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Batch cultivation of marine microalgae Nannochloropsis oculata and Tetraselmis suecica in treated municipal wastewater toward bioethanol production
Journal of Cleaner Production 150 (2017) 40e46
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Batch cultivation of marine microalgae Nannochloropsis oculata and Tetraselmis suecica in treated municipal wastewater toward bioethanol production * € Zubaidai Reyimu, Didem Ozçimen Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Yildiz Technical University, Davutpasa Campus, 34220, Esenler, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 December 2016 Received in revised form 16 February 2017 Accepted 26 February 2017
Algae have come into prominence over the last decade as a commercial biofuel feedstock due to their high production efficiencies compared to first and second feedstocks. However, algal investment is not economically feasible currently due to the operational and capital cost. There is still need for innovations for both high productivity and green productions. In order to decrease the cost of the algal processes, usage of some wastes as cultivation medium for algal productions and novel biofuel production methods should be considered according to green chemistry principals. In this study, seawater and wastewater supplied from Istanbul Water and Sewerage Administration (ISKI) were used and combined at different ratios to be utilized as a growth medium for Nannochloropsis oculata and Tetraselmis suecica microalgae strain under the same growth conditions, and its effect on cell proliferation and growth kinetics were investigated. It was found that, both N. oculata and T. suecica can tolerate and utilize the wastewater and, the specific growth rate of the cultures can up to 0.5430 d1 (75% of wastewater) for N. oculata and 0.4778 d1 (25% of wastewater) for T. suecica. Different concentrations show different results for the growth of two species due to the effect of higher concentrations of the fundamental sources on growth stage and change of ionic composition of the culture medium. To evaluate bioethanol production performance of these two strains, samples which included maximum carbohydrate content as well as control groups were chosen for further studies. The results showed that T. suecica is much suitable for ethanol production using municipal wastewater as a culture medium. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Nannochloropsis oculata Tetraselmis suecica Wastewater Microalgae Bioethanol
1. Introduction Meeting the increasing energy demand has been one of the important problems to be solved over last decade due to the expansion of world population and increase in industrial prosperity. The major energy demand is still supplied by conventional fossil fuels such as oil, coal, and natural gas (Li et al., 2014). According to Singh et al., fossil fuels accounted for 88% of the global primary energy consumption (Singh et al., 2011). However, rising prices and environmental problems such as acid rains and global warming lead countries to take major precaution to overcome this problem. Therefore, it is urgent to develop and produce new, environmentally friendly biofuel to meet world’s increasing need.
* Corresponding author. € E-mail address: [email protected] (D. Ozçimen). http://dx.doi.org/10.1016/j.jclepro.2017.02.189 0959-6526/© 2017 Elsevier Ltd. All rights reserved.
In recent years, there has been a trend towards the increased commercialization of various renewable energy sources. First generation biofuels such as bioethanol from wheat, biogas from corn, and biodiesel from rapeseed oil have been criticized for low land-use efficiency, increasing demand on arable land, and poor carbon balance (Andersson et al., 2014) and the production of first generation biomass also leads to the rising of food price. These increasing concerns about the first generation feedstock has raised attention to second-generation biofuels. Second generation biofuels are generally produced from waste biomass sources such as lignocellulosic forest wastes, agricultural food production residues, waste and non-edible oils (Nigam and Singh, 2011; Bhuiya et al., 2014) Second generation biofuel production has several advantages such as consuming wastes, making use of abandoned land, and promote rural development and the production of secondgeneration biofuels resolve the food versus fuel debate in the production of first generation biofuels (Nigam and Singh, 2011).
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On the other hand, their harvesting and purification and need of various pre-treatments made their production quite challenging and not economical (Singh et al., 2011). Algae can be considered as the third generation of biofuel feedstock considering the challenges of the first and second feedstock faced for renewable energy production. In recent years, microalgae related studies is growing and becoming popular due to the fact that microalgae need less cultivation area than terrestrial crops and also can be cultivated in saline water, brackish water or even wastewater. In cultivation process, culture media and culture conditions are the main aspects to be considered. In order to increase productivity of microalgae, nutrients such as nitrate, urea, ammonium, vitamins, phosphorous, nitrogen, iron, manganese, selenium, cobalt, nickel, and zinc are required in optimal level and as well as growth conditions, yet this may become more costly (Singh et al., 2015) than other feedstock. However, there are also some drawbacks about using algae for biofuel production. Several researches are carried out about the life cycle assessment of microalgal productions to reveal its economic, social, energy and environmental impacts and associated challenges (Wang et al., 2016). The most crucial problem is its low sufficiency to produce algae on large commercial scale because of high production costs (Vassilev and Vassileva, 2016). Huge investments are required in growing algae as flat lands and abundant water are essential (Bungay, 2004). In order to decrease the cost of the algal processes, usage of some wastes such as municipal wastewater, food industry wastes, and forestry wastewater as cultivation medium for algal productions should be utilized. Today, microalgae cultivation in wastewater is considered to be the most-effective way of biofuel production (Cheah et al., 2016), and the use of microalgae for treatment and recycling of wastewater has gained a lot of interest due to their capability of carbon dioxide fixation. Wastewater can be potentially a sustainable growth medium for algal biomass. As it is well known, wastewater includes plentiful of carbon, nitrogen and phosphorous which can easily cause eutrophication (Ramachandra et al., 2013). At the same time, growth of microalgae needs these elements to produce carbohydrates, lipids and proteins to produce different types of biofuel (He et al., 2013). In this study, to test influence of wastewater on different types of microalgae and its productivity on biofuel production, two different types of microalgae were cultivated with various concentrations of wastewater. N. oculata and T. suecica were cultivated at different ratios of municipal wastewater and seawater as a growth medium under the same growth condition, and the optimum combination ratio of wastewater was determined according to growth kinetic results. In addition to this, carbohydrate content and dry weight of microalgal species were also measured in order to evaluate bioethanol productivity of these two microalgae strains which were cultivated under optimal wastewater medium. 2. Experimental section
