04. Santoso.cdr Vol.14, No.3, September 2020, p 108-116 DOI: 10.5454/mi.14.3.4 Effect of Hydrocarbon-Polluted Seawater on the Cell Density of Microalgae Scenedesmus vacuolatus Shihira & Krauss 1 1 CLARA ALVERINA SANTOSO , NOVERITA DIAN TAKARINA , HANIES AMBARSARI *, 2 1 1 NINING BETAWATI PRIHANTINI , SITARESMI AND 1 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Kampus UI Depok 16424, Indonesia; 2 Center of Environmental Technology (PTL), Agency for the Assessment and Application of Technology (BPPT), Building 820 Geostech, PUSPIPTEK, Setu, South Tangerang City, Banten 15314, Indonesia. Study the effect of hydrocarbon-polluted seawater on the cell density of microalgae Scenedesmus vacuolatus has been carried out in this study. Hydrocarbon pollution derived from the oil in the sea can inhibit the photosynthesis process of microalgae. This might impact the density of microalgae cells. The purposes of this study are to determine the effect of the concentration of hydrocarbon-polluted seawater on the density of Scenedesmus vacuolatus microalgae cells and to determine the optimum treatment to reduce total petroleum hydrocarbons (TPH) levels. A sampling of hydrocarbon-polluted seawater was taken at Kali Adem port, Jakarta. The treatment done in this research used a walne medium with the addition of 25% hydrocarbon-polluted seawater (A), 50% (B), 75% (C), and 100% (D). Control is Walne medium with sterile seawater that was not from the Kali Adem port. The results showed the highest average density of Scenedesmus vacuolatus cells was in the control sample. This can be seen from the results of the average cell density at the peak time of 29.48 x 105 cells / mL, as well as the log phase length of Scenedesmus vacuolatus. Measurement of TPH levels showed decreases of TPH in all treatments. The optimum treatment to reduce TPH levels is treatment B with a reduction percentage of 70.62%.. Key words: Kali Adem port, Scenedesmus vacuolatus, total petroleum hydrocarbon (TPH), Walne medium Penelitian mengenai pengaruh air laut tercemar hidrokarbon terhadap kepadatan sel mikroalga Scenedesmus vacuolatus telah dilakukan. Pencemaran hidrokarbon yang berasal dari minyak di laut dapat menghambat proses fotosintesis mikroalga. Hal tersebut dapat berdampak pada kepadatan sel mikroalga. Penelitian ini bertujuan untuk mengetahui pengaruh konsentrasi air laut tercemar hidrokarbon terhadap kepadatan sel mikroalga Scenedesmus vacuolatus, serta mengetahui perlakuan yang optimum untuk menurunkan kadar total petroleum hidrokarbon (TPH). Pengambilan sampel air laut tercemar hidrokarbon dilakukan di pelabuhan Kali Adem, Jakarta. Perlakuan dalam penelitian adalah medium Walne dengan penambahan air laut tercemar hidrokarbon 25% (A), medium Walne dengan penambahan air laut tercemar hidrokarbon 50% (B), medium Walne dengan penambahan air laut tercemar hidrokarbon 75% (C), dan medium Walne dengan penambahan air laut tercemar hidrokarbon 100% (D). Kontrol yang digunakan adalah medium Walne dengan air laut steril yang bukan berasal dari pelabuhan Kali Adem. Hasil penelitian menunjukkan rata-rata kepadatan sel Scenedesmus vacuolatus tertinggi yaitu pada perlakuan kontrol. Hal tersebut dapat dilihat dari hasil rata-rata kepadatan sel pada masa puncak sebesar 29,48 x 105 sel/mL, serta panjang fase log dari Scenedesmus vacuolatus. Hasil pengukuran kadar TPH menunjukkan terdapat penurunan TPH pada seluruh perlakuan. Perlakuan optimum untuk menurunkan kadar TPH yaitu perlakuan B dengan persen penurunan sebesar 70,62%. Kata kunci: Medium Walne, Pelabuhan Kali Adem, Scenedesmus vacuolatus, total petroleum hidrokarbon (TPH) MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-21 75791377; Email: - hanies.ambarsari@bppt.go.id impacts on the environment and living things (Wibowo 2018). Microalgae are photosynthetic organisms that require sunlight and CO fixation to photosynthesis 2 (Muchammad et al. 2013). Contact between the oil layer and microalgae on the surface of the water in addition to having an impact on the photosynthesis process