Bioresource Technology xxx (2014) xxx–xxx
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Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae S. Venkata Mohan 1,⇑, M. Prathima Devi 1 Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Dual mode cultivation strategy for
maximization of biomass and lipid productivity. Salinity stress showed positive impact on lipid synthesis. Diverse fatty acid profile was detected with the function of salinity stress. Carbohydrate profile showed direct correlation with the lipid productivity pattern.
a r t i c l e
i n f o
Article history: Received 3 January 2014 Received in revised form 13 February 2014 Accepted 16 February 2014 Available online xxxx Keywords: Neutral lipid Fatty acid methyl esters (FAME) Saturated fatty acids Carbohydrates Wastewater treatment
a b s t r a c t Influence of salinity as a stress factor to harness biodiesel was assessed during dual mode cultivation of microalgae by integrating biomass growth phase (BGP) and salinity induced lipid induction phase (LIP). BGP was evaluated in mixotrophic mode employing nutrients (NPK) and carbon (glucose) source while LIP was operated under stress environment with varying salt concentrations (0, 0.5, 1 and 2 g NaCl/l). Salinity stress triggered both biomass growth and lipid synthesis in microalgae significantly. BGP showed higher increments in biomass growth (2.55 g/l) while LIP showed higher lipid productivity (1 g NaCl/l; total/neutral lipid, 23.4/9.2%) than BGP (total/neutral lipid, 15.2/6%). Lower concentrations of salinity showed positive influence on the process while higher concentrations showed marked inhibition. Salinity stress also facilitated in maintaining saturated fatty acid methyl esters in higher amounts which associates with the improved fuel properties. Efficient wastewater treatment was observed during BGP operation indicating the assimilation of carbon/nutrients by microalgae. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are considered to be the efficient photosynthetic organisms that have been suggested as a potential feedstock for the production of biofuels and bioenergy (Klein et al., 2013; Alcantara et al., 2013). Microalgae produce biofuels and other ⇑ Corresponding author at: Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India. Tel.: +91 40 27191664. E-mail address: [email protected] (S. Venkata Mohan). 1 Academy of Scientific and Innovative Research (AcSIR).
chemical by harvesting sunlight and fixing CO2. The fixed CO2 within these cells under stress conditions leads to the formation of lipids that can be transesterified to produce biodiesel (Rawat et al., 2013; Venkata Mohan et al., 2013). Lipids are the secondary metabolites of microalgae synthesized during stress conditions (Solovchenko, 2012; Devi et al., 2013; Venkata Mohan et al., 2013). Lipids, in the form of triacylglycerides typically provide a storage function in the cell that enables microalgae to endure adverse environmental conditions (Giorgos and Elias, 2013; Kalpesh et al., 2012). Studies have indicated that the lipid content of microalgae can be enhanced by changing the cultivation conditions and
http://dx.doi.org/10.1016/j.biortech.2014.02.103 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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subjecting them to diverse stress conditions (Pittman et al., 2011; Devi and Venkata Mohan, 2012; Asulabh et al., 2012; Chandra et al., 2014). Among the cultivation methods, two-stage systems are of intense interest where, optimum conditions are applied in the first phase aiming at the maximization of biomass production, while in the second phase, stress conditions are applied to facilitate the accumulation of lipids (Giorgos and Elias, 2013; Devi et al., 2012; Devi and Venkata Mohan, 2012. The major stress conditions applied to enhance lipid accumulation are temperature, light intensity, pH, salinity, mineral salts and nutrients (Takagi, 2006; Ifeanyi et al., 2011; Devi et al., 2012). Salinity is an intricate stress which influences various physiological and bio-chemical mechanisms associated with the growth and development of microalgae. Salinity stress can also lead to increment in the lipid content of microalgae due to its crucial role in causing changes in the fatty acid metabolism (Kalita et al., 2011). Under high salinity stress, many organisms including microalgae alter their metabolism to adapt to the extreme environment (Kan et al., 2012). The ability of microalgae to survive in saline environment under the influence of osmotic stress has received considerable attention which can also affect cell growth and lipid formation (Asulabh et.al., 2012). Fluctuations in the salt content of the growth medium has also been found to alter the lipid composition of microalgae (Kalpesh et al., 2012). As, algae are inhabitants of biotopes characterized by varying salinities, they have gained significance in salt tolerance studies domain and have served as model organisms for better understanding of salt acclimation in more complex physiological processes (Talebi et al., 2013; Alkayal et al., 2011). When cells are exposed to salinity, specific processes such as, restoration of turgor pressure, regulation of the uptake and export of ions through the cell membrane, and accumulation of osmo-protecting solutes and stress proteins gets activated leading to new steady state growth (Talebi et al., 2013; Allakhverdiev et al., 2000). These mechanisms in turn, generates stress inside the algal cells causing increment in the total lipid content which act as a reserve energy material until favourable conditions arise (Talebi et al., 2013; Asulabh et.al., 2012). Dunaliella sp. provide the best example of microalgae that can tolerate high salt concentrations (Azachi et al., 2002). Their ability to increase the biomass growth and lipid content under salinity stress makes them one of the suitable candidates to study the effects of salinity on microalgae (Kalpesh et al., 2012). Although many species of microalgae including marine heterotrophic strains are tolerant to great variations of salinity, their chemical and fatty acid composition can vary with respect to salt stress (Kalpesh et al., 2012; Kirroliaa et al., 2011). The effect of salt concentration was evaluated to explore the potential of marine resources confined with specific species of microalgae (Ifeanyi et al., 2011; Takagi, 2006). When photosynthetic organisms are exposed to salt stress, the fatty acids of membrane lipids are desaturated leading to increment in the proportion of unsaturated fatty acids (Asulabh et al., 2012). On the other hand, increment in the saturated fatty acids and decrement in polyunsaturated fatty acids under high-salt stress has also been reported (Kan et al., 2012). Algae produce some metabolites to protect them from salt injury and also to adjust to the surrounding osmotica (Rao et al., 2007). In the present study, the role of salinity as a stress factor on microalgae lipid synthesis towards biodiesel production was evaluated in dual mode cultivation viz., biomass growth phase (BGP) followed by lipid induction phase (LIP). BGP was evaluated in mixotrophic mode employing domestic wastewater as substrate to induce growth while LIP was operated in autotrophic mode by varying the concentrations of sodium chloride. Changes in the biomass growth, chlorophyll components, lipid productivities and fatty acid compositions were studied at the end of both BGP and
LIP operations. Wastewater treatment capability of the system was also evaluated at the end of BGP operation.
