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The effects of salinity on the growth and biochemical properties of Chlamydomonas mexicana GU732420 cultivated in municipal wastewater a

a

b

c

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El-Sayed Salama , Reda A.I. Abou-Shanab , Jung Rae Kim , Sangho Lee , Seong-Heon Kim , d

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Sang-Eun Oh , Hyun-Chul Kim , Hyun-Seog Roh & Byong-Hun Jeon

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Department of Environmental Engineering, Yonsei University, Wonju, Gangwon-do 220-710, South Korea b

School of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, South Korea c

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GESTRA Engineering, Inc., 1626 W Fond Du Lac Ave, Milwaukee, WI 53205, USA

d

Department of Biological Environment, Kangwon National University, Chuncheon-si, Gangwon-do, South Korea Published online: 10 Jan 2014.

To cite this article: El-Sayed Salama, Reda A.I. Abou-Shanab, Jung Rae Kim, Sangho Lee, Seong-Heon Kim, Sang-Eun Oh, Hyun-Chul Kim, Hyun-Seog Roh & Byong-Hun Jeon (2014) The effects of salinity on the growth and biochemical properties of Chlamydomonas mexicana GU732420 cultivated in municipal wastewater, Environmental Technology, 35:12, 1491-1498, DOI: 10.1080/09593330.2013.871350 To link to this article: http://dx.doi.org/10.1080/09593330.2013.871350

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Environmental Technology, 2014 Vol. 35, No. 12, 1491–1498, http://dx.doi.org/10.1080/09593330.2013.871350

The effects of salinity on the growth and biochemical properties of Chlamydomonas mexicana GU732420 cultivated in municipal wastewater El-Sayed Salamaa , Reda A.I. Abou-Shanaba , Jung Rae Kimb , Sangho Leec , Seong-Heon Kima , Sang-Eun Ohd , Hyun-Chul Kima , Hyun-Seog Roha and Byong-Hun Jeona,∗ a Department of Environmental Engineering, Yonsei University, Wonju, Gangwon-do 220-710, South Korea; b School of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, South Korea; c GESTRA Engineering, Inc., 1626 W Fond Du Lac Ave, Milwaukee, WI 53205, USA; d Department of Biological Environment, Kangwon National University, Chuncheon-si, Gangwon-do, South Korea

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(Received 3 June 2013; accepted 27 November 2013 ) A freshwater microalga Chlamydomonas mexicana was grown on municipal wastewater with different levels of salinity up to 400 mmol/L NaCl, and the biochemical properties were characterized after 10 days of cultivation. C. mexicana showed the higher specific growth rates for 100 and 200 mmol/L NaCl. Nitrogen was completely removed within 10 days as a result of algal growth promoted by the addition of 200–400 mmol/L NaCl. Phosphorus removal increased from 77–84% as the concentration of NaCl increased from 100 to 400 mmol/L. The highest removal of total inorganic carbon (66%) was obtained with the addition of 200 mmol/L NaCl. The lipid content increased from 17% to 38% as the concentration of NaCl increased from 0 to 400 mmol/L. The total fatty acid content and glycerol yield of C. mexicana increased 1.8- and 4-fold in wastewater amended with NaCl, respectively. Fatty acids accumulated in the algal biomass were mainly composed of palmitic (27–29%), γ -linolenic (27–30%), and linolelaidic acids (16–18%). The optimal condition for fatty acids production in C. mexicana was observed when the municipal wastewater was amended with 100–200 mmol/L NaCl with a simultaneous removal of nutrients. Keywords: sodium chloride; Chlamydomonas mexicana; wastewater; glycerol; fatty acids

1. Introduction Biofuel production from photosynthetic microorganisms is considered an effective strategy for production of renewable energy.[1] Microalgae are classified as eukaryotic and prokaryotic photosynthetic microorganisms that live in an aquatic environment and have relatively simple requirements for growth, thus the mass cultivation of these organisms for industrial applications is feasible.[2] There is much interest in using microalgae for oil production due to the high lipid content of some species and the fact that lipids are the best substrate to produce biodiesel.[3–5] Moreover, biodiesel has many advantages compared with petroleumoriginated diesel. Biodiesel is, in principal, a renewable, biodegradable, less-toxic energy carrier, and an essentially sulphur- and aromatics-free fuel.[6] The most significant barriers for economical biodiesel production using microalgae are the technological and engineering aspects, such as the high cost of algae cultivation and the fuel transformation process, difficulties in keeping stable cultures for extended periods, a poor understanding of the regulation of triacylglycerol (TAG) accumulation, and the lack of an efficient genetic tool to find and develop oil-accumulating algae species.[7] Growing ∗ Corresponding

