH meter is sensitive to high oil content. The pH was a crucial factor 4
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influencing metabolic effect in microalgae cells. pH 7 was used as the initial point for all analysis as at the neutral pH of most freshwater habitats (pH 6–8), proton acidity is the most important component while mineral or organic acidity are minor contributors [41]. Two pHdependent conceptual models were proposed for test organisms based on observed responses to variable pH values (Fig. 2a to 2e). These include Model-I: decreasing toxicity with increasing pH (positive correlation), Model-II: increasing toxicity with decreasing pH (negative correlation) [42]. The effect of pH on microalgae growth as shown in Fig. 2a to 2e plays a crucial role as the pH decreases at 120 h exposure, the survival rate decreases. The growth rates for all five organic and inorganic matter exhibited a similar pattern over the pH range (around pH 3.0–10.0) with the optimum pH values for growth obtained between pH 7.0 and 9.0. By comparing Fig. 1 and Fig. 2, algae growth rates decreased when the pH values drop below pH 5. In general, pH values of 7–9 might be most conducive to increased algae production and minimizing invading organisms [43]. The pH of acetic acid, formic acid, propionic acid and copper drop below neutral pH after 24 h exposure except for butyric acid. Butyric acid showed a slightly increase in pH after 24 h exposure and decrease its pH after 48 h exposure. The same pattern can be observed for acetic acid, formic acid and propionic acid after 72 h, which the pH was increasing and started to fall back after 96 h exposure. However, butyric acid and copper continuoes to increase its pH until 120 h of exposure. The mix-culture microalgae used in this experiment seems to withstand low pH condition after manage to regain back their neutral pH after 72 h. However, at higher concentration of butyric acid (316 and 366 mg/ L) propionic acid (650 and 800 mg/L) and copper (1.2 and 1.4 mg/L), the pH maintains in acidic condition (< pH 5) after 72 h. According to [44], the nature of the freshwater microalgae are robust microorganism, and it has a wide range of pH adaptation. They can increase tolerance to acidic and alkaline pH as an adaptive response to widely fluctuating pH levels that occur commonly in many productive freshwater environments [45]. The homeostasis maintained by proton efflux and influx mechanism of microalgae is based on the cell to the environment communication [44,46]. Microalgae can regulate their internal pH through proton exchange, so whatever the extracellular pH may be, the cytoplasmic pH always remains near neutral [44]. Besides, microalgae cell wall and production of extracellular polysaccharide to neutralize basic conditions can protect microalgae cell from pH damage [44,47]. In this study, the reduction of pH values does affect the survival rate of mixed microalgae culture. The previous report has shown that butyric acid at concentrations of 1–5 mM was found to be highly toxic corresponded to the pH values around 4–5. However, it was relatively harmless at pH 6. On the other hand, propionic acid was highly toxic within pH range around 4–6 [48]. The results obtained from this study support previous findings that the complex forms of the organic acids are more toxic. This is due to the drop of pH values which corresponded to the low pKa value in the media. However, some microalgae favor acidic condition, which they may utilize this condition for better growth and survival. These observations suggest that pH assessment over growth rates possess resistance and enhanced growth rate.
substances have negative slope graph except for acetic acid, which has a positive slope. The negative linear fit line shows that increasing substance concentration reduces survival rate whereas positive fit line shows vice versa. From Fig. 3, the EC50 indicated that AA, BA, FA and PA promote microalgae growth at both short and long-term exposure (survival rate > 50 % at all selected concentrations) whereas, copper and oil grease suppress microalgae growth (survival rate < 50 %) during shortterm exposure. This might be due to the sensitivity differences in uptake rates across the plasma membrane and internal binding mechanisms of microalgae towards toxicants [49]. AA seems to have a high survival rate (> 100 %) at short-term exposure and being highest (113.83 % survival rate) at a concentration of 4100 mg/L (P < 0.05) during 96 h exposure as it is proven to be more easily consumed by several microorganisms by previous research. As stated by [50], acetate is preferred during the early exponential phase for microalgae growth and their presence as an organic compound supplies nutrient assimilation for microalgae carbon skeleton. The metabolic oxidation for AA and BA are very much the same except for butyrate uptake by microalgae is much slower and can reduce the microalgae growth when both are present. It was found that at much higher concentrations (above 500 ppm), the y-butyric acids were found to be highly toxic, whereas the acetic acids have an inhibitory effect on growth [51]. This explains why the survival rate in AA are much higher than BA during long-term exposure. Fifty percent effective dose (the ecological dose resulting in a 50 % decrease in activity), EC50 values in BA, FA, and PA significantly decreases (P < 0.05) from 24 hours–96 hours, 896.68 mg/L to 498.84 mg/L, 6108.79 mg/L to 2412.28 mg/L and 1798.34 mg/L to 828.55 mg/L respectively. Conversely, the EC50 value for AA Table 2, Cu and O &G significantly increases (P < 0.05) from 91.93 mg/L to 1947.56 mg/ L, 0.1766 mg/L to 0.7485 mg/L and 6402.14 mg/L to 8901.83 mg/L, respectively through 24 hours–96 hours incubation (Fig. 3). During the 24 h and 96 h exposure, majority all of the organic matters survival rate above 50 % whereas majority survival rate for O&G and Cu fall below 50 % (Table 2),(P < 0.05). Microalgae survive better in organic