will also have an impact on its density. Microalgae flexibility in morphology, physiology, and life cycle gives the ability to survive in polluted conditions (Amirlatifi et al. 2013). Response and tolerance of exposure to pollutant levels in each Port is one of the places with the most human activities, especially transportation. The number of transportation activities by ships causes common pollution in the port area is oil pollution. Oil pollution to the sea is the release of liquid petroleum hydrocarbon pollutants which mainly come from human activities (Priyadarshani et al. 2011). Petroleum hydrocarbon pollution in the sea has many negative Volume 14, 2020 Microbiol Indones 109 type of the microalgae are different. One of microalgae genus that is adaptive in oil-polluted waters is the Scenedesmus. The genus microalgae Scenedesmus can adapt to oil-contaminated environments through acclimatization of physiology and genetic mutations (Martinez et al. 2013). Scenedesmus is a green microalga that can be found in fresh to brackish water (Phinyo et al. 2017). Generally, the genus Scenedesmus has an ellipsoidal cell shape and forms a coenobitic with a multiple of 4 (Guiry and Guiry 2019). Scenedesmus vacuolatus is a species of the genus Scenedesmus which has a different shape. These microalgae are round in shape, do not have a prominent apex, and do not have cell-to-cell connections like the genus Scenedesmus in general. S. vacuolatus is very tolerant and adaptive to the environment. Lewis and Flechtner in 2014, found S. vacuolatus in macrobiotic crust in deserts (Lewis and Flechtner 2004). In addition, these microalgae are environmentally tolerant with a wide salinity range (Anand et al. 2019). Hydrocarbon compounds in water can trigger eutrophication and cause an abundance of certain types of microalgae, resulting in changes in community structure. Besides affecting the structure of the microalgae community, hydrocarbons also affect the size and physiology of microalgae cells. In phytoplankton exposed to hydrocarbons, the cell size is reduced in line with the increase in petroleum hydrocarbon content (Nair et al. 2014). The response of each microalgae to pollutants, especially petroleum hydrocarbons is different. Al Obaidy and Lami (2014) conducted researched on chlorophyll A levels in Microcystis flos-aquae (Wittr.) Kircher and Nostoc carneum Agardh grown on a medium containing crude oil. In Microcystis flos-aquae (Wittr.) Kircher levels of chlorophyll A decreased, whereas in Nostoc carneum Agardh levels of chlorophyll A tended to increase (Al Obaidy and Lami 2014). Microalgae are capable of contributing to the degradation of environmental pollutants either by directly altering pollutants, or by increasing the degradation potential of existing microbial (Semple et al. 1999). Several chemicals can be found in total petroleum hydrocarbons (TPH) hexane, jet fuel, mineral oil, benzene, toluene, xylene, naphthalene, fluorene, and other petroleum products. One of the benzene derivatives that can be degraded by microalgae is phenol. Phenol can be degraded via the meta-cleavage pathway. The meta-cleavage pathway in phenol degradation produces the final products, namely pyruvate and acetaldehyde. Pyruvate can be metabolized in the Krebs cycle, and acetaldehyde can be converted to acetyl-CoA with the help of acetaldehyde dehydrogenase (AcDH) (Patel et al. 2017). The study aims to determine the effect of concentrations of hydrocarbon polluted seawater on the density of Scenedesmus vacuolatus microalgae cells, and determine the optimum treatment to reduce levels of total petroleum hydrocarbons (TPH). MATERIALS AND METHODS Design Experiment. This study used five variations of treatment with three repetitions; Control (0%), A (25%), B (50%), C (75%), and D (100%). This research used 2-litter gallons with the composition of Scenedesmus vacuolatus microalgae, Walne nutrient and vitamin solutions, hydrocarbon-polluted seawater samples, and also sterile seawater (not from the port) as a solvent. Growth Medium for Microalgae. The growth medium that will be used for the growth of Scenedesmus vacuolatus in the study is Walne medium. The first stage of making 1 liter Walne medium is to prepare distilled water as a solvent for trace metal solutions, vitamin solutions, and nutrient solutions. The next stage is the weighing of trace metal solution materials namely ZnCl (2.1 g), CoCl .6H O (2 2 2 2 g), (NH )6Mo O .4H O (0.9 g), and CuSO .5H O (2 g). 