algae in domestic and/or municipal wastewater, which would require minimum energy input, could reduce cultivation costs. Wastewater is a readily available source of water and a source of the major nutrients required by algae proliferation.[8] Thus, wastewater has been considered a potential sustainable growth medium for algal feedstock.[9,10] Microalgal biomass can be a source of proteins, carbohydrates, and lipids, and has received substantial attention in research for chemical production and bioenergy.[4,5,11,12] The total lipid content of microalgae is in the range of 1– 70% of the dry cell weight.[13] The biochemical properties of microalgae can be affected by culture conditions [14–17] with salinity as one of the most influential parameters.[18] Most studies exploring the salinity impact on algal growth have mainly focused on marine algae.[19–21] However, a limited number of studies have focused on the impact of salinity in culture media to investigate the extent and mechanism of lipid production by freshwater microalgae. It has been reported that the total lipid content of freshwater microalgae Botryococcus braunii and Scenedesmus obliquus cultivated in synthetic media increased when the salinity of the growth media increased,[22–25] while the

author. Emails: [email protected], [email protected]

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opposite conclusion was drawn by Ruangsomboon.[23] There has been a lack of research related to the impact of salinity on freshwater microalgal species grown in wastewater for biomass and lipid production with simultaneous nutrient removal. Therefore, the main objective of this research was to evaluate the effect of salinity on the growth rate, biomass, lipid content, glycerol production, and fatty acid composition of freshwater microalga Chlamydomonas mexicana cultivated in municipal wastewater. Nutrient removal from municipal wastewater as a result of algal growth was quantitatively studied. 2. Materials and methods 2.1. Sampling and characterization of wastewater Raw municipal wastewater was collected from a primary sedimentation basin at the Wonju wastewater treatment plant in South Korea. Samples were directly filtered through 0.2 μm nylon membrane filters to remove microorganisms and suspended solids. Physicochemical properties of the prefiltered wastewater were analysed (Table 1). Total inorganic carbon (TIC) was measured with a Shimadzu TOC-VCPH analyzer. Total nitrogen (TN), total phosphorus (TP), and total chemical oxygen demand (TCOD) were measured using a Hach Kit (Hach DR/4000, Loveland, CO, 2− USA). Chloride (Cl− ), nitrate (NO− 3 ), and sulphate (SO4 ) were determined by the single-column ion chromatography (761 compact IC, Metrohm Ltd., Switzerland). Metal ions were analysed using an ELAN DRC II inductively coupled plasma-mass spectrophotometer (PerkinElmer Sciex, USA). The salinity of the wastewater was adjusted by adding NaCl (up to 400 mmol/L). Wastewater amended with different concentrations of NaCl was used as culture media. The total salinity and conductivity of wastewater were measured using an Orion 115A+ conductivity meter Table 1. Physicochemical municipal wastewater.

characteristics

Parameter pH TN (mg/L) TP (mg/L) TIC (mg/L) TCOD (mg/L) Chloride (mg/L) Nitrate–nitrogen (NO3 –N, mg/L) Sulphate (mg/L) Calcium (mg/L) Sodium (mg/L) Potassium (mg/L) Magnesium (mg/L) Boron (mg/L)

of

Value 8.0 ± 0.1 18 ± 0.3 1.4 ± 0.1 33.4 ± 0.3 63 ± 0.9 68 ± 0.2 7.3 ± 0.1 30 ± 1 29 ± 0.3 23 ± 0.2 8.8 ± 0.1 4.0 ± 0.1 0.4 ± 0.1

Note: Concentrations of total iron, manganese, and barium accounted for ≤ 0.1 mg/L, and concentrations of zinc, aluminium, and copper were also insignificant (≤ 0.04 mg/L).

(Thermo Electron Corporation, USA), and the solution pH was measured with a pH meter (Orion 290A). Data were presented as mean ± standard deviation (SD) of triplicate experiments. 2.2.