and sterilized before utilization. Total carbon and nitrogen analysis of wastewater was given in Table 1. f/2 medium which was used for the cultivation of control group was supplied from Sigma-Aldrich. Sodium hydroxide was used for pre-treatments and 96% grade of ethanol was used to determine the concentration of bioethanol. Sulfuric acid (98% concentrated), phenol and D-glucose were used for determination of carbohydrate content. Luria Broth (LB) medium was used for the yeast growth. All these chemicals were supplied from Merck. 2.2. Biomass cultivation The study was carried out in 250 ml Erlenmeyer flasks containing fresh N. oculata and T. suecica microalgae strain as well as different concentration of wastewater medium for each sample. The batch cultures of N. oculata and T. suecica were grown at different ratio of wastewater and filtered autoclaved seawater (FASW) (0% (control), 25%, 50%, 75% and 100% wastewater in seawater solution). Seawater used in the study may contain different microorganisms, impurities (jellyfish, mussel, sand) and/ or different types of microalgae. In order to prevent the contamination, seawater was firstly filtered and then autoclaved in this study. As for the control group, only f/2 medium was used. Cultures were incubated in a shaking incubator at the temperature of 24 ± 2 C and 150 rpm mixing rate. Continuous illumination was provided by 18 W- fluorescent tubes (1300 lm per fluorescent tubes). pH of the culture medium was maintained between 7.5 and 8.5 during 14 days of cultivation. After 14 days, all samples were dried and milled to obtain a fine powder and weighing for determining the biomass productivity of different concentration of wastewater. After drying and milling, samples that present highest carbohydrate content as well as control groups of N. oculata and T. suecica were chosen for bioethanol production. 2.3. Bioethanol production To convert algal biomass into bioethanol, alkaline pre-treatment was performed to degrade the biomass and increase the yeast’s the accessibility to carbohydrate structures for fermentation. In this process, 0.75% (w/v) concentration of NaOH was added to the algal biomass and stirred at 500 rpm for 10 min. Then the four samples were incubated at the temperature of 120 C for 30 min in an oven (Harun et al., 2010a). After incubation, samples were cooled down to room temperature and centrifuged for 10 min at 4500 rpm. The supernatants were collected for bioethanol production. Along with this process, yeast S. cerevisiae was prepared in the same way as reported in (Harun et al., 2010a,b). After all the preparation and pretreatment process, 3% (v/v) of the yeast culture was inoculated into the pre-treated four different microalgae biomass samples. Fermentation was carried out at 30 C in a shaking incubator set to 150 rpm for 48 h. 2.4. Analytical methods
2.1. Materials N. oculata and T. suecica were obtained from Algal Biotechnology Laboratory of Yıldız Technical University, TURKEY. Municipal wastewater used for the cultivation of microalgal cultures, was collected from Istanbul Water and Sewerage Administration (ISKI)
Table 1 TOC analysis of ISKI wastewater.
ISKI WW
41
Total carbon concentration
Total nitrogen concentration
7,67 mg/l ± 655,4 mg/l
3,77 mg/l ± 117,4 mg/l
During 14 days of cultivation, measurements of optical density and cell density of the cultures were used to monitor the algal growth. Optical density was measured by using UVevisible spectrophotometer at 680 nm for N. oculata and 620 nm for T. suecica, respectively (Doan et al., 2011; Lim et al., 2006; Gu et al., 2012a, 2012b). Cell densities of N. oculata and T. suecica were measured using hemocytometer under microscope during 14 days of cultivation (Bastidas, 2009). Determination of the dry weight and measurement of carbohydrate content were carried out on the 14th day of cultivation (Alsull and Omar, 2012). Phenol-sulfuric acid method was used for total carbohydrate analysis.
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Productivity of N. oculata and T. suecica were expressed as the specific growth rate (m) and doubling time (td) by using Equations (1) and (2) from the cell density change during specific time period of exponential phase (Widdel, 2007).
m¼
Nt ln N0 t
td ¼
0:693
m
(1)
(2)
To evaluate bioethanol concentration YL 6100 GC gas chromatography was used. Samples that obtained during the bioethanol fermentation process (24 h and 48 h) were taken and prepared for GC instrument for further analysis. Samples were filtered using 0.45 mm filters to avoid blocking in column. The GC gas chromatograph contains flame ionization detector (FID) and 30 m 0.32 mm x 0.25 mm ZB-FFAP column oven. The temperature of injector, detector and oven were maintained at 150, 250 and 100 C, respectively. Hydrogen was used as carrier gas. Bioethanol concentration was calculated using calibration curve that prepared by the different concentration of bioethanol standards (0.1%e10% (v/v)) (Inan, 2014). Total carbohydrate and GC analysis were carried out in triplicate. The results obtained from each experiment were the average of these data. 3. Results and discussion 3.1. Effect of municipal wastewater on N. oculata and T. suecica After 14 days of cultivation, the cell density and the optical density of N. oculata and T. suecica cultivated at different concentrations of wastewater were shown in Figs. 1e2. The growth curve of N. oculata, shows that, the maximum cell density was reached at 75% of wastewater medium on the 7th day (up to 12.75 106 cells/ ml). As for control group, 25% wastewater and 50% wastewater, algal growth started earlier, almost without lag-phase, than the other 75% and 100% wastewater cultivation groups. It can be commented that microalgae groups which were cultivated with the low nutrient levels of the wastewater may well adapted to the culture medium because of the proper amount of nutrients were given (Brennan and Owende, 2010). As for 75% and 100% wastewater medium, the nutrient levels were much higher, so it may take some time to adapt to the abundant nutrients at lag-phase and then microalgae species grow faster with high growth rate because of the adequate nutrient supply. On the other hand, it was seen that the growth curve of T. suecica was not so steady compared to N. oculata samples. It may be due to T. suecica and N. oculata showed different reaction to the compounds in wastewater. As for T. suecica, the logarithmic phases of the control group and culture that cultivated with 25% of wastewater started earlier than the other microalgae groups. It can be reported that, the higher the wastewater concentration is, the later the logarithmic phase starts for T. suecica. It can be explained that the growth of T. suecica may be highly affected by the changes in nutrient level or culture condition (Gouveia, 2011), and the higher nutrient level may hindered the strain from entering into the logarithmic phase because of intensive amount of nutrient prompt change in medium conditions. F/2 medium which was used for control group, was prepared with the stock solutions of 75 g/L dH2O NaNO3, 5 gr/L dH2O NaH2PO4H2O with the molar concentrations in final medium as 8.82 104M and 3.62 105M, respectively. There was no carbon source in F/2 medium and also seawater has not enough carbon and other elements in ppm concentration in its