4 7 24 2 4 2 The trace metal solution is dissolved into 100 mL distilled water and labeled. Weigh the ingredients of the vitamin solution, namely vitamin B12 (10 mg), vitamin B1 (10 mg), and biotin (200 µg). The vitamin solution ingredients are dissolved into 100 mL distilled water and labeled. The next stage is weighing the ingredients of nutrient solutions, namely FeCl .6H O (1.3 g), 3 2 MnCl .4H O (0.36 g), H BO (33.6 g), EDTA - 2Na (45 2 2 3 3 g), NaH PO .2H O ( 20 g), NaNO (100 g). The nutrient 2 4 2 3 solution ingredients are dissolved into 1 L of distilled water and 1 ml of trace metal solution is added then labeled. One mL of nutrient solution has been made and then dissolved into 1 L of sea water that has been sterilized using a vacuum filter. The solution was sterilized using an autoclave at 121 °C for 15 minutes at a pressure of 0.2 MPa. After sterilization, add aseptic a 0.1 mL vitamin solution. Observation of Microalgae Culture Colors. Microalgae culture colors were observed using Faber Castell's standard color table. Observations were made every day during the study. Measurement of Microalgae Cell Density. Cell size in starter culture, control culture, treatments A, B, C, and D were observed using a microscope at 1000x magnification. Microalgae cell density calculations are p e r f o r m e d u s i n g a n i m p r o v e d n e u b a u e r haemacytometer then observed under a microscope. The observations are then calculated using the following formula: 4 d = 10 x Q cell/mL � d: cell density 2� Q: average cell count per 1 mm (Andersen 2005: 249) Measurement of Total Petroleum Hydrocarbon (TPH). Measurement of total petroleum hydrocarbon content using gravimetric methods according to SNI 6989.10: 2011 standard. The measurement of TPH concentration is divided into four parts, namely oil and fat extraction, distillation, calculation of oil and fat content, and calculation of mineral oil content (total petroleum hydrocarbons). Measurement of Environmental Parameters. o Physical parameters observed were temperature ( C), salinity (‰), light intensity (lux), and culture color. Retrieval of temperature data is taken using a multiparameter water quality meter [Aquaread]. Measurement of light intensity is done with a lux meter [Uni-T]. Salinity levels were measured by using a refractometer [ATC Brix]. Chemical parameters observed were pH level, and dissolved oxygen (mg/L). Sample analysis was performed aiming to determine the level of chemical content in the water sample before and after the experiment. The pH data collection was carried out using pH paper, dissolved oxygen was measured using a multiparameter water quality meter device. All of these factors were observed every day during the study then recorded. Statistical Methods. The data obtained were tested using the Saphiro Wilk normality test followed by the Kruskal Wallis non-parametric statistical test (α <0.05). These data were tested to determine whether or not there was a significant difference in the concentration of seawater contaminated with hydrocarbons on the density of microalgae cells Scenedesmus vacuolatus. RESULTS Observation of Microalgae Culture Colors. Figure 1 shows the change of microalgae cultures color during research. The color of the Scenedesmus vacuolatus culture in the control, A, B, C, and D medium on day 2 to day 9 experienced the same changes. On the 12th day of observation, the treatment medium C and D experienced a darker color change, namely moss green. The macroscopic appearance of the cultures on the control, A, and B treatment mediums were the same as the 9th day, namely