Microalga and growth conditions

A microalgal strain (C. mexicana GU732420) was previously isolated from a freshwater lake at Yonsei University, Wonju, South Korea.[4] This culture was selected for a study based on its high growth rate and oil content. Four different culture media with various NaCl concentrations (100, 200, 300, and 400 mmol/L) were prepared using the prefiltered municipal wastewater for cultivation of C. mexicana. Prefiltered wastewater without added NaCl was used as a control. The initial optical densities (OD) of the algal suspension were adjusted to an absorbance of 1.5 at 680 nm. Two millilitres of algal suspension was used as initial inoculums. C. mexicana was cultivated using 500-mL conical flasks containing 150 mL prefiltered wastewater under white fluorescent light illumination at 45– 50 μmol photon m2 /s at 27◦ C for 10 days while shaking at 150 rpm. 2.3.

Determination of microalgal biomass and nutrient removal The biomass concentration of the culture was monitored by determining an OD at a wavelength of 680 nm (OD680 ) using a spectrophotometer (DR/4000, Hach). The OD680 was obtained by subtracting the light absorbance from uninoculated wastewater. The OD680 was then converted to a dry weight (DCW) concentration [26] using a linear relationship between OD680 and dry cell weight (g/L), which was obtained after an extensive data analysis in this study and is given by: dry cell weight(g/L) = 0.6857 × OD680 + 0.0041(R2 = 0.9975). The specific growth rate (μ) was calculated using the equation, μ = (lnX2 −ln X1 )/(t2 − t1 ), where X2 and X1 are the DCW at times t2 and t1 , respectively. The yield coefficient (Y , mg/mg) was calculated using, YX /S = dX /dS, where X and S are the DCW and amount of substrate (nitrogen) consumed, respectively. TN and TP were determined at 0 and 10 days of cultivation using a Hach Kit (DR/4000, Hach). TIC concentrations were determined at 0 and 10 days using a Shimadzu TOC-VCPH analyzer, Japan. Percentages of nutrients removal were calculated by dividing the difference between the initial and final values by the initial value, and then multiplying by 100.[27] Blank experiments (wastewater without inoculums) were placed in the shaker under the same conditions. Data were presented as mean ± SD of triplicate experiments.

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2.4.

Lipid extraction and fatty acid methyl ester composition Lipids were extracted from the fresh microalga biomass (0.2 g/L) using the methods described by Bligh and Dyer.[28] In brief, lipids extracted with a mixture of chloroform and methanol (1:2, v/v) were transferred into a tube, sonicated (Sonic 420, South Korea) at the maximum intensity for 30 min, and then incubated overnight at 27◦ C while shaking at 150 rpm. Sonication for 30 min after adding 1.25 mL chloroform to the tube followed by centrifugation at 3900 rpm for 10 min after adding 1.25 mL deionized water to the content was conducted to separate and subsequently collect the chloroform layer. A second extraction was performed by adding 2.5 mL chloroform and vortexing the tube. The chloroform layer was gently collected from the bottom of the tube, washed using 5 mL of 5% NaCl solution, evaporated in a dry oven at 50◦ C, and the residues (i.e. crude lipid) were gravimetrically measured. Data were presented as mean ± SD of triplicate experiments. Fatty acids were analysed using a modification of the method described by Lepage and Roy.[29] A 2-mL mixture of chloroform and methanol (2:1, v/v) was freshly prepared and used to completely dissolve at least 10 mg crude lipid and 1 mL chloroform containing nonadecanoic acid (500 mg/L) as an internal standard and then transmethylation reagents were added. The contents were transferred to a 10-mL Pyrex tube, incubated at 100◦ C for 10 min, and separated into two phases by adding 1 mL de-ionized water. After 10 min of vigorous mixing and centrifugation at 4000 rpm for another 10 min, the chloroform layer was transferred from the bottom of the tube to another tube using a hypodermic disposable polypropylene syringe and subsequently filtered using 0.2-μm polyvinylidene fluoride syringe microfilters. Fatty acid methyl esters (FAMEs) in the extracted liquid were quantified using an Agilent 6890 gas chromatograph (Agilent Technologies, USA) equipped with a flame ionization detector and a HP-INNO wax capillary column.

2.5.