composition. In comparison with F/2 medium, wastewater has significant amount of carbon and nitrogen source. Increasing concentration of wastewater causes the positive effect on N. oculata growth and the negative effect on T. suecica growth. The use of nitrogen changes the pH of microalgae culture. If the microalgae use ammonium as nitrogen source, the Hþ ion is released and the pH decreases. Reaction is given with the equation below:
NH4þ þ 7:6CO2 þ 17:7H2 O / C7:6 H8:1 O2:5 N þ 7:6O2 þ 15:2H2 O þ Hþ (3) High amount of ammonium usage can stop microalgae growth causing a decrease in the culture pH as pH < 6. In the case of utilizing nitrate, pH of the medium increases with the nitrate consumption. Theoretically, when 1 mol of nitrate is used, 1 mol of OH is produced. Reaction is given with the equation below: NO 3 þ 5:7CO2 þ 5:4H2 O/C5:7 H9:8 O2:3 N þ 8:25O2 þ OH
(4)
There is a linear relationship between nitrate usage, and the pH of the medium and microalgae growth. If nitrate is used excessively, the pH may increase to over 10 and may affect microalgae growth (Rashid et al., 2014). Similar results can be seen in the literature. It was proven that, optimum growth conditions for T. suecica was bregas found at high salinity and low NaNO3 concentrations (Fa et al., 1984). The maximum cell density of T. suecica was observed with control group on 7th day (6.25 105 cells/ml), which maybe due to T. suecica is not so adapted to the wastewater medium when compared to N. oculata. 3.2. Specific growth rate and cell doubling time According to Table 2, as it was expected, the specific growth rate of N. oculata in different concentration of wastewater was consistently higher than the specific growth rate of T. suecica. In the similar studies, it was reported that the growth rate of N. oculata was measured as 0.39 d1 at the temperature of 25 ± 1 C under continuous illumination, and the growth rate of T. suecica as 0.19 d1 at the temperature of 20 C under a constant photon flux €€ density, respectively (S¸irin and Sillanpa a, 2015; Giordano et al., 2015). In this study, however, the maximum specific growth rate of N. oculata and T. suecica reached to 0.5430d1 in 75% of wastewater medium and 0.4778 d1 in 25% of wastewater medium, respectively. Therefore, it can be concluded that 75% and 25% concentrations of wastewater are the ideal concentrations for cultivating N. oculata and T. suecica for high growth rate and low doubling time. 3.3. Dry weight and carbohydrate content The results in Table 3 showed that the dry weight of all groups of N. oculata were higher than the dry weight of T. suecica. Furthermore, when dry weights of N. oculata were measured, it was found that there were white unknown ingredients in powder. Especially in control group, there were much more white ingredients than any other groups and its dry weight showed the highest result (2.41 g/L) among all groups. In the literature, the other studies also mentioned that there were observed ash and slag deposits. This can be explained as the presence of inorganic component in the biomass can influence the formation of ash (Hamidi et al., 2014). In another study, it is claimed that there wasn’t any differences for the ash content in N. oculata cultivated with agricultural fertilizer, aquacultural fertilizer and f/2 media, and the content was ranged ~ a-Torres et al., 2012; Marinho-Soriano from 26% to 27% (Campan
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(a) N. oculata
140
127.5
Cell density (Cells/ml)
120 100 80 60 40 20 0 0
2
4
6
8
10
12
14
Culture dura on (Day) control
25%wastewater
75%wastewater
100%wastewater
50%wastewater
(b) T. suecica 7
6.25
Cell density (Cells/ml)
6 5 4 3 2 1 0 0
2
4
6
8
10
12
14
Culture dura on (Day) control
25%wastewater
75%wastewater
100%wastewater
50%wastewater
Fig. 1. Cell densities versus culture period (day) for N. oculata (a) and T. suecica (b) in control and different wastewater concentration (25%, 50%, 75% and 100%).
et al., 2006). This suggests that the ash content of N. oculata must be taken into consideration because it may directly influence the carbohydrate concentration in dry weight. On the other hand, the
milled fine powder of T. suecica, can be regarded as pure green and such ash content was observed when taking its dry weight into consideration (Reyimu, 2016). The maximum dry weight was
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(a) N. oculata 0.6
Absorbance(680nm)
0.5 0.4 0.3 0.2 0.1 0 0
2
4
6
8
10
12
14
Culture period (Day) control
25%wastewater
75%wastewater
100%wastewater
50%wastewater
(b) T. suecica 0.18 0.16 Absorbance(620nm)
0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0
2
4
6
8
10
12
14
Culture period (Day) control
25% wastewater
75%wastewater
100%wastewater
50%wastewater
Fig. 2. Growth curve of the N. oculata (a) and T. suecica (b) in control and different wastewater concentration (25%, 50%, 75% and 100%).
obtained from cultures grown in 75% of wastewater. This results matches with a similar study implemented by Beuckels et al. that microalgae can adjust the N and P concentrations of their biomass depending on their supply of nutrients in the medium, so that it
may result in a low dry weight when the supply of a nutrient is low (Hamidi et al., 2014; Beuckels et al., 2015). In this experimental study, as 75% of wastewater contains higher N and P, so that it accumulate much more biomass than other groups. According to
€ Z. Reyimu, D. Ozçimen / Journal of Cleaner Production 150 (2017) 40e46 Table 2 Specific growth rate (m) and doubling time (td) of N. oculata and T. suecica. N. oculata
0% (Control) 25% WW 50% WW 75% WW 100% WW
T. suecica
m (d1)
td (d)
m/(d1)
td (d)
0.3698 0.3095 0.1248 0.5430 0.3793
1.874 2.239 5.553 1.276 1.837
0.1488 0.4778 0.1352 0.2824 0.1105
4.657 1.450 5.126 2.454 6.271