matter because they serve as carbon sources to support microalgae growth [18] while inorganic matter like copper always suppresses their growth by messing their metabolic pathway. Growth inhibition in microalgae is mainly related to free ions and its sensitivity varies among microalgae [52]. As explained by [53], the effect of Cu towards microalgae depends on the composition of culture medium, experimental protocol and species used. Therefore, direct comparison of sensitivity among microalgae due to copper exposure is difficult but can be minimized at least by using a similar experimental protocol. Cu often causes the generation of reactive species (RS) which cause algal growth and photosynthetic inhibition [54]. It is proven that the increasing level of RS can lead to severe cellular injury or death to cells [55] [52,55] reported that copper in ionic form is able to inhibit the synthesis of D-aminolevulinic acid and protochlorophyllide reductase, peroxidative breakdown of pigments and membrane lipids by reactive oxygen species at higher concentration thus causes chlorophyll-α reduction. From the results obtained, algae have high toxicity tolerance due to its microorganism characteristics, such as has a thicker cell wall being eukaryotic cell. Some microalgae can tolerate at a very high concentration of AA. However, BA also promotes the growth of microalgae at a very much lower concentration as compared to AA. Therefore, it is somewhat to say that AA and BA could be used as a microalgae enhancer. It is suggested that if the microalgae are to be cultured in wastewater containing both AA and BA, the algal biomass can be manipulated either by increasing the initial mix culture biomass or by manipulating the initial acetate: butyrate ratio [51]. Based on the comparison of previous studies on toxicity level [56,57], and shows that using mixed culture as test microorganism has better toxicity fortitude towards toxicants chemical substance as compared to using single
3.3. Half maximal effective concentration (EC50) Microalgae are particularly suitable for toxicity testing because they possess the majority of the same biochemical pathways as higher organisms. Half maximal toxicity concentration assessment on test organism are expressed in EC50 value for 24 h and 96 h. The 24 h incubation could be used to measure short term toxicity, while long-term toxicity test incubation can be tested using 96 h incubation period [35]. From the results achieved in this study, microalgae showed different survival rate and responses towards the organic and inorganic compounds used in this experiment. Fig. 3 linear line graph shows that all 5
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Fig. 2. Concentrations (mg/L) of the toxicants as function of initial pH over time (hr).
culture. Mixed cultures consist of community dynamics where the competitive advantage of more tolerant ones resulted in a change in community composition and survival rate.
4. Conclusion The analysis of the data on toxic effects of organic and inorganic toxicants showed that BA, FA and PA promote the growth of microalgae at a lower level, whereas microalgae are more tolerant towards AA at a 6
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Fig. 3. Toxicity of selected toxicants to a mixed algae population: (a) 24 h; (b) 96 h of exposure at 50 % survival rate. Data are the average of results from triplicate parallel experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
higher level. Oil and grease can be harmful to microalgae growth at high concentration, but copper on the other hand, suppress microalgae growth even at low concentration. Because of the dynamicity of microalgae, it was hard to determine the actual value of the inhibition test. This protocols also tend to have large variability in test result concerning the variation of physicochemical and biological parameters.
This study shows that generally, no single toxicity test can be characterized as being more sensitive than the others. In assessing the toxicity of pollutants to the microorganism, attention must therefore be focused on the specific physicochemical abiotic factors of the recipient environment, which may mediate or potentiate the toxicity of the contaminant.
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Table 2 Algae survival rate of acetic, butyric, formic, propionic acid, oil and grease and copper at 24 and 96 h. Toxicants (mg/L)
AA
BA
FA
PA
Cu
Oil & Grease
[6]
Survival Rate (%)
3000 3200 3400 3600 3800 4100 66 116 216 266 316 366 300 600 900 1200 1500 1800 50 200 350 500 650 800 0.1 0.3 3.0 6.0 9.0 12.0 4500 5500 6500 7500 8500 9500
24 h
96 h
100.03 101.21 104.34 103.95 102.35 106.15 97.99 99.43 108.83 101.72 81.67 77.80 99.97 99.41 100.02 96.83 96.82 83.00 109.76 111.21 106.52 93.05 89.20 87.77 47.43 36.71 32.87 28.85 27.70 18.98 43.28 66.30 44.48 46.36 52.10 41.45
84.53 80.82 81.71 84.84 90.59 113.83 107.56 117.42 127.41 127.97 65.60 51.79 77.90 80.47 80.46 76.74 72.62 48.27 103.53 112.38 108.77 72.89 55.47 54.78 73.46 59.44 44.27 38.92 18.53 2.92 97.06 87.50 79.11 63.66 55.85 43.16
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Acknowledgement
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The author want to thanks Universiti Kebangsaan Malaysia-Yayasan Sime Darby (UKM-YSD) Chair for Sustainable Development: Zero Waste Technology for the scholarship of J. Jasni during this study. We would like to thank Universiti Kebangsaan Malaysia for funding this work through Geran Galakan Penyelidik Muda (GGPM-2017-060) and Geran Universiti Penyelidikan (GUP-2018-092). We also thank the Ministry of Science and Technology of Taiwan for funding (grant numbers: MOST 105-2622-E-035-005- CC2; MOST 106-2632-E-035-001-; MOST 1062221-E-035-077-). Thanks, are also due to the Precision Instrument Support Center of Feng Chia University and Makmal Berpusat i-CRIM of UKM for providing the fabrication and measurement facilities.
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