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2.6. Statistical analysis One-way analysis of variance (ANOVA) was used to examine the differences among average values. GraphPad Prism version 5.0 for Windows (GraphPad Software, Inc., USA) was used for all statistical analyses, and differences in the variables were considered significant at the P < 0.05 level of confidence.

3. Results and discussion 3.1. Effect of NaCl on algal growth and biomass, and consequent removal of inorganic constituents from wastewater Significantly improved microalgal growth was observed (0.67 ± 0.02 and 0.58 ± 0.03 g/L) after 10 days of incubation in wastewater amended with 100 and 200 mmol/L NaCl, respectively. When NaCl concentration increased from 300 to 400 mmol/L, the algal biomass decreased (Figure 1). Freshwater microalga, S. obliquus, can grow in synthetic media amended with 600 mmol/L NaCl and the highest dry biomass (0.63 g/L) was obtained at 50 mmol/L after 15 days of cultivation.[25] The absence of plasmolysed cells suggested that the salt stress of cells under these conditions counterbalanced the drop in the osmotic potential of the surrounding medium.[25] Sodium ion acts through osmolytes that are sequestered in the vacuole to maintain turgor pressure in plants at high salinity.[31] Consequently, algae that have a tolerance for salt stress are able to optimize their growth.[32] Sodium ion is required to facilitate photosynthesis in microalgae through inorganic nutrients uptake, intracellular pH regulation, and alkalotolerance.[33,34] Our result was consistent with earlier reports that the growth of microalgae is substantially inhibited by low or excess salinity due to alterations of basic biosynthetic functions such as photosynthesis and photorespiration.[20,25,31] Microalgae play an important role in biomass production and biological treatment of wastewater.[35–37] The growth of

Determination of glycerol production

The production of glycerol for osmoregulation of C. mexicana under salt stress conditions was identified and quantified using algal biomass. The harvested biomass was washed twice with deionized water. Algal cells were treated with liquid nitrogen, sonicated at high power (Branson Ultrasonic Cleaner 8510, USA) and centrifuged at 3900 rpm for 15 min to remove cell debris.[30] Glycerol extraction was done twice and the supernatant was pooled. The glycerol content of the cell extracts and glycerol produced in the culture media were enzymatically determined using the glycerol kinase method (KIT method, Cat. F6428, Sigma, USA).

Figure 1. Effect of NaCl concentration on the dry biomass of C. mexicana cultivated in municipal wastewater (No NaCl was added to wastewater for 0 mmol/L condition).

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Figure 2. Effect of NaCl concentration on nutrient removal by C. mexicana cultivated in municipal wastewater. There was a significant difference among cultures at P < 0.05 (TN, P < 0.0001; TP, P < 0.0002; and TIC, P < 0.0001). No NaCl was added to wastewater for 0 mmol/L condition.

Chlamydomonas mirabilis cultivated in sewage mixed with effluents of industrial wastewater was 0.21 g/L with substantial removal of N (from 39.4 to 16.5 mg/L) and P (from 4.65 to 3.05 mg/L).[35] The growth of Chlamydomonas sp. in industrial wastewater increased,[36] and the biomass yield of Chlamydomonas reinhardtii was 0.8 g/L/day when cultured in municipal wastewater with 55% removal of N and 15.4% of P.[37] Removal of TN from wastewater was always higher than TP removal after 10 days of cultivation regardless of NaCl dose. TN was completely removed with addition of 200–400 mmol/L NaCl (Figure 2). TP was removed by 68% without NaCl addition, which increased from 77 to 84% with an increase in NaCl from 100 to 400 mmol/L. The highest removal of TIC (66%) was obtained after adding 200 mmol/L NaCl to wastewater (Figure 2). In the absence of C. mexicana (blank experiment), removals of N (17%), P (14%), and TIC (3%) were obtained after 10 days. Inorganic N/P species are required for growth of all algae.[38] El-Sayed [32] reported that the rise of the N/P removal rate was closely related to the given salinity level and all N/P were uptaken by freshwater green microalga Scenedesums sp. which was cultivated for 8 days in growth media with different salinities (up to 100% seawater). High specific growth rate (0.12–0.11 l/day) and yield coefficient (37–31 mg/mg) of C. mexicana were achieved in wastewater amended with 100–200 mmol/L NaCl, respectively (Figure 3). Yeesang and Cheirsilp [18] found that the freshwater microalga Botryococcus sp. survived under high-salinity and nitrogen-rich culture conditions. Indeed, P assimilation in algae is markedly influenced by a certain level of pH, Na+ , K+ , Mg2+ , and/or various heavy metals.[39] However, Rao et al. [22] concluded that the decrease in phosphate was due to the utilization by freshwater microalga B. braunii, which was not influenced by salinity concentration.