Table 3, after 14 days of cultivation, T. suecica accumulated much more carbohydrate than N. oculata. This is also mentioned in other studies that, T. suecica is the microalgae species that tend to accumulate high carbohydrate content and its carbohydrate can range from 21% to 64% of their cell dry weight (Lam and Lee, 2015). In this study, the highest carbohydrate content in wastewater group of T. suecica was measured in control group (27% dry weight) followed by 100% of wastewater (6.52% dry weight). This is promising when considering T. suecica with this concentration of wastewater as an optimum feedstock for bioethanol production. As for N. oculata, however, maximum carbohydrate concentration in wastewater group was found in 75% concentration of wastewater sample (2.39% dry weight). The result was low when compared to other studies that this may be because the existence of ash content in dry biomass which influence the overall carbohydrate content. 3.4. Bioethanol production According to the BP statistical review of world energy-2016 report (BP, 2016), it can be seen that, ethanol production of the
45
world is higher than biodiesel production. The increase of the production of ethanol in last decade is quite remarkable, and due to the global warming and high oil prices, it is considered that the production of ethanol will continue to increase. Although there are a lot of studies on biodiesel production from different type of microalgae species in the literature, researches on bioethanol production from microalgae are less. For this reason, bioethanol production from the microalgae species was studied considering the world’s renewable energy trends. According to Table 3 results, N. oculata and T. suecica samples that showed highest carbohydrate content in wastewater sample (N. oculata cultivated in 75% of wastewater medium and T. suecica cultivated in 100% of wastewater medium) and the control groups were chosen to compare their bioethanol productivity. Fig. 3 shows that bioethanol yield of N. oculata and T. suecica ranges from 0.41% to 7.26%. When comparing ethanol yield of N. oculata and T. suecica, it is clear that T. suecica showed better ethanol yield both at 24 h and 48 h than N. oculata samples. This is due to the fact that T. suecica accumulated much more carbohydrate than N. oculata during cultivation. In Fig. 3, it is observed that both at 24 h and 48 h of fermentation periods, the bioethanol yield of N. oculata cultivated in 75% of wastewater sample was higher than control group that and the yield increased from 1.21% to 3.68%. The 75% of wastewater group also showed highest carbohydrate yield, this results were expected. As for T. suecica, ethanol yield resulted in higher in control group than 100% of wastewater group. The yield increased from 2.46% to 7.26% at 24 and 48 h of fermentation. This is mostly because of the control group achieved to accumulated highest amount of carbohydrate (27% of dry weight) regardless of its lower dry weight (0.14 g/L). In 100% concentration of wastewater group, although it did not showed highest ethanol yield, it is still
Table 3 Carbohydrate content and dry weight of N. oculata and T. suecica on the 14th day of cultivation. N. oculata
Control 25% ww 50% ww 75% ww 100% ww
T. suecica
CH content (% dry weight)
Dry weight(g/L)
CH content (% dry weight)
Dry weight(g/L)
1 ± 0.3% 2.26 ± 0.2% 2.30 ± 0.4% 2.39 ± 0.1% 2.11 ± 0.2%
2.41 1.19 1.28 1.285 0.975
27 ± 0.1% 4.24 ± 0.1% 4.54 ± 0.1% 3.42 ± 0.2% 6.52 ± 0.2%
0.14 0.76 0.855 1.055 0.48
N.oculata
T.suecica Bioethanol yield (%)
Bioethanol yield (%)
5 4 3 2 1 0 24 h 48 h Fermenta on hours Control group
75% of wastewater
8 7 6 5 4 3 2 1 0 24 h
48 h
Fermenta on hours Control group 100% of wastewater
Fig. 3. Bioethanol yield of N. oculata and T. suecica during 24 h and 48 h of fermentation.
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higher when comparing with both control and 75% of wastewater sample of N. oculata. As in stoichiometric aspects, it can be commented that, the higher carbohydrate content will increase the bioethanol yield. 4. Conclusion This study demonstrates that for effective cultivation microalgae species such as N. oculata and T. suecica, municipal wastewater can be utilized at a specific concentration as a medium. According to specific growth rate, after 14 days of cultivation, it was found that 75% concentration of wastewater can be an ideal concentration for N. oculata medium, while 25% concentration of wastewater was optimum for T. suecica. When N. oculata and T. suecica were compared, adaptation of N. oculata to wastewater medium resulted better than T. suecica according to their growth rate profile. After 14 days of cultivation, bioethanol productivities of two species were also investigated. The highest bioethanol yield of N. oculata was obtained from the microalgae cultivated in 75% of wastewater media as 3.68%. The results showed that N. oculata cultivated at 75% of wastewater represents highest carbohydrate content; hence its ethanol concentration was higher than control group. On the other hand, in comparison with N. oculata, the highest carbohydrate content of T. suecica was found in the control group. The second highest carbohydrate content of T. suecica samples was obtained from the cultures grown at 100% concentration of wastewater medium. The highest bioethanol yield was 7.26% which was obtained from the control group of T. suecica. When comparing ethanol concentration of N. oculata and T. suecica, it was obvious that, T. suecica samples showed higher ethanol yield than N. oculata. These results indicated that T. suecica was more suitable for ethanol production using wastewater as a culture medium, as had higher carbohydrate content than N. oculata. Using wastewater for microalgal production also may not seem economically due to the sterilization because of containing different chemical contents and different bacterial microorganisms. However, some of the wastewater from advanced biological wastewater treatment plants of ISKI, is used as industrial water and used for irrigation of recreation areas after the final disinfection process. A significant portion of the energy required to operate the treatment plants is also utilized from this wastewater treatment process. Also improved filtration systems can be applied for large-scale productions. In conclusion, these results suggested that municipal wastewater from ISKI can be utilized as growth medium for N. oculata and, as well as T. suecica and it can be a remarkable solution for waste utilization and bioethanol production can be improved with further studies. Acknowledgement Zubaidai Reyimu gratefully acknowledges Turks Abroad and Related Communities for the scholarship programme. References Alsull, M., Omar, W.M.W., 2012. Responses of Tetraselmis sp. and Nannochloropsis sp. isolated from Penang National Park coastal waters, Malaysia, to the combined influences of salinity, light and nitrogen limitation. In: International Conference on Chemical, Ecology and Environmental Sciences (ICEES 2012). Andersson, V., Viklund, S.B., Hackl, R., Karlsson, M., Berntsson, T., 2014. Algae-based biofuel production as part of an industrial cluster. Biomass Bioenerg. 71, 113e124, 2014. Bastidas, O., 2009. Technical NoteeNeubauer Chamber Cell Counting. www. researchgate.net/publictopics.PublicPostFileLoader.html? id¼53c6a939d4c11856478b45d2&key¼89353f79-aa75e4e19-8161d900619b728d (accessed 18 April 2016). Beuckels, A., Smolders, E., Muylaert, K., 2015. Nitrogen availability influences phosphorus removal in microalgae-based wastewater treatment. Water Res. 77, 98e106.