2. Methods 2.1. Microalgae Microalgae culture collected in pre-monsoon season from a lentic water body (Nacharam Cheruvu, Hyderabad) receiving domestic effluents was used as inoculum (Venkata Mohan et al., 2011). Prior to experimentation, the culture was washed and pelletized by centrifugation (3000 rpm; 10 min; 30 °C) to remove associated debris. 2.2. Experimental methodology Experiments were designed and operated in dual mode viz., biomass growth phase (BGP, mixotrophic) followed by salinity stress induced lipid induction phase (LIP, autotrophic). In BGP, microalgae were grown mixotrophically in culture-tub (working volume, 40 L; depth, 10 cm; surface area, 0.23 m2) and maintained as an open-pond system. The culture-tub was fed with domestic sewage (COD, 400 mg/l; TDS, 750 mg/l; nitrates, 115 mg/l) with additional supplementation of carbon (500 mg glucose/l) and nutrients (500 mg NaNO3/l; 500 mg Na2PO4/l) to accelerate algal biomass growth by adjusting pH to 8.2. After 8 days of growth period, the cultures were harvested by siphoning wastewater from the tub. The dewatered culture was used as inoculum for the second phase (LIP) of the experiments at where, three levels of sodium chloride viz., 0.5, 1.0 and 2.0 g NaCl/l along with a control condition without NaCl were operated to assess the influence of natural stress (NS). Prior to start-up, 20 ml of microalgal biomass (2.14 g/l) was inoculated to 160 ml of tap water and closed with cotton plugs. Before inoculation, the pH was adjusted to 8.2 using 3 N NaOH. LIP-NS condition was operated with tap water alone. Experiments were carried out under 12 h (light):12 h (dark). In the light phase, flasks were mounted on a temperature controlled shaking incubator (120 rpm) in the presence of a fluorescent light (0.074 mol/m2 s). In dark phase, the light source was turned off to facilitate dark conditions while the rest of the operating conditions remained same. After LIP, the resulting biomass was separated by centrifugation (5000 rpm; 5 min at 28 °C) and the algal biomass pellet was subjected to solar drying followed by blending in to powder form. The blended powder was further disrupted using sonicator (20 kHz) for 30 min (Power Sonic 410) and the extracted lipid was used for analysis. All the experiments were carried out in triplicates and the results presented here represent an average of three independent operations. 2.3. Extraction of lipids Extraction of total lipids was carried out using Bligh and Dyer method employing chloroform:methanol (2:1 v/v) as solvents while neutral lipids were extracted with n-Hexane employing solvent extraction procedure using Soxhlet apparatus (Lee et al., 2010; Venkata Mohan et al., 2011). Prior to extraction, the dried algae-biomass was sonicated (30 min; 20 kHz) in requisite solvent / solvent mixture (chloroform:methanol/n-Hexane) and transferred to thimbles made with Whattman filter paper No. 1 for neutral lipids where as the biomass was directly placed in to the reaction vials for total lipids extraction. The solvent mixture was refluxed for 5 h (30 °C) and concentrated in a rotavapor followed by vacuum drying with a temperature controlled oil bath (120 °C) and cooled to room temperature (Devi and Venkata Mohan, 2012).
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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2.4. Fatty acid methyl esters (FAME) Lipid analysis was performed at the end of BGP and LIP by concentrating the biomass (5000 rpm; 5 min at 30 °C). Microalgae lipid (100 mg) with methanol–sulphuric acid (2%) mixture (40:1) were refluxed for 4 h by monitoring the reaction by thin layer chromatography (TLC) using n-Hexane and ethyl acetate (EA) mixture (90:10) as mobile phase (Venkata Mohan et al., 2011). Reaction was continued till the oil spot disappeared on the TLC plate. After the reaction time (4 h), the contents were washed with 25 ml of water, the aqueous layer was extracted with EA (2 25 ml) and pooled. The extract was dried over anhydrous Na2SO4 and concentrated under vacuum. The dried FAME was analyzed using GC-FID (Venkata Mohan and Devi, 2012). After conversion of fatty acids to methyl esters, the concentrated sample was used for the detection of FAME composition by GC with FID (Nucon-5765) through capillary column [Valcobond (VB) 30 mm (0.25 mm 0.25 lm)] using nitrogen as carrier gas (1 ml/min). The temperature of the oven was initially maintained at 140 °C (for 5 min), later increased to 240 °C at a ramp of 4 °C/min for 10 min. The injector and detector temperatures were maintained at 280 and 300 °C respectively with a split ratio of 1:10. FAME composition was compared with the standard FAME mix (C8–C22; LB66766, SUPELCO). 2.5. Analyses Biomass was estimated on every day during BGP and LIP based on optical density (OD, 650 nm). Chlorophyll quantification was performed by taking 2 ml of algal cells followed by concentration and disruption using sonication (20 kHz) for 7 min and extracted with 2 ml of 90% acetone. The extract was centrifuged at 3000g (5 min) and OD of the supernatant was measured to quantify
a
Biomass (at 650nm;g/l)
6
LIP
BGP
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4 LIP-NS LIP - 0.5 g NaCl/l LIP - 1 g NaCl/l LIP - 2 g NaCl/l
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24 48 72 96 120 144 168 192 216 240 264 288 312 336 360
Chlorophyll concentration (µg/mg)
Cultivation time (h) 16
b
Total chlorophyll Chl a Chl b
12
8
4
0 BGP
LIP - NS
LIP - 0.5 g NaCl/l
LIP - 1 g NaCl/l
LIP - 2 g NaCl/l
Experimental Variation Fig. 1. Biomass (a) and chlorophyll concentrations (b) noticed at the BGP and LIP of the study.