Figure 3. Kinetic coefficients of C. mexicana cultivated in municipal wastewater under salt stress (no NaCl was added to wastewater for 0 mmol/L condition).

3.2.

Salinity impacts on the biochemical characteristics of C. mexicana 3.2.1. Effect of NaCl on lipid content Lipids from microalgae are mainly composed of ethyl esters of glycerol and fatty acids, which are suitable for producing biodiesel.[40] Fatty acid profile and lipid productivity have been used as potential indicators of biodiesel productivity.[41] Table 4 indicates that the amount of lipids accumulated in C. mexicana was dependent on the concentration of NaCl in municipal wastewater, which coincided with the previously reported works using synthetic growth media.[22,42] There was a significant correlation (P < 0.0119) between NaCl concentration and lipid content (Table 4). The Lipid content in the algal biomass increased from 17 to 38% as the concentration of NaCl increased from 0 to 400 mmol/L. Under unfavourable environments, many algae species synthesize a large amount of storage lipids such as TAGs.[24] The lipid content was not correlated with the growth of microalga. High lipid content was observed at high concentrations of NaCl, which was possibly due to salt stress and nitrogen deficiency in culturing medium.[43] As stated in Section 3.1, TN was completely removed after 10 days of cultivation under 200–400 mmol/L of NaCl (Figure 2). Our observation was in good agreement with previous studies showing the effect of salinity on the lipid content for different microalgae cultivated in synthetic media.[25,44] Kong et al. [37] reported that the lipid content of C. reinhardtii increased up to 26% when it was cultivated in wastewater for 10 days. However, the lipid content decreased to 17% after C. reinhardtii was grown in artificial media. In this study, the lipid content of C. mexicana was 17% after cultivation in wastewater for 10 days without adding salt and increased up to 28–38% due to the amendment with 100–400 mmol/L NaCl. Jiang et al. [45] found that the lipid content (28%) of marine microalga (Nannochloropsis sp.) was enhanced when the alga was cultured in 50% seawater/50% wastewater, while the lipid content (23%) of the same alga decreased after it was cultivated

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Table 2. Comparison of salinity effects on the lipid content for different microalgal species cultivated in municipal wastewater and synthetic media. Strain

Salinity (g/L)

C. mexicanaa

C. reinhardtiia S. obliquusa

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Nannochloropsis oculatab

Nannochloropsis sp.b

0.2 (0.5) 6.1 (11) 11.8 (20) 19.7 (32) 25.1 (41) – – 5.8 11.6 17.5 10 15 20 25 – –

Medium

Lipid (%)

Reference This study

F2 medium

17 28 29 33 38 17 26 15 22 36 29

F2 medium Seawater/ wastewater (1:1)

31 33 35 23 28

Municipal wastewater

Artificial medium Wastewater Chu 13 medium

[37] [25] [44]

[45]

Note: Values in parentheses represent the conductivity (mS/cm) of wastewater. a Freshwater microalgae. b Marine microalgae. Table 3. Effects of NaCl concentrations in municipal wastewater on fatty acid composition of C. mexicana and results of one-way ANOVA analysis (F-ratios). NaCl (mmol/L) Fatty acid

0a

100

Fatty acid composition (wt%) C16:0 28.7 ± 0.7 27.7 ± 0.1 C17:1 11.3 ± 0.3 13.6 ± 1.1 C18:1n9c 6.2 ± 0.8 5.6 ± 0.4 C18:2n6t 18.6 ± 0.5 18.2 ± 0.0 C18:3n6 29.8 ± 0.1 29 ± 0.1 Other 5.4 ± 0.7 5.9 ± 0.6 Total (%) 100 ± 3.1 100 ± 2.3 a Without addition ∗ P < 0.03. ∗∗ P < 0.001. ∗∗∗ P < 0.009.