grass green. On the 13th day of observation, all test cultures were the same color, namely grass green. The macroscopic color change of the culture on the 14th day only occurred in the control medium, A, and B treatment mediums, which were leaf green, while the C and D treatment mediums were still the same color as the previous day (T ). On the 16th day of observation, the culture colors 13 on control medium, A, and B were still the same color as the previous day (T ), namely leaf green (leaf 14 green), while the treatment medium C and D changed to sap green. The macroscopic appearance of the culture in the control medium, A, B, C, and D treatment mediums were the same color on the 19th to the 21st day, namely sap green. Overall, the color changes in the C and D cultures occurred more rapidly than in other cultures. This can be seen on the 12th and 16th days, the color of C and D cultures changed to a darker green. Measurement of Microalgae Cell Size. Observation of Scenedesmus vacuolatus cell size was observed through the size of cells in starter cultures, control cultures, and each treatment. Microscopic observations of Scenedesmus vacuolatus cells cultured in starter cultures, control cultures, treatment A, and treatment B had a cell size range between 4—6 µm. The size of S. vacuolatus cells cultured in treatment C and D ranged from 4—5 µm. Overall, there is no significant difference in size for each treatment. In all treatments, the size of S. vacuolatus cells ranged from 4 - 6 µm. The difference in smaller cell size was only found in C and D medium. Measurement of Microalgae Cell Density. The cell density of the starter culture of Scenedesmus vacuolatus entered into the test culture was 958,000 -1 cells mL in 1,800 mL of medium. The results of the mean cell density of Scenedesmus vacuolatus are listed in Table 2. These results are the average of three repetitions for each variation of the treatment medium. The growth curve of Scenedesmus vacuolatus is shown in Figure 2. The average density of Scenedesmus vacuolatus cells on days 1 to 9 in the medium which was given hydrocarbon-polluted sea water (A, B, C, and D) was higher than in the control medium. The growth curve of Scenedesmus vacuolatus after day 9 showed that the cell density increased significantly. After the 9th to 21st day, the average density of Scenedesmus vacuolatus 110 SANTOSO ET AL. Microbiol Indones Fig 1 Macroscopic color appearance of the Scenedesmus vacuolatus culture from day 2 – 21. Volume 14, 2020 Microbiol Indones 111 Fig 2 Microscopic observation of Scenedesmus vacuolatus on starter culture (a), control medium (b), treatment medium A (c), medium B (d), medium C (e), and medium D (f). Fig 3 Growth curve of Scenedesmus vacuolatus. a b c d e f 112 SANTOSO ET AL. Microbiol Indones cells in the control medium was higher than the other mediums.The growth continued until it reached the peak period on the 14th day for control, A, B, and C medium, while the culture in D medium reached its peak on the 16th day. The growth rate of Scenedesmus vacuolatus began to decline after day 14 in control, A, B, and C, whereas in culture D it occurred after day 16. This is indicated by a decrease in the cell density curve. Kruskal Wallis statistical test showed that there was no significant difference in the density of microalgae Scenedesmus vacuolatus cells to variations in the concentration of sea water contaminated with hydrocarbons. The increase or decrease in S. vacuolatus cell density was not directly affected by the differences in the treatment given. Measurement of Total Petroleum Hydrocarbon (TPH). The total petroleum hydrocarbon (TPH) content shows the oil concentration in each treatment. The total petroleum hydrocarbon content can be determined from the residual weight of the oil after the extraction process. The TPH level in treatment medium A at the beginning of the study was 719.38 mg, and at the end of the study was 266.65 mg. The decrease in TPH levels in treatment medium A was 62.93%. The TPH level in the B treatment