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chlorophyll concentration (Devi et al., 2013). Chemical oxygen demand (COD; closed refluxing titrimetric method), VFA, nitrates, phosphates pH and ORP were analyzed according to the Standard Methods (APHA, 1998). Total soluble carbohydrate content (TSCC) in the algal cells was determined using Anthrone sulphuric acid method. Radiance during the experimental run was measured with a lux meter (Extech).
3. Results and discussion 3.1. Salt concentration vs. microalgae biomass growth Dual mode cultivation viz., biomass growth phase (BGP, mixotrophic) followed by salinity stress induced lipid accumulation phase (LIP, autotrophic) documented positive influence on the biomass growth of microalgae and lipid synthesis (Fig. 1a). BGP operated under mixotrophic mode of nutrition showed increment in the biomass growth from 2.14 to 4.69 g/l over 8 days of cultivation. The mixotrophic nutrition facilitates higher biomass growth through carbon utilization (Farooq et al., 2013; Venkata Mohan et al., 2013). The second phase (LIP) prevailing under autotrophic mode with variable salt concentrations showed positive influence on biomass growth compared to the natural stress (control) operation. Presence of salt-tolerant enzymes may also function over a wide range of salinities leading to increments in biomass growth of microalgae (Talukdar et al., 2012). Maximum biomass growth was observed in 1 g NaCl/l (6.12 g/l) operation which might be attributed to the enough salt concentration that supported growth and metabolic activities of microalgae (Britta and Kautsky, 2003). The prevalence of salt tolerant species in the culture might also have attributed for the growth. Lower biomass growth was observed with 2 g NaCl/l (5.55 g/l) operation which might be due to the hyper-saline conditions and the non-adaptability of the culture to high salinity (Vazquez and Arredondo, 1991). The salinity induced growth decrement might also be attributed to the accumulation of reactive oxygen species (Kalita et al., 2011). With 0.5 g NaCl/l operation, biomass growth of 5.35 g/l was observed which might be due to the hypo-saline conditions leading to the insufficient availability of salt concentrations that is required by microalgae for its growth and metabolic activities. LIP-NS operation with natural stress environment documented lower biomass growth (4.95 g/l) among the experimental variations studied. Chlorophyll pigment is considered as one of the indices for the biomass growth of microalgae. Total chlorophyll and its components estimated at the end of BGP and LIP operations showed some interesting observations pertaining to the relation among growth, concentrations and components of chlorophyll (Fig. 1b). Total chlorophyll concentration and biomass growth patterns during BGP documented linearity inferring the increments in chlorophyll concentration with increasing biomass growth. On the contrary, the total chlorophyll content estimated during LIP showed decrement with increasing biomass growth. The chlorophyll content noticed at the end of BGP (12.5 lg/mg) reduced after LIP (8.2 lg/mg (1 g NaCl/l); 5.5 lg/mg (2 g NaCl/l); 4.3 lg/mg (0.5 g NaCl/l); 4.1 lg/mg (LIP-NS)). Higher chlorophyll content at the end of BGP might be attributed to mixotrophic non-saline photosynthetic activity. Decrement in chlorophyll content after LIP might be attributed to saline stress caused due to limitations in photosynthetic electron transport and partial stomatal closure in saline environments (Asulabh et.al., 2012; Zhang et al., 2010). Chlorophyll components showed distinct variation with both the phase operation and as a function of salinity. Relatively higher Chl a (9.25 lg/mg) with lesser Chl b (3.25 lg/mg) was noticed at the end of BGP operation reflecting the increments in biomass growth. On the contrary, higher Chl b and lesser Chl a was observed
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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Lipid content (mg/g of biomass)
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Experimental Variation
Lipid productivity (%)
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stress operation depicting the effect of salinity on lipid composition. The lipid synthesis noticed with 2 g NaCl/l (TL/NL, 19.1/ 7.3%) operation indicates the possibility of microalgae adaptation to higher salt concentrations. BGP (TL/NL, 15.2/6%) also showed lipid synthesis along with biomass growth. The absence of nutrient and carbon during LIP-NS operation might have led to lesser lipid synthesis than BGP. Lipid productivity correlated well with the chlorophyll compositions where, LIP operations documented higher Chl b directing towards lipid accumulation and higher Chl a in BGP operations promoting biomass growth. The ratio of neutral to total lipids (N/T) illustrated some interesting observations. In spite of lower lipid productivity, the N/T value of BGP operation showed comparatively higher ratio equivalent to 1 g NaCl/l (0.39) operation followed by 2 g NaCl/l (0.38), 0.5 g NaCl/l (0.31) and LIP-NS (0.35). It is evident from the above results that the salt stress has definite influence on the lipid profile during synthesis.