200

300

400

29.2 ± 0.2 15.8 ± 0.1 5.1 ± 0.1 17.1 ± 0.1 27 ± 0.2 5.7 ± 0.4 100 ± 1.1

29.4 ± 0.4 16.3 ± 0.6 4.1 ± 0.7 17.1 ± 0.1 27.4 ± 1 5.8 ± 0.6 100 ± 3.5

28.2 ± 0.0 17.2 ± 0.2 3.8 ± 0.3 16.5 ± 0.1 28.5 ± 0.4 5.8 ± 0.2 100 ± 1.3

F-ratio 6.01∗ 33.2∗∗ 7.3∗ 26.9∗∗ 11.9∗∗∗ – –

of NaCl.

in synthetic media for 7 days (Table 2). Outstanding survivability of freshwater microalgae has been reported even when cultivated in highly saline growth media.[23] Zhila et al. [24] demonstrated that a growth medium with high salinity increased microalgae cellular content by improving the formation of TAG. 3.2.2. Effect of NaCl on fatty acid composition Salinity fluctuation leads to variations in the fatty acid profiles of algae, which can influence the functions of algal cell membranes and metabolic processes.[46] The degree of saturation of membrane fatty acids is an important parameter in the adaptation of algae to environmental conditions.[24] The composition of fatty acids in C. mexicana in which the percentages of palmitic (C16:0), heptadecenoic (C17:1),

oleic (C18:1n9c), linolelaidic (C18:2n6t), and γ -linolenic acids (C18:3n6) are plotted as a function of NaCl dose in Table 3. Under our experimental conditions, the influence of salinity was important and statistically significant and variations in the fatty acid composition of the alga (C. mexicana) were mainly due to the salinity of the medium (Table 3). Both C16:0 and C18:3n6 were major forms, accounting for 27–29% and 27–30% of total fatty acid, respectively, under the examined NaCl levels (0–400 mmol/L). C18:2n6t (18 ± 1%) was the next most abundant and the amount of C17:1 was similar to that of C18:2n6t after adding 200–400 mmol/L NaCl. C18:1n9c ranged from 4 to 6%. Saturated fatty acid (palmitic acid) accounted for 27 ± 2% of total FAME regardless of NaCl concentration, while unsaturated fatty acid (heptadecenoic acid) increased from 11 to 17% as the concentration of NaCl increased from 0 to

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400 mmol/L, which was the most remarkable variation in the composition of fatty acids. The fatty acid profile generally depends on the physiological state of algae and the level of salinity.[22,24] Changes in the fatty acid profile in response to elevated salinity of the medium are inevitable to keep the membrane fluid and prevent its destruction.[24] The other fatty acids accounted for approximately 5 ± 1% in all experimental variations (Table 3). Under salt stress conditions, unsaturated fatty acids accounted for approximately 70% of total fatty acids. Our observation was consistent with the previously reported work in which the accumulation of palmitic and oleic acids in a freshwater microalgal strain (B. braunii) improved with stepped NaCl doses to the growth medium.[22] Rao et al. [22] also found that B. braunii changed its fatty acid profiles in response to elevated salinity to retain membrane fluid and prevent its destruction. A review of literature reveals that saturated acid components in algal biomass are in the range of 25–45%, while unsaturated fatty acids account for 50–55% of total fatty acids.[47] Thus, the ratio of unsaturated to saturated fatty acids in algae oils ranges between 1 and 2, which is somewhat similar to that of plant oils (such as palm).[48] The quality of biodiesel produced from algae biomass can be expected to be highly competitive with that produced from palm oil. Algal FAMEs were composed predominantly of unsaturated fats, indicating that algae biomass may be preferable for cold weather use due to a typically lower gel point.[49]