medium at the beginning of the study was 1438.76 mg, and at the end of the study was 422.69 mg. The decrease in TPH levels in the B treatment medium was 70.62%. The TPH level in the C treatment medium at the beginning of the study was 2158.14 mg, and at the end of the study was 840.71 mg. The decrease in TPH levels in the C treatment medium was 61.04%. The TPH level in the D treatment medium at the beginning of the study was 2586.89 mg, and at the end of the study was 1597.91 mg. The decrease in TPH levels in the D treatment medium was 38.23%. The highest decrease in total petroleum hydrocarbons was culture B, amounting to 70.62%. The results of measuring TPH levels in each treatment medium at the beginning and end of the study are listed in Table 3. The results of Kruskal Wallis's non-parametric statistical test (attachment 6) showed that there was no significant difference in the density of microalgae Scenedesmus vacuolatus cells to variations in the concentration of sea water contaminated with hydrocarbons. in 1 D A Y C e ll D e n sity (c e ll.m L-1) (1 05) C o n tro l (0 % ) A (2 5 % ) B (5 0 % ) C (7 5 % ) D (1 0 0 % ) 0 9 ,5 8 9 ,5 8 9 ,5 8 9 ,5 8 9 ,5 8 1 1 ,1 9 1 ,2 5 1 ,2 1 1 ,7 8 2 ,2 1 2 1 ,2 3 1 ,7 8 1 ,8 3 3 ,3 6 3 ,7 5 5 3 ,7 9 3 ,3 9 3 ,9 6 5 ,0 5 4 ,9 6 6 4 ,7 5 5 ,2 2 5 ,2 3 6 ,5 3 7 ,2 6 7 6 ,3 9 6 ,5 9 6 ,5 2 8 ,2 2 8 ,8 8 8 7 ,1 2 7 ,6 5 7 ,6 7 8 ,8 1 9 ,3 4 9 7 ,8 8 9 ,1 0 8 ,0 7 9 ,0 8 1 0 ,5 5 1 2 1 7 ,9 3 1 0 ,7 3 1 0 ,3 2 9 ,8 7 1 1 ,2 2 1 3 2 0 ,6 8 1 7 ,4 8 1 5 ,3 8 1 7 ,6 4 1 4 ,1 5 1 4 2 9 ,4 8 1 8 ,5 9 1 6 ,3 5 1 9 ,9 7 2 0 ,5 1 1 6 2 2 ,8 5 1 6 ,8 1 1 4 ,2 7 1 5 ,9 8 2 3 ,2 5 1 9 1 7 ,0 2 1 4 ,9 2 1 3 ,3 7 1 6 ,3 2 1 7 ,4 4 2 1 1 3 ,8 5 1 2 ,7 7 1 2 ,1 7 1 3 ,2 3 1 2 ,5 4 1 Treatment Total Petroleum Hydrocarbon (TPH) Level Beginning (mg) End (mg) % Residue % Decreasing Control (0%) 0 0 0 0 A (25%) 719,38 266,65 37,07 62,93 B (50%) 1438,76 422,69 29,38 70,62 C (75%) 2158,14 840,71 38,96 61,04 D (100%) 2586,89 1597,91 61,77 38,23 Table 1 Average cell density of Scenedesmus vacuolatus Table 2 Average of total petroleum hydrocarbon (TPH) level Volume 14, 2020 Microbiol Indones 113 seawater does not directly affect the density of microalgae cells, but this study shows that S. vacuolatus can reduce the hydrocarbon levels contained in oil in sea water. Measurement of Environmental Parameters. Observation of the environmental conditions of the test culture during the study was carried out by measuring temperature, pH, salinity, dissolved oxygen in the medium, and light intensity. Observation of the temperature of the test culture was carried out using a multiparameter tool. The results of the culture o temperature observations ranged from 27.3 - 28.3 C. Observation of the salinity of the test cultures was carried out using a refractometer. The results of the salinity measurement during the study on treatment K and A were 28 ‰, while in treatment B, C, and D were 30 ‰. This difference occurs because the composition of solvent seawater is more in treatment K and A. The initial salinity measurement of solvent seawater is 28 ppt. Dissolved oxygen observation in the test culture was carried out using a multiparameter device. -1 Dissolved oxygen data ranged between 7,1 - 9,5 mg L . Observation of pH of the test culture was carried out using pH paper. The result of pH measurement in the test culture during the study was 7. Observation of light intensity at the place where the test culture was placed was carried out using a lux meter. The results of observations of light intensity in this study ranged from 7200 - 7900 lux. DISCUSSION The results of culture color observations in the study were compared to the Faber castell color table. Overall, the color changes in cultures C and D occur more quickly than in other cultures. This can be seen on the 12th and 16th day the colors of the C and D cultures change to a denser green. These changes can occur due to greater levels of oil pollutants. Exposure to oil pollutants in high concentrations has an impact on decreased levels of