Total Neutral
16 12 8 4 0 BGP
LIP-NS
LIP - 0.5 g NaCl/l
LIP - 1 g NaCl/l
LIP - 2 g NaCl/l
Experimental variation Fig. 2. Distinctions noticed in the (a) lipid content and (b) lipid productivity profiles during the study.
at the end of LIP (0.5 g NaCl/l (Chl a, 1.15 lg/mg; Chl b, 3.15 lg/ mg); 1 g NaCl/l (Chl a, 3.1 lg/mg; Chl b, 5.1 lg/mg); 2 g NaCl/l (Chl a, 2.25 lg/mg; Chl b, 3.25 lg/mg); LIP-NS (Chl a, 1.05 lg/mg; Chl b, 3.05 lg/mg)) showing specific increase of Chl b with salinity. Replacement of the methyl groups in chlorophyll with formyl groups under stress conditions might also be attributed to the increased Chl b content (Venkata Mohan and Devi, 2012). In spite of the lesser Chl a content, LIP operations showed biomass growth depicting the influence of salinity on growth rather than on chlorophyll. 3.2. Salinity vs. algal-lipid Experimental variations illustrated significant influence on the lipid content and lipid productivity of microalgae (Fig. 2a and b). Stress induced by changes in salt content has been shown to affect the total and neutral lipid fractions within microalgal cells (Rawat et al., 2013; Kalpesh et al., 2012). Experimental variations with induced salinity stress depicted higher lipid synthesis compared to the BGP and LIP-NS operations. Lipid composition showed significant variations in terms of total lipid (TL) and neutral lipid (NL), owing to the phenomenon, that microalgae growing under varying salinities undergo changes in lipid composition (Kalpesh et al., 2012). Maximum lipid synthesis was observed with 1 g NaCl/l (TL/NL, 23.4/9.2%) operation which might be due to optimum availability of salt concentration that facilitated efficient lipid synthesis. The presence of NaCl leads to the oxidative stress causing increment in the triacylglycerol content (Takagi, 2006; Asulabh et.al., 2012; Kan et al., 2012). The lipid synthesis observed at 0.5 g NaCl/l (TL/NL, 17.5/5.5%) was higher than the natural stress condition studied (LIP-NS; TL/NL, 11.8/4.2%) which again supports the hypothesis that salinity induces lipid synthesis. Increment in neutral lipid fraction was specifically observed with LIP-saline
3.2.1. Carbohydrates conversion Cellular carbohydrate (CHO) concentration was measured at the end of BGP and LIP to understand the role of carbohydrates conversion on lipid production. Carbohydrate concentrations were in accordance with lipid productivity profiles. The concentration of carbohydrate increased as a function of biomass growth and reached 320.5 mg/g of biomass from 80.3 mg/g of biomass by the end of BGP. The observed pattern indicates the gradual increment of CHO concentration that was well supported by the increasing biomass concentrations during BGP. After LIP, all the operations documented a decrement in CHO concentration indicating the conversion of accumulated carbohydrates to lipids. The pattern was well visualised by the lipid productivities noticed after LIP operation. Maximum CHO concentration observed at the end of LIP was higher with non-saline operation (LIP-NS, 275.5 mg/g of biomass) followed by 0.5 g NaCl/l (255.2 mg/g of biomass), 2 g NaCl/l (210.1 mg/g of biomass) and 1 g NaCl/l (132.4 mg/g of biomass). Maximum CHO conversion efficiency was noticed with 1 g NaCl/l (58.68%) followed by 2 g NaCl/l (34.48%), 0.5 g NaCl/l (20.37%) and LIP-NS (14.04%). 3.2.2. Total fatty acid composition Fatty acid composition (after transesterification) was studied at the end of BGP and LIP operations to enumerate the variation in the distribution of fatty acids (Fig. 3a). Supplementation of salt concentrations not only increased the lipid productivity but also caused changes in the fatty acid profile. Microalgal lipids are generally characterized by both saturated and unsaturated fatty acids. Fatty acids derived at all the operations showed higher SFAs than USFAs indicating less risk of combustion characteristics and ignition delay (Benjumea et al., 2011). Among the conditions, 1 g NaCl/l operation showed higher saturation profile (SFAs, 10; USFAs, 3) accounting for the highest saturation fraction of 3.3 while 2 g NaCl/l operation depicted the presence of 9 SFAs and 5 USFAs with a saturation fraction of 1.8. The 0.5 g NaCl/l operation with 8 SFAs and 3 USFAs showed a saturation fraction of 2.6 while BGP showed the presence of 7 SFAs and 5 USFAs with saturation fraction of 1.4. The LIP-NS operation documented 8 SFAs and 4 USFAs accounting for a saturation fraction of 2. Salinity showed marked improvement in the saturation fraction besides causing changes in the distribution of fatty acids. Ratio of SFAs and total number of fatty acids was higher with 1 g NaCl/l operation (0.58) followed by 2 g NaCl/l (0.52), 0.5 g NaCl/ l and LIP-NS (0.47) and BGP (0.41). The conditions with highest lipid productivity showed maximum ratio except with the BGP and LIP-NS operations. Results suggested the positive influence of salinity on increasing the saturation fraction of algal-oil and enhancing its fuel properties. Exposure of photosynthetic organisms to salt stress resulted in increment in the proportion of saturated fatty acids (Kan et al., 2012).