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Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia for biodiesel production. Bioresour. Technol. 129, 177e181. Inan, B., 2014. Utilization of Algae for Bioethanol Production. Master’s Thesis. Yıldız Technical University. Lam, M.K., Lee, K.T., 2015. Bioethanol production from microalgae. In: Kim, S. (Ed.), Handbook of Marine Microalgae-Biotechnology Advances. Elsevier Academic Press, Amsterdam, pp. 197e209. Li, K., Liu, S., Liu, X., 2014. An overview of algae bioethanol production. Int. J. Energ Res. 38 (8), 965e977. Lim, C.Y., Yoo, Y.H., Sidharthan, M., Ma, C.W., Bang, I.C., Kim, J.M., Lee, K.S., Park, N.S., Shin, H.W., 2006. Effects of copper(I) oxide on growth and biochemical compositions of two marine microalgae. J. Environ. Biol. 27 (3), 461e466. Marinho-Soriano, E., Fonseca, P.C., Carneiro, M.A.A., Moreira, W.S.C., 2006. Seasonal variation in the chemical composition of two tropical seaweeds. Bioresour. Technol. 97 (18), 2402e2406. Nigam, P.S., Singh, A., 2011. Production of liquid biofuels from renewable resources. Prog. Energy Combust. Sci. 37 (1), 52e68. Ramachandra, T.V., Madhab, M.D., Shilpi, S., Joshi, N.V., 2013. Algal biofuel from urban wastewater in India: scope and challenges. Renew. Sustain. Energy Rev. 21, 767e777. Rashid, N., Rehman, M.S.U., Sadiq, M., Mahmood, T., Han, J.I., 2014. Current status, issues and developments in microalgae derived biodiesel production. Renew. Sustain. Energy Rev. 40, 760e778, 2014. Reyimu, Z., 2016. Cultivation of Nannochloropsis Oculata and Tetraselmis Suecica in Treated Municipal Wastewater toward Microalgal Bioethanol Production. Master’s Thesis. Yıldız Technical University. Singh, A., Nigam, P.S., Murphy, J.D., 2011. Renewable fuels from algae: an answer to debatable land based fuels. Bioresour. Technol. 102 (1), 10e16. Singh, P., Gupta, S.K., Guldhe, A., Rawat, I., Bux, F., 2015. Microalgae isolation and basic culturing techniques. In: Kim, S. 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Batch cultivation of marine microalgae Nannochloropsis oculata and Tetraselmis suecica in treated municipal wastewater toward bioethanol production * € Zubaidai Reyimu, Didem Ozçimen Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Yildiz Technical University, Davutpasa Campus, 34220, Esenler, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 December 2016 Received in revised form 16 February 2017 Accepted 26 February 2017
Algae have come into prominence over the last decade as a commercial biofuel feedstock due to their high production efficiencies compared to first and second feedstocks. However, algal investment is not economically feasible currently due to the operational and capital cost. There is still need for innovations for both high productivity and green productions. In order to decrease the cost of the algal processes, usage of some wastes as cultivation medium for algal productions and novel biofuel production methods should be considered according to green chemistry principals. In this study, seawater and wastewater supplied from Istanbul Water and Sewerage Administration (ISKI) were used and combined at different ratios to be utilized as a growth medium for Nannochloropsis oculata and Tetraselmis suecica microalgae strain under the same growth conditions, and its effect on cell proliferation and growth kinetics were investigated. It was found that, both N. oculata and T. suecica can tolerate and utilize the wastewater and, the specific growth rate of the cultures can up to 0.5430 d1 (75% of wastewater) for N. oculata and 0.4778 d1 (25% of wastewater) for T. suecica. Different concentrations show different results for the growth of two species due to the effect of higher concentrations of the fundamental sources on growth stage and change of ionic composition of the culture medium. To evaluate bioethanol production performance of these two strains, samples which included maximum carbohydrate content as well as control groups were chosen for further studies. The results showed that T. suecica is much suitable for ethanol production using municipal wastewater as a culture medium. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Nannochloropsis oculata Tetraselmis suecica Wastewater Microalgae Bioethanol
1. Introduction Meeting the increasing energy demand has been one of the important problems to be solved over last decade due to the expansion of world population and increase in industrial prosperity. The major energy demand is still supplied by conventional fossil fuels such as oil, coal, and natural gas (Li et al., 2014). According to Singh et al., fossil fuels accounted for 88% of the global primary energy consumption (Singh et al., 2011). However, rising prices and environmental problems such as acid rains and global warming lead countries to take major precaution to overcome this problem. Therefore, it is urgent to develop and produce new, environmentally friendly biofuel to meet world’s increasing need.
* Corresponding author. € E-mail address: [email protected] (D. Ozçimen). http://dx.doi.org/10.1016/j.jclepro.2017.02.189 0959-6526/© 2017 Elsevier Ltd. All rights reserved.
In recent years, there has been a trend towards the increased commercialization of various renewable energy sources. First generation biofuels such as bioethanol from wheat, biogas from corn, and biodiesel from rapeseed oil have been criticized for low land-use efficiency, increasing demand on arable land, and poor carbon balance (Andersson et al., 2014) and the production of first generation biomass also leads to the rising of food price. These increasing concerns about the first generation feedstock has raised attention to second-generation biofuels. Second generation biofuels are generally produced from waste biomass sources such as lignocellulosic forest wastes, agricultural food production residues, waste and non-edible oils (Nigam and Singh, 2011; Bhuiya et al., 2014) Second generation biofuel production has several advantages such as consuming wastes, making use of abandoned land, and promote rural development and the production of secondgeneration biofuels resolve the food versus fuel debate in the production of first generation biofuels (Nigam and Singh, 2011).