carbohydrates and microalgae protein (Lewis & flechtner 2004). Oil pollutants in seawaters contain many fractions of compounds which can be grouped into hydrocarbon and non-hydrocarbon fractions. At higher concentrations of oil pollutants, the more non-hydrocarbon fractions on the medium. The non-hydrocarbon fraction includes several inorganic compounds such as nitrogen, sulfur, phosphorus, iron and some trace elements (Anand et al. 2019). High nitrogen content can interfere with the process of formation of photosynthetic pigments in microalgae. That is because the deactivation process in photosynthetic pigment activity (Valotton et al. 2008). The results of measurements of the average density of Scenedesmus vacuolatus cells on days 1 to 9 in the medium given sea water contaminated with hydrocarbons (culture A, B, C, and D) are higher than in the control medium. This is caused by the ability of microalgae to accumulate and use oil (petroleum hydrocarbons) as a source of organic compounds (Al Obaidy & Lami 2014). Growth continues until it reaches its peak on the 14th day for cultures on medium K, A, B, and C, while culture on medium D reaches the peak period on the 16th day. This can occur because of the higher concentration of petroleum hydrocarbons in the medium D. Provision of crude oil to microalgae culture can prolong the growth phase and produce high biomass production (Semple et al. 1999). After the 9th day to the 21st day, the average density of Scenedesmus vacuolatus cells in the control medium was higher than that of the entire medium with the addition of hydrocarbon polluted sea water. This can occur due to the impact of residual petroleum hydrocarbons that are unable to accumulate or are degraded by microalgae. Petroleum hydrocarbons can inhibit the growth of microalgae by reducing the ability to absorb CO , 2 photosynthesis, respiration, and cell division (Patel et al. 2017). The growth rate of Scenedesmus vacuolatus begins to decrease after the 14th day in cultures K, A, B, and C, whereas in culture D occurs after the 16th day. This is indicated by the decrease in the cell density curve. This occurs due to reduced nutrition in the medium and the effect of residual oil (petroleum hydrocarbons) which cannot be accumulated by microalgae. Petroleum hydrocarbons can be toxic to microalgae when forming thick oil layers around organisms, inhibiting gas diffusion, and destruction of cell membranes due to continuous hydrocarbon uptake (El-Dib et al. 2001). The highest decrease in total petroleum hydrocarbons was in culture B, by 70.62%. This shows that Scenedesmus vacuolatus can reduce TPH levels optimally at 50% petroleum hydrocarbon concentration. Microalgae degrades hydrocarbons by accumulating and transforming the compound (Phinyo et al. 2017). Some chemicals that can be found in total petroleum hydrocarbons (TPH) are hexane, jet fuel, mineral oil, benzene, toluene, xylene, naphthalene, fluorene, and other petroleum products (EPA 2017). One of the hydrocarbons that can be degraded by microalgae is phenol which is a derivative of benzene compounds. Phenols can be degraded by microalgae through the 114 SANTOSO ET AL. Microbiol Indones meta-cleavage pathway. The degradation process produces the final product, namely acetaldehyde, pyruvate and carbon dioxide. The three end products can be used in photosynthesis and microalgae cell respiration. Pyruvate can be metabolized in the Krebs cycle, and acetaldehyde can be converted to acetyl-CoA with the help of acetaldehyde dehydrogenase (AcDH) (Patel et al. 2017). The temperature range in this study is included in the optimal temperature range for the growth of the o genus Scenedesmus which is 25 - 30 C. The maximum o growth rate of the genus Scenedesmus occurs at 30 C. Temperature fluctuations in culture are caused by changes in the temperature of the surrounding environment. Temperature affects the chemical composition of cells, nutrient uptake and CO , and 2 microalgae growth rate. Salinity measurement results in studies are higher than the optimum