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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BGP LIP - NS LIP - 0.5 g NaCl/l LIP - 1 g NaCl/l LIP - 2 g NaCl/l
a 40
(%)
Fatty acids based on weight
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30 20 10 0
Fatty acids Fatty acids (% on weight basis)
40
Palmitic acid Stearic acid Elaidic acid
b 30
20
10
0 BGP
LIP - NS
LIP - 0.5 g NaCl/l
LIP - 1 g NaCl/l
LIP - 2 g NaCl/l
Experimental Variation Fig. 3. (a) Total fatty acid profile; (b) weight based percentage of palmitic, stearic and elaidic acids detected during the study.
The proportion of palmitic (C16:0), stearic (C18:0) and elaidic acids (C18:1n9t) varied with the experimental variations showing a significantly higher ratio under salinity stress (Fig. 3b). Palmitic acid was observed to have two to three folds higher concentration over stearic and elaidic acids. 1 g NaCl/l operation documented
relatively higher concentration of palmitic acid (38.4%) followed by 0.5 g NaCl/l (34.8%), BGP (20.3%), 2 g NaCl/l (18.5%) and LIP-NS (17.5%). Subsequent to palmitic acid, higher quantity of stearic acid was noticed in 0.5 g NaCl/l (15.8%) operation followed by 1 g NaCl/l (13.5%), 2 g NaCl/l (11%), LIP-NS (7.2%) and BGP (5.05%). Significant amounts of elaidic acid was observed in BGP operation (8.4%)
100 VFA
Nitrates
Phosphates
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80
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60 8.4
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Removal efficiency (%)
9.4 COD
40
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48
72
96
120
144
168
Cultivation time (h)
6.9 0
24 48 72 96 120 144 168 192 216 240 264 288 312 336 360
Cultivation time (h) Fig. 4. COD, VFA, nitrates and phosphate removal patterns noticed during the study.
Fig. 5. Distinctions in the pH profiles recorded at BGP and LIP of the study.
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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followed by LIP-NS (6.5%), 1 g NaCl/l (3.3%), 2 g NaCl/l (3.06%) and 0.5 g NaCl/l (2.6%). The variations noticed at each operation might be attributed to the differences in the physiological state of the algae (Xin et al., 1997). Fatty acids derived during the study possess good applied value with diverse fuel characteristics that are desirable in the preparation of biofuels, formation of methyl esters and combustion fuels for engines. The SFAs and USFAs without biofuel properties can also be used as ingredients and excipients for several medicinal and industrial purposes (Venkata Mohan et al., 2011). 3.3. Wastewater treatment Wastewater treatment in terms of COD, volatile fatty acids (VFA), nitrates and phosphates removal was evaluated during BGP (Fig. 4). COD removal efficiency increased gradually and reached a maximum on the 8th day of mixotrophic operation (85.5%). Syntrophism assumes significantly when mixed cultures are used as biocatalyst. Algae along with many other living forms survive in the culture microenvironment developing a mutual dependence on each other and cause a reduction in the organic pollutants (Venkata Mohan et al., 2011; Devi et al., 2012). Similar to COD removal efficiency, VFA removal pattern during BGP was also found to increase till the end of BGP operation (58.1%). The removal pattern documented good agreement with the biomass growth profiles suggesting the fact that wastewater based carbon, forms main substrate for metabolism towards biomass growth as part of mixotrophic mode of cultivation. Nutrient removal monitored as nitrates and phosphates during the BGP documented increased removal patterns. The phosphorus and nitrogen gets utilized for the growth of microalgae. Among the nutrients evaluated, nitrates documented higher removal (75%) compared to phosphates (70%). In nitrification process, ammonia gets oxidized to nitrite and then to nitrate while in denitrification, the nitrate will be taken up by algae and either be converted to organic nitrogen in their cell tissues (algae assimilation) or will be reduced to elemental nitrogen (N2) and lost as a gas (Devi et al., 2013). The observed nutrients/carbon removal suggests that microalgae can effectively utilise nutrients/carbon for its growth with simultaneous reduction leading to the phenomenon of biological waste remediation. 3.4. Bioprocess monitoring Redox condition was regularly monitored during the BGP and LIP operations (Fig. 5). Initial pH for both BGP and LIP was adjusted to 8.2, which later showed slight variations during the experimental run and shifted towards basic (BGP, 8.9; LIP-NS, 8.7) and towards neutral (LIP, 2 g NaCl/l, 7.1; 1 g NaCl/l, 7.3; 0.5 g NaCl/l, 7.5). The shift towards basic microenvironment in BGP denotes the consumption of wastewater based carbon to form bicarbonates while in LIP-NS, the presence of accumulated bicarbonates in the first phase and the absence of salinity in the second phase might have led to the basic environment. Neutral pH patterns noticed during LIP saline stress operations might be due to the salt injury/osmotic stress caused due to the prevalence of saline environment (Rao et al., 2007). In spite of the neutral pH in LIP, biomass concentrations were found to increase, depicting the adaptation of the biocatalyst to saline environment. Oxidation-reduction potentials (ORP) recorded during the BGP and LIP showed good correlation with pH. BGP ( 165.1 mV) and LIP-NS ( 172.3 mV) operation showed negative values. In spite of the neutral pH, saline stress operations also registered negative ORP (2 g NaCl/l, 60.5 mV; 1 g NaCl/l, 62.5 mV; 0.5 g NaCl/l, 65.2 mV). The shift of ORP towards negative value supports the oxidation of carbon and its utilization in the metabolic pathway of microalgae which