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On the other hand, their harvesting and purification and need of various pre-treatments made their production quite challenging and not economical (Singh et al., 2011). Algae can be considered as the third generation of biofuel feedstock considering the challenges of the first and second feedstock faced for renewable energy production. In recent years, microalgae related studies is growing and becoming popular due to the fact that microalgae need less cultivation area than terrestrial crops and also can be cultivated in saline water, brackish water or even wastewater. In cultivation process, culture media and culture conditions are the main aspects to be considered. In order to increase productivity of microalgae, nutrients such as nitrate, urea, ammonium, vitamins, phosphorous, nitrogen, iron, manganese, selenium, cobalt, nickel, and zinc are required in optimal level and as well as growth conditions, yet this may become more costly (Singh et al., 2015) than other feedstock. However, there are also some drawbacks about using algae for biofuel production. Several researches are carried out about the life cycle assessment of microalgal productions to reveal its economic, social, energy and environmental impacts and associated challenges (Wang et al., 2016). The most crucial problem is its low sufficiency to produce algae on large commercial scale because of high production costs (Vassilev and Vassileva, 2016). Huge investments are required in growing algae as flat lands and abundant water are essential (Bungay, 2004). In order to decrease the cost of the algal processes, usage of some wastes such as municipal wastewater, food industry wastes, and forestry wastewater as cultivation medium for algal productions should be utilized. Today, microalgae cultivation in wastewater is considered to be the most-effective way of biofuel production (Cheah et al., 2016), and the use of microalgae for treatment and recycling of wastewater has gained a lot of interest due to their capability of carbon dioxide fixation. Wastewater can be potentially a sustainable growth medium for algal biomass. As it is well known, wastewater includes plentiful of carbon, nitrogen and phosphorous which can easily cause eutrophication (Ramachandra et al., 2013). At the same time, growth of microalgae needs these elements to produce carbohydrates, lipids and proteins to produce different types of biofuel (He et al., 2013). In this study, to test influence of wastewater on different types of microalgae and its productivity on biofuel production, two different types of microalgae were cultivated with various concentrations of wastewater. N. oculata and T. suecica were cultivated at different ratios of municipal wastewater and seawater as a growth medium under the same growth condition, and the optimum combination ratio of wastewater was determined according to growth kinetic results. In addition to this, carbohydrate content and dry weight of microalgal species were also measured in order to evaluate bioethanol productivity of these two microalgae strains which were cultivated under optimal wastewater medium. 2. Experimental section
and sterilized before utilization. Total carbon and nitrogen analysis of wastewater was given in Table 1. f/2 medium which was used for the cultivation of control group was supplied from Sigma-Aldrich. Sodium hydroxide was used for pre-treatments and 96% grade of ethanol was used to determine the concentration of bioethanol. Sulfuric acid (98% concentrated), phenol and D-glucose were used for determination of carbohydrate content. Luria Broth (LB) medium was used for the yeast growth. All these chemicals were supplied from Merck. 2.2. Biomass cultivation The study was carried out in 250 ml Erlenmeyer flasks containing fresh N. oculata and T. suecica microalgae strain as well as different concentration of wastewater medium for each sample. The batch cultures of N. oculata and T. suecica were grown at different ratio of wastewater and filtered autoclaved seawater (FASW) (0% (control), 25%, 50%, 75% and 100% wastewater in seawater solution). Seawater used in the study may contain different microorganisms, impurities (jellyfish, mussel, sand) and/ or different types of microalgae. In order to prevent the contamination, seawater was firstly filtered and then autoclaved in this study. As for the control group, only f/2 medium was used. Cultures were incubated in a shaking incubator at the temperature of 24 ± 2 C and 150 rpm mixing rate. Continuous illumination was provided by 18 W- fluorescent tubes (1300 lm per fluorescent tubes). pH of the culture medium was maintained between 7.5 and 8.5 during 14 days of cultivation. After 14 days, all samples were dried and milled to obtain a fine powder and weighing for determining the biomass productivity of different concentration of wastewater. After drying and milling, samples that present highest carbohydrate content as well as control groups of N. oculata and T. suecica were chosen for bioethanol production. 2.3. Bioethanol production To convert algal biomass into bioethanol, alkaline pre-treatment was performed to degrade the biomass and increase the yeast’s the accessibility to carbohydrate structures for fermentation. In this process, 0.75% (w/v) concentration of NaOH was added to the algal biomass and stirred at 500 rpm for 10 min. Then the four samples were incubated at the temperature of 120 C for 30 min in an oven (Harun et al., 2010a). After incubation, samples were cooled down to room temperature and centrifuged for 10 min at 4500 rpm. The supernatants were collected for bioethanol production. Along with this process, yeast S. cerevisiae was prepared in the same way as reported in (Harun et al., 2010a,b). After all the preparation and pretreatment process, 3% (v/v) of the yeast culture was inoculated into the pre-treated four different microalgae biomass samples. Fermentation was carried out at 30 C in a shaking incubator set to 150 rpm for 48 h. 2.4. Analytical methods
2.1. Materials N. oculata and T. suecica were obtained from Algal Biotechnology Laboratory of Yıldız Technical University, TURKEY. Municipal wastewater used for the cultivation of microalgal cultures, was collected from Istanbul Water and Sewerage Administration (ISKI)
Table 1 TOC analysis of ISKI wastewater.
ISKI WW
41
Total carbon concentration
Total nitrogen concentration
7,67 mg/l ± 655,4 mg/l
3,77 mg/l ± 117,4 mg/l
During 14 days of cultivation, measurements of optical density and cell density of the cultures were used to monitor the algal growth. Optical density was measured by using UVevisible spectrophotometer at 680 nm for N. oculata and 620 nm for T. suecica, respectively (Doan et al., 2011; Lim et al., 2006; Gu et al., 2012a, 2012b). Cell densities of N. oculata and T. suecica were measured using hemocytometer under microscope during 14 days of cultivation (Bastidas, 2009). Determination of the dry weight and measurement of carbohydrate content were carried out on the 14th day of cultivation (Alsull and Omar, 2012). Phenol-sulfuric acid method was used for total carbohydrate analysis.
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Productivity of N. oculata and T. suecica were expressed as the specific growth rate (m) and doubling time (td) by using Equations (1) and (2) from the cell density change during specific time period of exponential phase (Widdel, 2007).
m¼
Nt ln N0 t
td ¼
0:693
m
(1)
(2)