salinity range for Scenedesmus growth Salinity affects the ability of Scenedesmus cells to accumulate oil. In high salinity, the genus Scenedesmus is able to accumulate oil up to 36% dry weight (Kaewkannetra et al. 2012). According to Latala's (1991) study, the genus Scenedesmus can live up to 25 ‰ salinity. In this study, S. vacuolatus was grown in medium with sea water with salinity up to 30‰. This shows that S. vacuolatus is an adaptive microalgae in an environment with salinity stress. Fluctuations in dissolved oxygen in the test culture are influenced by the density of Scenedesmus vacuolatus cells. It is also related to the oil layer in the medium and petroleum hydrocarbons that are accumulated by microalgae (Papa et al. 2009). The optimal pH range for growth of S. vacuolatus is 6.5 - 8.5. At pH more than 8.5 the growth rate will slow down (Neuwoener & Escher 2011). At pH 4.8 the growth rate of the genus Scenedesmus will stop (Nalewajko et al. 1997). Light intensity in this study is included in the optimum range for Scenedesmus growth. The genus Scenedesmus is able to grow at the lowest light intensity of 2,500 lux (Latiffi et al. 2017). Scenedesmus vacuolatus species are able to grow optimally to a light intensity of 49,200 lux (Carbone et al. 2017). AUTHORS’ CONTRIBUTIONS The main contributors were CAS and HA, while the others were the supporting contributors. ACKNOWLEDGEMENTS We acknowledge all the members of Department of Biology Universitas Indonesia, and Center of Environmental Technology (PTL), Agency for the Assessment and Application of Technology (BPPT) for their cooperation and assistance. This work was supported by a grant from Insentif Riset Sistem Inovasi Nasional (INSINAS) 2020, Ministry of Research, Technology, and Higher Education on behalf of HA. REFERENCES Al Obaidy AHMJ, Lami MH. 2014. The toxic effects of crude oil in some freshwater cyanobacteria. Journal of Environmental Protection. 5(5): 359-367. Amirlatifi F, Soltani N, Saadatmand S, Shokravi S, Dezfulian M. 2013. Crude oil-induced morphological and physiological responses in Cyanobacterium microchaete tenera ISC13. International Journal of Environmental Research. 7(4): 1007-1014. Ammar SH. 2015. Cultivation of microalgae Chlorella vulgaris in airlift photobioreactor for biomass production using commercial NPK nutrients. Al- Khawarizmi Engineering Journal. 12(1): 90-99. Anand V, Kashyap M, Samadhiya K, Ghosh A, Kiran B. 2019. Salinity driven stress to enhance lipid production in Scenedesmus vacuolatus: A biodiesel trigger?. Biomass and Bioenergy. 127(105252): 1-8. Andersen RA. 2005. Algal culturing techniques. Elsevier Academic Press, New York: 578 hlm. Carbone DA, Olivieri G, Pollio A, Melkonian M. 2017. Growth and biomass productivity of Scenedesmus vacuolatus on a twin layer system and a comparison with other types of cultivations. Applied microbiology and biotechnology. 101(23-24): 8321-8329. El-Dib MA, Abou-Waly HF, El-Naby AH. 2001. Fuel oil effect on the population growth, species diversity and chlorophyll (a) content of freshwater microalgae. International Journal of Environmental Health Research. 11(2): 189-197. Environmental Protection Agency (EPA). 2017. What are T o t a l P e t r o l e u m H y d r c a r b o n s ( T P H ) ? . https://www3.epa.gov/region1/eco/uep/tph.html. 1hlm. Diakses pada 1 November 2019 19.05. Guiry MD, Guiry GM. 2019. Algae Base. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; Diakses pada 28 Oktober 2019 pkl. 21.00. Ibrahim MBM, Gamila HA. 2004. Algal bioassay for Volume 14, 2020 Microbiol Indones 115 evaluating the role of algae in bioremediation of crude oil: II. freshwater phytoplankton assemblages. Bulletin of environmental contamination and toxicology. 73(6): 971-978. Ifeanyi VO, Ogbulie JN. 2016. Biodegradation of crude oil by microalgae Microcystis flos-aquae. Nigerian Journal of Microbiology. 30(2): 3459-3463. Kaewkannetra P, Enmak P, Chiu T. 2012. The Effect of CO 2 and Salinity on� the Cultivation of Scenedesmus obliquus for Biodiesel Production. Biotechnology and� bioprocess engineering. 