is well associated with the increased biomass growth and increased substrate degradation noticed during BGP operation (Syrett et al., 1963). 4. Conclusions Experimental data documented the positive effect of salinity stress on lipid synthesis of microalgae. BGP showed effective biomass growth pattern with simultaneous substrate degradation and nutrient removal while LIP documented good lipid productivity associated with higher concentrations of Chl b supporting the biosynthesis of lipids. FAME profile related to fuel properties was found to be influenced by salinity. Integration of BGP with LIP (saline stress) illustrated the positive response of microalgae towards biodiesel production. The study also focussed on the flexibility of microalgae to utilize wastewater through mixotrophic mode for harnessing biofuels with simultaneous wastewater remediation. Acknowledgements Authors duly acknowledge the Director, CSIR-IICT for encouragement and support in carrying out this work. M.P.D. acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi for providing research fellowship. Research was supported by Council of Scientific and Industrial Research in the form of 12th Plan Task Force Project on ‘Photo-biological process to produce bioenergy through carbon sequestration and wastewater utilization-Biomass to Energy (BioEn, CSC-0116). References Alcantara, C., García-Encina, P.A., Munoz, R., 2013. Evaluation of mass and energy balances in the integrated microalgae growth-anaerobic digestion process. Chem. Eng. J. 221, 238–246. Alkayal, F., Albion, R.L., Tillett, R.L., Hathwaik, L.T., Lemos, M.S., Cushman, J.C., 2011. Expressed sequence tag (EST) profiling in hyper saline shocked Dunaliella salina reveals high expression of protein synthetic apparatus components. Plant Sci. 179, 437–449. Allakhverdiev, I.S., Sakamoto, A., Nishiyama, Y., Inaba, M., Murata, N., 2000. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp.. Plant Physiol. 123 (3), 1047–1056. American Public Health Association (APHA)American Water Works Association (AWWA), 1998. Standard Methods for the Examination of Water and Wastewater. Water Environment Federation, Washington, DC. Asulabh, K.S., Supriya, G., Ramachandra, T.V., 2012. Effect of salinity concentrations on growth rate and lipid concentration in Microcystis sp., Chlorococcum sp. and Chaetoceros sp. In: LAKE: National Conference on Conservation and Management of Wetland Ecosystems, pp. 27–32. Azachi, M., Sadka, A., Fisher, M., Goldshlag, P., Gokhman, I., Zamir, A., 2002. Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina. Plant Physiol. 129, 1320–1329. Benjumea, P., John, R.A., Andrés, F.A., 2011. Effect of the degree of unsaturation of biodiesel fuels on engine performance, combustion characteristics, and emissions. Energy Fuels 25, 77–85. Britta, E.T., Kautsky, L., 2003. Review on toxicity testing with marine macroalgae and the need for method standardization exemplified with copper and phenol. Mar. Pollut. Bull. 46, 171–181. Chandra, R., Rohit, M.V., Swamy, Y.V., Venkata Mohan, S., 2014. Regulatory function of organic carbon supplementation during growth and nutrient stress phases of mixotrophic microalgae cultivation on lipid synthesis. Bioresour. Technol. http://dx.doi.org/10.1016/j.biortech.2014.02.102. Devi, M.P., Swamy, Y.V., Venkata Mohan, S., 2013. Nutritional mode influences lipid accumulation in microalgae with the function of carbon sequestration and nutrient supplementation. Bioresour. Technol. 142, 278–286. Devi, M.P., Venkata Mohan, 2012. CO2 supplementation to domestic wastewater enhances microalgae lipid accumulation under mixotrophic microenvironment: effect of sparging period and interval. Bioresour. Technol. 112, 116–123. Devi, M.P., Subhash, G.V., Venkata Mohan, S., 2012. Heterotrophic cultivation of mixed microalgae for lipid accumulation and wastewater treatment during sequential growth and starvation phases. Effect of nutrient supplementation. J. Renew. Energy 43, 276–283. Farooq, W., Lee, Y.C., Ryu, B.G., Kim, B.H., Kim, H.S., Choi, Y.E., Yang, J.W., 2013. Twostage cultivation of two Chlorella sp. strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity. Bioresour. Technol. 132, 230–238.
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Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae S. Venkata Mohan 1,⇑, M. Prathima Devi 1 Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Dual mode cultivation strategy for
maximization of biomass and lipid productivity. Salinity stress showed positive impact on lipid synthesis. Diverse fatty acid profile was detected with the function of salinity stress. Carbohydrate profile showed direct correlation with the lipid productivity pattern.
a r t i c l e
i n f o
Article history: Received 3 January 2014 Received in revised form 13 February 2014 Accepted 16 February 2014 Available online xxxx Keywords: Neutral lipid Fatty acid methyl esters (FAME) Saturated fatty acids Carbohydrates Wastewater treatment
a b s t r a c t Influence of salinity as a stress factor to harness biodiesel was assessed during dual mode cultivation of microalgae by integrating biomass growth phase (BGP) and salinity induced lipid induction phase (LIP). BGP was evaluated in mixotrophic mode employing nutrients (NPK) and carbon (glucose) source while LIP was operated under stress environment with varying salt concentrations (0, 0.5, 1 and 2 g NaCl/l). Salinity stress triggered both biomass growth and lipid synthesis in microalgae significantly. BGP showed higher increments in biomass growth (2.55 g/l) while LIP showed higher lipid productivity (1 g NaCl/l; total/neutral lipid, 23.4/9.2%) than BGP (total/neutral lipid, 15.2/6%). Lower concentrations of salinity showed positive influence on the process while higher concentrations showed marked inhibition. Salinity stress also facilitated in maintaining saturated fatty acid methyl esters in higher amounts which associates with the improved fuel properties. Efficient wastewater treatment was observed during BGP operation indicating the assimilation of carbon/nutrients by microalgae. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are considered to be the efficient photosynthetic organisms that have been suggested as a potential feedstock for the production of biofuels and bioenergy (Klein et al., 2013; Alcantara et al., 2013). Microalgae produce biofuels and other ⇑ Corresponding author at: Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India. Tel.: +91 40 27191664. E-mail address: [email protected] (S. Venkata Mohan). 1 Academy of Scientific and Innovative Research (AcSIR).