several organic solutes and osmolytes.[50] Compatible osmolytes such as proline, sugars, polyols, amino acids, and glycerol are synthesized in response to salt stress.[31,51] In this study, the glycerol content rapidly increased upon adding NaCl up to 100 mmol/L, but an insignificant further increase was found when the concentration of NaCl in the culture media increased to 400 mmol/L (Table 4). Gustavs et al. [52] suggested that organisms growing in habitats with high osmotic stress tend to accumulate energetically cheaper organic osmolytes. Metabolic accumulation of glycerol in eukaryotic algae with elevated salinity was described as an osmoregulation mechanism.[49] Algal cells have developed many adaptive strategies in response to different abiotic stresses (such as salinity, dehydration, cold temperature, and excessive osmotic pressure) making use of different mechanisms including changes in morphological/developmental patterns as well as physiological/biochemical processes.[53] It was observed that the glycerol was not released into the medium, which suggested that most of the glycerol produced and retained in the algal biomass is for maintaining osmotic balance in cells and forming TAG which led to increasing lipid content.[24] The lipid content in algal biomass is strongly dependent on salinity in the culture media due to the accumulation of small organic molecules (e.g. glycerol) in response to osmotic pressure.[54] These results indicate that inorganic carbon was converted to glycerol through either a photosynthetic pathway or metabolic degradation during photosynthesis in C. mexicana, which resulted in improved lipid production from municipal wastewater with increasing NaCl concentrations (Figure 2 and Table 4). C. mexicana cultivated in municipal wastewater for 10 days showed an excellent potential for biomass and lipid productivity compared to other microalgae species (Table 5).

3.2.3. Synthesis of glycerol for osmoregulation in C. mexicana Adaptation to salt stress in algae is associated with metabolic adjustments that lead to the accumulation of

Table 4. Salinity effects on biomass yield, lipid, total fatty acid, and glycerol yield of C. mexicana cultivated in municipal wastewater. NaCl Biomass yield Lipid content Lipid productivity Total fatty acid Glycerol yield (mmol/L) (g/L) (%) (g/L) (mg/g) (g/L) 0a 100 200 300 400 a Without

0.08 ± 0.02 0.67 ± 0.03 0.58 ± 0.03 0.30 ± 0.04 0.21 ± 0.02

17 ± 0.7 28 ± 0.9 29 ± 0.5 33 ± 0.9 38 ± 0.5

0.014 ± 0.01 0.185 ± 0.03 0.168 ± 0.04 0.099 ± 0.04 0.079 ± 0.01

174 ± 6 301 ± 2 307 ± 3 319 ± 5 346 ± 3

20 ± 3 88 ± 9 100 ± 1 110 ± 6 107 ± 4

addition of NaCl.

Table 5. Comparison for biomass and lipid productivity of C. mexicana with other microalgae species cultivated in different synthetic media and wastewater. Strain

Medium

Nannochloropsis sp. F2 medium Scenedesmus sp. BG11 medium C. mexicana Piggery wastewater Municipal wastewater

Drsy biomass Lipid productivity Cultivation time (g/L) (g/L) (days) Reference 0.31 0.66 1.20 0.67

0.034 0.110 0.310 0.185

20 15 20 10

[55] [56] [57] This study

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Environmental Technology 4. Conclusion A freshwater microalga (C. mexicana) showed salt adaptation by regulating its biochemical properties in municipal wastewater. Higher biomass production (0.58 g/L) and lipid content (38%) were achieved with up to 200 and 400 mmol/L NaCl, respectively. The highest lipid productivities of 0.185 and 0.168 g/L of C. mexicana were obtained at 100 and 200 mmol/L NaCl, respectively. Additional NaCl in the wastewater resulted in increased glycerol yield in C. mexicana (fourfold). Removal of TN, TP, and TIC improved with the addition of NaCl to wastewater. High specific growth rate and yield coefficient of C. mexicana were achieved in wastewater amended with 100– 200 mmol/L NaCl. Some freshwater microalgae can be cultivated in high-salinity water (i.e. a river mouth where freshwater and seawater meet). This study demonstrated that C. mexicana is suitable for lipid/glycerol production and simultaneous nutrient removal from municipal wastewater supplemented with 100–200 mmol/L NaCl.

Funding This work was supported by the Senior Researchers Program (the National Research Foundation of Korea [2010–0026904]), Human Resource Development Project for Energy from Waste & Recycling (Korea Ministry of Environment), and Academic-Industrial Common Technology Development Project (Administration of the Small & Medium Business [2013-C0103527]).

[10]

[11]

[12] [13] [14] [15]

[16]

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The effects of salinity on the growth and biochemical properties of Chlamydomonas mexicana GU732420 cultivated in municipal wastewater.

A freshwater microalga Chlamydomonas mexicana was grown on municipal wastewater with different levels of salinity up to 400 mmol/L NaCl, and the bioch...
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