To evaluate bioethanol concentration YL 6100 GC gas chromatography was used. Samples that obtained during the bioethanol fermentation process (24 h and 48 h) were taken and prepared for GC instrument for further analysis. Samples were filtered using 0.45 mm filters to avoid blocking in column. The GC gas chromatograph contains flame ionization detector (FID) and 30 m 0.32 mm x 0.25 mm ZB-FFAP column oven. The temperature of injector, detector and oven were maintained at 150, 250 and 100 C, respectively. Hydrogen was used as carrier gas. Bioethanol concentration was calculated using calibration curve that prepared by the different concentration of bioethanol standards (0.1%e10% (v/v)) (Inan, 2014). Total carbohydrate and GC analysis were carried out in triplicate. The results obtained from each experiment were the average of these data. 3. Results and discussion 3.1. Effect of municipal wastewater on N. oculata and T. suecica After 14 days of cultivation, the cell density and the optical density of N. oculata and T. suecica cultivated at different concentrations of wastewater were shown in Figs. 1e2. The growth curve of N. oculata, shows that, the maximum cell density was reached at 75% of wastewater medium on the 7th day (up to 12.75 106 cells/ ml). As for control group, 25% wastewater and 50% wastewater, algal growth started earlier, almost without lag-phase, than the other 75% and 100% wastewater cultivation groups. It can be commented that microalgae groups which were cultivated with the low nutrient levels of the wastewater may well adapted to the culture medium because of the proper amount of nutrients were given (Brennan and Owende, 2010). As for 75% and 100% wastewater medium, the nutrient levels were much higher, so it may take some time to adapt to the abundant nutrients at lag-phase and then microalgae species grow faster with high growth rate because of the adequate nutrient supply. On the other hand, it was seen that the growth curve of T. suecica was not so steady compared to N. oculata samples. It may be due to T. suecica and N. oculata showed different reaction to the compounds in wastewater. As for T. suecica, the logarithmic phases of the control group and culture that cultivated with 25% of wastewater started earlier than the other microalgae groups. It can be reported that, the higher the wastewater concentration is, the later the logarithmic phase starts for T. suecica. It can be explained that the growth of T. suecica may be highly affected by the changes in nutrient level or culture condition (Gouveia, 2011), and the higher nutrient level may hindered the strain from entering into the logarithmic phase because of intensive amount of nutrient prompt change in medium conditions. F/2 medium which was used for control group, was prepared with the stock solutions of 75 g/L dH2O NaNO3, 5 gr/L dH2O NaH2PO4H2O with the molar concentrations in final medium as 8.82 104M and 3.62 105M, respectively. There was no carbon source in F/2 medium and also seawater has not enough carbon and other elements in ppm concentration in its
composition. In comparison with F/2 medium, wastewater has significant amount of carbon and nitrogen source. Increasing concentration of wastewater causes the positive effect on N. oculata growth and the negative effect on T. suecica growth. The use of nitrogen changes the pH of microalgae culture. If the microalgae use ammonium as nitrogen source, the Hþ ion is released and the pH decreases. Reaction is given with the equation below:
NH4þ þ 7:6CO2 þ 17:7H2 O / C7:6 H8:1 O2:5 N þ 7:6O2 þ 15:2H2 O þ Hþ (3) High amount of ammonium usage can stop microalgae growth causing a decrease in the culture pH as pH < 6. In the case of utilizing nitrate, pH of the medium increases with the nitrate consumption. Theoretically, when 1 mol of nitrate is used, 1 mol of OH is produced. Reaction is given with the equation below: NO 3 þ 5:7CO2 þ 5:4H2 O/C5:7 H9:8 O2:3 N þ 8:25O2 þ OH
(4)
There is a linear relationship between nitrate usage, and the pH of the medium and microalgae growth. If nitrate is used excessively, the pH may increase to over 10 and may affect microalgae growth (Rashid et al., 2014). Similar results can be seen in the literature. It was proven that, optimum growth conditions for T. suecica was bregas found at high salinity and low NaNO3 concentrations (Fa et al., 1984). The maximum cell density of T. suecica was observed with control group on 7th day (6.25 105 cells/ml), which maybe due to T. suecica is not so adapted to the wastewater medium when compared to N. oculata. 3.2. Specific growth rate and cell doubling time According to Table 2, as it was expected, the specific growth rate of N. oculata in different concentration of wastewater was consistently higher than the specific growth rate of T. suecica. In the similar studies, it was reported that the growth rate of N. oculata was measured as 0.39 d1 at the temperature of 25 ± 1 C under continuous illumination, and the growth rate of T. suecica as 0.19 d1 at the temperature of 20 C under a constant photon flux €€ density, respectively (S¸irin and Sillanpa a, 2015; Giordano et al., 2015). In this study, however, the maximum specific growth rate of N. oculata and T. suecica reached to 0.5430d1 in 75% of wastewater medium and 0.4778 d1 in 25% of wastewater medium, respectively. Therefore, it can be concluded that 75% and 25% concentrations of wastewater are the ideal concentrations for cultivating N. oculata and T. suecica for high growth rate and low doubling time. 3.3. Dry weight and carbohydrate content The results in Table 3 showed that the dry weight of all groups of N. oculata were higher than the dry weight of T. suecica. Furthermore, when dry weights of N. oculata were measured, it was found that there were white unknown ingredients in powder. Especially in control group, there were much more white ingredients than any other groups and its dry weight showed the highest result (2.41 g/L) among all groups. In the literature, the other studies also mentioned that there were observed ash and slag deposits. This can be explained as the presence of inorganic component in the biomass can influence the formation of ash (Hamidi et al., 2014). In another study, it is claimed that there wasn’t any differences for the ash content in N. oculata cultivated with agricultural fertilizer, aquacultural fertilizer and f/2 media, and the content was ranged ~ a-Torres et al., 2012; Marinho-Soriano from 26% to 27% (Campan
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(a) N. oculata
140
127.5
Cell density (Cells/ml)
120 100 80 60 40 20 0 0
2
4
6
8
10
12
14
Culture dura on (Day) control
25%wastewater
75%wastewater
100%wastewater
50%wastewater
(b) T. suecica 7
6.25
Cell density (Cells/ml)
6 5 4 3 2 1 0 0
2
4
6
8
10
12
14
Culture dura on (Day) control
25%wastewater
75%wastewater
100%wastewater
50%wastewater
Fig. 1. Cell densities versus culture period (day) for N. oculata (a) and T. suecica (b) in control and different wastewater concentration (25%, 50%, 75% and 100%).
et al., 2006). This suggests that the ash content of N. oculata must be taken into consideration because it may directly influence the carbohydrate concentration in dry weight. On the other hand, the
milled fine powder of T. suecica, can be regarded as pure green and such ash content was observed when taking its dry weight into consideration (Reyimu, 2016). The maximum dry weight was
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(a) N. oculata 0.6
Absorbance(680nm)
0.5 0.4 0.3 0.2 0.1 0 0
2
4
6
8
10
12
14
Culture period (Day) control
25%wastewater
75%wastewater
100%wastewater
50%wastewater
(b) T. suecica 0.18 0.16 Absorbance(620nm)
0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0
2
4
6
8
10
12
14
Culture period (Day) control
25% wastewater
75%wastewater
100%wastewater
50%wastewater
Fig. 2. Growth curve of the N. oculata (a) and T. suecica (b) in control and different wastewater concentration (25%, 50%, 75% and 100%).