chemical by harvesting sunlight and fixing CO2. The fixed CO2 within these cells under stress conditions leads to the formation of lipids that can be transesterified to produce biodiesel (Rawat et al., 2013; Venkata Mohan et al., 2013). Lipids are the secondary metabolites of microalgae synthesized during stress conditions (Solovchenko, 2012; Devi et al., 2013; Venkata Mohan et al., 2013). Lipids, in the form of triacylglycerides typically provide a storage function in the cell that enables microalgae to endure adverse environmental conditions (Giorgos and Elias, 2013; Kalpesh et al., 2012). Studies have indicated that the lipid content of microalgae can be enhanced by changing the cultivation conditions and
http://dx.doi.org/10.1016/j.biortech.2014.02.103 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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subjecting them to diverse stress conditions (Pittman et al., 2011; Devi and Venkata Mohan, 2012; Asulabh et al., 2012; Chandra et al., 2014). Among the cultivation methods, two-stage systems are of intense interest where, optimum conditions are applied in the first phase aiming at the maximization of biomass production, while in the second phase, stress conditions are applied to facilitate the accumulation of lipids (Giorgos and Elias, 2013; Devi et al., 2012; Devi and Venkata Mohan, 2012. The major stress conditions applied to enhance lipid accumulation are temperature, light intensity, pH, salinity, mineral salts and nutrients (Takagi, 2006; Ifeanyi et al., 2011; Devi et al., 2012). Salinity is an intricate stress which influences various physiological and bio-chemical mechanisms associated with the growth and development of microalgae. Salinity stress can also lead to increment in the lipid content of microalgae due to its crucial role in causing changes in the fatty acid metabolism (Kalita et al., 2011). Under high salinity stress, many organisms including microalgae alter their metabolism to adapt to the extreme environment (Kan et al., 2012). The ability of microalgae to survive in saline environment under the influence of osmotic stress has received considerable attention which can also affect cell growth and lipid formation (Asulabh et.al., 2012). Fluctuations in the salt content of the growth medium has also been found to alter the lipid composition of microalgae (Kalpesh et al., 2012). As, algae are inhabitants of biotopes characterized by varying salinities, they have gained significance in salt tolerance studies domain and have served as model organisms for better understanding of salt acclimation in more complex physiological processes (Talebi et al., 2013; Alkayal et al., 2011). When cells are exposed to salinity, specific processes such as, restoration of turgor pressure, regulation of the uptake and export of ions through the cell membrane, and accumulation of osmo-protecting solutes and stress proteins gets activated leading to new steady state growth (Talebi et al., 2013; Allakhverdiev et al., 2000). These mechanisms in turn, generates stress inside the algal cells causing increment in the total lipid content which act as a reserve energy material until favourable conditions arise (Talebi et al., 2013; Asulabh et.al., 2012). Dunaliella sp. provide the best example of microalgae that can tolerate high salt concentrations (Azachi et al., 2002). Their ability to increase the biomass growth and lipid content under salinity stress makes them one of the suitable candidates to study the effects of salinity on microalgae (Kalpesh et al., 2012). Although many species of microalgae including marine heterotrophic strains are tolerant to great variations of salinity, their chemical and fatty acid composition can vary with respect to salt stress (Kalpesh et al., 2012; Kirroliaa et al., 2011). The effect of salt concentration was evaluated to explore the potential of marine resources confined with specific species of microalgae (Ifeanyi et al., 2011; Takagi, 2006). When photosynthetic organisms are exposed to salt stress, the fatty acids of membrane lipids are desaturated leading to increment in the proportion of unsaturated fatty acids (Asulabh et al., 2012). On the other hand, increment in the saturated fatty acids and decrement in polyunsaturated fatty acids under high-salt stress has also been reported (Kan et al., 2012). Algae produce some metabolites to protect them from salt injury and also to adjust to the surrounding osmotica (Rao et al., 2007). In the present study, the role of salinity as a stress factor on microalgae lipid synthesis towards biodiesel production was evaluated in dual mode cultivation viz., biomass growth phase (BGP) followed by lipid induction phase (LIP). BGP was evaluated in mixotrophic mode employing domestic wastewater as substrate to induce growth while LIP was operated in autotrophic mode by varying the concentrations of sodium chloride. Changes in the biomass growth, chlorophyll components, lipid productivities and fatty acid compositions were studied at the end of both BGP and
LIP operations. Wastewater treatment capability of the system was also evaluated at the end of BGP operation.