17(3): 591-597.Latala A. 1991. Effects of Salinity, Temperature and Light on the Growth and Morphology of Green Planktonic Algae. Oceanologia. 31: 119-138. Latiffi A, Atikah N, Mohamed R, Saphira R M, Apandi NM, Tajuddin RM. 2017. Experimental assessment on effects of growth rates microalgae Scenedesmus sp. in different conditions of pH, temperature, light intensity and photoperiod. In Key Engineering Materials. 744: 546-551. Lewis LA, Flechtner VR. 2004. Cryptic species of Scenedesmus (Chlorophyta) from dessert soil communities of Western North America 1. Journal of phycology. 40(6): 1127-1137. Martinez CD, Mateos-Sanz A, Lopez-Rodas V, Costas E. 2011. Adaptation of microalgae to a gradient of continuous petroleum contamination. Aquatic Toxicology. 101(2), 342-350. Muchammad A, Kardena E, Rinanti A. 2013. Pengaruh i n t e n s i t a s c a h a y a t e r h a d a p p e n y e r a p a n g a s karbondioksida oleh mikroalga tropis Ankistrodesmus sp. dalam fotobioreaktor. Jurnal Teknik Lingkungan. 19(2): 103-111. Nair A, Desai I, Suresh B. 2014. Impact of petroleum hydrocarbons on the communiy structure of plankton in the coastal water of Saurashtra. Electronic Journal of Environmental Sciences. 136-157. Nalewajko C, Colman B, Olaveson M. 1997. Effects of pH on growth, photosynthesis, respiration, and copper t o l e r a n c e o f t h r e e S c e n e d e s m u s s t r a i n s . � Environmental and Experimental Botany. 37(2-3), 153-160. Neuwoehner J, Escher BI. 2011. The pH-dependent toxicity of basic pharmaceuticals in the green algae Scenedesmus vacuolatus can be explained with a toxicokinetic ion-trapping model. Aquatic toxicology. 101(1), 26—275. Papa RD, Wu JT, Baldia S, Cho C, Cruz MA, Saguiguit A, Aquino R. 2009. Blooms of the colonial green algae, Botryococcus braunii Kützing, in Paoay Lake, Luzon Island, Philippines. Phil. J. Syst. Biol. 2: 21—31. Patel A, Sartaj K, Arora N, Pruthi V, Pruthi PA. 2017. Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast. Journal of Hazardous Materials. 340: 47-56. Petsas AS, Vagi MC. 2017. Effects on the photosynthetic activity of algae after exposure to various organic and inorganic pollutants: Review. Chlorophyll. 37-77. Phinyo K, Pekkoh J, Peerapornpisal Y. 2017. Distribution and ecological habitat of Scenedesmus and related genera in some freshwater resources of Northern and North-Eastern Thailand. Biodiversitas Journal of Biological Diversity. 18(3): 1092-1099. Piehler MF, Winkelmann V, Twomey LJ, Hall NS, Currin CA, Paerl HW. 2003. Impact of diesel fuel exposure on the microphytobenthic community of an intertidal sand flat. J Exp Mar Biol Ecol. 297: 219-237. Priyadarshani I, Sahu D, Rath B. 2011. Microalgal bioremediaton: Current practices and perspectives. J Biochem Tech. 3(3): 299-304. Ramadass K, Megharaj M, Venkateswarlu K, Naidu R. 2015. Toxicity and oxidative stress induced by used and unused motor oil on freshwater microalga, Pseudokirchneriella subcapitata. Environmental Science and Pollution Research. 22(12): 8890-8901. Semple KT, Cain RB, Schmidt S. 1999. Biodegradation of aromatic compounds by microalgae. FEMS Microbiology letters. 170(2): 291-300. Singh AK, Gaur JP. 1990. Effects of petroleum oils and their paraffinic, asphaltic, and aromatic fractions on photosynthesis and respiration of microalgae. Ecotoxicology and environmental safety. 19(1): 8-16. SNI 6989.10:2011. Air dan air limbah – Bagian 10: Cara uji minyak nabati dan minyak mineral secara gravimetri. 13 hlm. Vallotton N, Moser D, Eggen RI, Junghans M, Chèvre N. 2008. S-metolachlor pulse exposure on the alga Scenedesmus vacuolatus: Effects during exposure and the subsequent recovery. Chemosphere. 73(3): 395- 400. Wetherell DF. 1963. Osmotic equilibration and growth of Scenedesmus obliquus in saline media. Physiologia Plantarum. 16(1): 82–91. Wibowo M. 2018. Pemodelan sebaran pencemaran tumpahan minyak di perairan Cilacap. Jurnal Teknologi Lingkungan. 19(2): 191-202. 116 SANTOSO ET AL. Microbiol Indones Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9