obtained from cultures grown in 75% of wastewater. This results matches with a similar study implemented by Beuckels et al. that microalgae can adjust the N and P concentrations of their biomass depending on their supply of nutrients in the medium, so that it
may result in a low dry weight when the supply of a nutrient is low (Hamidi et al., 2014; Beuckels et al., 2015). In this experimental study, as 75% of wastewater contains higher N and P, so that it accumulate much more biomass than other groups. According to
€ Z. Reyimu, D. Ozçimen / Journal of Cleaner Production 150 (2017) 40e46 Table 2 Specific growth rate (m) and doubling time (td) of N. oculata and T. suecica. N. oculata
0% (Control) 25% WW 50% WW 75% WW 100% WW
T. suecica
m (d1)
td (d)
m/(d1)
td (d)
0.3698 0.3095 0.1248 0.5430 0.3793
1.874 2.239 5.553 1.276 1.837
0.1488 0.4778 0.1352 0.2824 0.1105
4.657 1.450 5.126 2.454 6.271
Table 3, after 14 days of cultivation, T. suecica accumulated much more carbohydrate than N. oculata. This is also mentioned in other studies that, T. suecica is the microalgae species that tend to accumulate high carbohydrate content and its carbohydrate can range from 21% to 64% of their cell dry weight (Lam and Lee, 2015). In this study, the highest carbohydrate content in wastewater group of T. suecica was measured in control group (27% dry weight) followed by 100% of wastewater (6.52% dry weight). This is promising when considering T. suecica with this concentration of wastewater as an optimum feedstock for bioethanol production. As for N. oculata, however, maximum carbohydrate concentration in wastewater group was found in 75% concentration of wastewater sample (2.39% dry weight). The result was low when compared to other studies that this may be because the existence of ash content in dry biomass which influence the overall carbohydrate content. 3.4. Bioethanol production According to the BP statistical review of world energy-2016 report (BP, 2016), it can be seen that, ethanol production of the
45
world is higher than biodiesel production. The increase of the production of ethanol in last decade is quite remarkable, and due to the global warming and high oil prices, it is considered that the production of ethanol will continue to increase. Although there are a lot of studies on biodiesel production from different type of microalgae species in the literature, researches on bioethanol production from microalgae are less. For this reason, bioethanol production from the microalgae species was studied considering the world’s renewable energy trends. According to Table 3 results, N. oculata and T. suecica samples that showed highest carbohydrate content in wastewater sample (N. oculata cultivated in 75% of wastewater medium and T. suecica cultivated in 100% of wastewater medium) and the control groups were chosen to compare their bioethanol productivity. Fig. 3 shows that bioethanol yield of N. oculata and T. suecica ranges from 0.41% to 7.26%. When comparing ethanol yield of N. oculata and T. suecica, it is clear that T. suecica showed better ethanol yield both at 24 h and 48 h than N. oculata samples. This is due to the fact that T. suecica accumulated much more carbohydrate than N. oculata during cultivation. In Fig. 3, it is observed that both at 24 h and 48 h of fermentation periods, the bioethanol yield of N. oculata cultivated in 75% of wastewater sample was higher than control group that and the yield increased from 1.21% to 3.68%. The 75% of wastewater group also showed highest carbohydrate yield, this results were expected. As for T. suecica, ethanol yield resulted in higher in control group than 100% of wastewater group. The yield increased from 2.46% to 7.26% at 24 and 48 h of fermentation. This is mostly because of the control group achieved to accumulated highest amount of carbohydrate (27% of dry weight) regardless of its lower dry weight (0.14 g/L). In 100% concentration of wastewater group, although it did not showed highest ethanol yield, it is still
Table 3 Carbohydrate content and dry weight of N. oculata and T. suecica on the 14th day of cultivation. N. oculata
Control 25% ww 50% ww 75% ww 100% ww
T. suecica
CH content (% dry weight)
Dry weight(g/L)
CH content (% dry weight)
Dry weight(g/L)
1 ± 0.3% 2.26 ± 0.2% 2.30 ± 0.4% 2.39 ± 0.1% 2.11 ± 0.2%
2.41 1.19 1.28 1.285 0.975
27 ± 0.1% 4.24 ± 0.1% 4.54 ± 0.1% 3.42 ± 0.2% 6.52 ± 0.2%
0.14 0.76 0.855 1.055 0.48
N.oculata
T.suecica Bioethanol yield (%)
Bioethanol yield (%)
5 4 3 2 1 0 24 h 48 h Fermenta on hours Control group
75% of wastewater
8 7 6 5 4 3 2 1 0 24 h
48 h
Fermenta on hours Control group 100% of wastewater
Fig. 3. Bioethanol yield of N. oculata and T. suecica during 24 h and 48 h of fermentation.
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higher when comparing with both control and 75% of wastewater sample of N. oculata. As in stoichiometric aspects, it can be commented that, the higher carbohydrate content will increase the bioethanol yield. 4. Conclusion This study demonstrates that for effective cultivation microalgae species such as N. oculata and T. suecica, municipal wastewater can be utilized at a specific concentration as a medium. According to specific growth rate, after 14 days of cultivation, it was found that 75% concentration of wastewater can be an ideal concentration for N. oculata medium, while 25% concentration of wastewater was optimum for T. suecica. When N. oculata and T. suecica were compared, adaptation of N. oculata to wastewater medium resulted better than T. suecica according to their growth rate profile. After 14 days of cultivation, bioethanol productivities of two species were also investigated. The highest bioethanol yield of N. oculata was obtained from the microalgae cultivated in 75% of wastewater media as 3.68%. The results showed that N. oculata cultivated at 75% of wastewater represents highest carbohydrate content; hence its ethanol concentration was higher than control group. On the other hand, in comparison with N. oculata, the highest carbohydrate content of T. suecica was found in the control group. The second highest carbohydrate content of T. suecica samples was obtained from the cultures grown at 100% concentration of wastewater medium. The highest bioethanol yield was 7.26% which was obtained from the control group of T. suecica. When comparing ethanol concentration of N. oculata and T. suecica, it was obvious that, T. suecica samples showed higher ethanol yield than N. oculata. These results indicated that T. suecica was more suitable for ethanol production using wastewater as a culture medium, as had higher carbohydrate content than N. oculata. Using wastewater for microalgal production also may not seem economically due to the sterilization because of containing different chemical contents and different bacterial microorganisms. However, some of the wastewater from advanced biological wastewater treatment plants of ISKI, is used as industrial water and used for irrigation of recreation areas after the final disinfection process. A significant portion of the energy required to operate the treatment plants is also utilized from this wastewater treatment process. Also improved filtration systems can be applied for large-scale productions. In conclusion, these results suggested that municipal wastewater from ISKI can be utilized as growth medium for N. oculata and, as well as T. suecica and it can be a remarkable solution for waste utilization and bioethanol production can be improved with further studies. Acknowledgement Zubaidai Reyimu gratefully acknowledges Turks Abroad and Related Communities for the scholarship programme. References Alsull, M., Omar, W.M.W., 2012. Responses of Tetraselmis sp. and Nannochloropsis sp. isolated from Penang National Park coastal waters, Malaysia, to the combined influences of salinity, light and nitrogen limitation. In: International Conference on Chemical, Ecology and Environmental Sciences (ICEES 2012). Andersson, V., Viklund, S.B., Hackl, R., Karlsson, M., Berntsson, T., 2014. Algae-based biofuel production as part of an industrial cluster. Biomass Bioenerg. 71, 113e124, 2014. Bastidas, O., 2009. Technical NoteeNeubauer Chamber Cell Counting. www. researchgate.net/publictopics.PublicPostFileLoader.html? id¼53c6a939d4c11856478b45d2&key¼89353f79-aa75e4e19-8161d900619b728d (accessed 18 April 2016). Beuckels, A., Smolders, E., Muylaert, K., 2015. Nitrogen availability influences phosphorus removal in microalgae-based wastewater treatment. Water Res. 77, 98e106.
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