2. Methods 2.1. Microalgae Microalgae culture collected in pre-monsoon season from a lentic water body (Nacharam Cheruvu, Hyderabad) receiving domestic effluents was used as inoculum (Venkata Mohan et al., 2011). Prior to experimentation, the culture was washed and pelletized by centrifugation (3000 rpm; 10 min; 30 °C) to remove associated debris. 2.2. Experimental methodology Experiments were designed and operated in dual mode viz., biomass growth phase (BGP, mixotrophic) followed by salinity stress induced lipid induction phase (LIP, autotrophic). In BGP, microalgae were grown mixotrophically in culture-tub (working volume, 40 L; depth, 10 cm; surface area, 0.23 m2) and maintained as an open-pond system. The culture-tub was fed with domestic sewage (COD, 400 mg/l; TDS, 750 mg/l; nitrates, 115 mg/l) with additional supplementation of carbon (500 mg glucose/l) and nutrients (500 mg NaNO3/l; 500 mg Na2PO4/l) to accelerate algal biomass growth by adjusting pH to 8.2. After 8 days of growth period, the cultures were harvested by siphoning wastewater from the tub. The dewatered culture was used as inoculum for the second phase (LIP) of the experiments at where, three levels of sodium chloride viz., 0.5, 1.0 and 2.0 g NaCl/l along with a control condition without NaCl were operated to assess the influence of natural stress (NS). Prior to start-up, 20 ml of microalgal biomass (2.14 g/l) was inoculated to 160 ml of tap water and closed with cotton plugs. Before inoculation, the pH was adjusted to 8.2 using 3 N NaOH. LIP-NS condition was operated with tap water alone. Experiments were carried out under 12 h (light):12 h (dark). In the light phase, flasks were mounted on a temperature controlled shaking incubator (120 rpm) in the presence of a fluorescent light (0.074 mol/m2 s). In dark phase, the light source was turned off to facilitate dark conditions while the rest of the operating conditions remained same. After LIP, the resulting biomass was separated by centrifugation (5000 rpm; 5 min at 28 °C) and the algal biomass pellet was subjected to solar drying followed by blending in to powder form. The blended powder was further disrupted using sonicator (20 kHz) for 30 min (Power Sonic 410) and the extracted lipid was used for analysis. All the experiments were carried out in triplicates and the results presented here represent an average of three independent operations. 2.3. Extraction of lipids Extraction of total lipids was carried out using Bligh and Dyer method employing chloroform:methanol (2:1 v/v) as solvents while neutral lipids were extracted with n-Hexane employing solvent extraction procedure using Soxhlet apparatus (Lee et al., 2010; Venkata Mohan et al., 2011). Prior to extraction, the dried algae-biomass was sonicated (30 min; 20 kHz) in requisite solvent / solvent mixture (chloroform:methanol/n-Hexane) and transferred to thimbles made with Whattman filter paper No. 1 for neutral lipids where as the biomass was directly placed in to the reaction vials for total lipids extraction. The solvent mixture was refluxed for 5 h (30 °C) and concentrated in a rotavapor followed by vacuum drying with a temperature controlled oil bath (120 °C) and cooled to room temperature (Devi and Venkata Mohan, 2012).
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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2.4. Fatty acid methyl esters (FAME) Lipid analysis was performed at the end of BGP and LIP by concentrating the biomass (5000 rpm; 5 min at 30 °C). Microalgae lipid (100 mg) with methanol–sulphuric acid (2%) mixture (40:1) were refluxed for 4 h by monitoring the reaction by thin layer chromatography (TLC) using n-Hexane and ethyl acetate (EA) mixture (90:10) as mobile phase (Venkata Mohan et al., 2011). Reaction was continued till the oil spot disappeared on the TLC plate. After the reaction time (4 h), the contents were washed with 25 ml of water, the aqueous layer was extracted with EA (2 25 ml) and pooled. The extract was dried over anhydrous Na2SO4 and concentrated under vacuum. The dried FAME was analyzed using GC-FID (Venkata Mohan and Devi, 2012). After conversion of fatty acids to methyl esters, the concentrated sample was used for the detection of FAME composition by GC with FID (Nucon-5765) through capillary column [Valcobond (VB) 30 mm (0.25 mm 0.25 lm)] using nitrogen as carrier gas (1 ml/min). The temperature of the oven was initially maintained at 140 °C (for 5 min), later increased to 240 °C at a ramp of 4 °C/min for 10 min. The injector and detector temperatures were maintained at 280 and 300 °C respectively with a split ratio of 1:10. FAME composition was compared with the standard FAME mix (C8–C22; LB66766, SUPELCO). 2.5. Analyses Biomass was estimated on every day during BGP and LIP based on optical density (OD, 650 nm). Chlorophyll quantification was performed by taking 2 ml of algal cells followed by concentration and disruption using sonication (20 kHz) for 7 min and extracted with 2 ml of 90% acetone. The extract was centrifuged at 3000g (5 min) and OD of the supernatant was measured to quantify
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chlorophyll concentration (Devi et al., 2013). Chemical oxygen demand (COD; closed refluxing titrimetric method), VFA, nitrates, phosphates pH and ORP were analyzed according to the Standard Methods (APHA, 1998). Total soluble carbohydrate content (TSCC) in the algal cells was determined using Anthrone sulphuric acid method. Radiance during the experimental run was measured with a lux meter (Extech).
3. Results and discussion 3.1. Salt concentration vs. microalgae biomass growth Dual mode cultivation viz., biomass growth phase (BGP, mixotrophic) followed by salinity stress induced lipid accumulation phase (LIP, autotrophic) documented positive influence on the biomass growth of microalgae and lipid synthesis (Fig. 1a). BGP operated under mixotrophic mode of nutrition showed increment in the biomass growth from 2.14 to 4.69 g/l over 8 days of cultivation. The mixotrophic nutrition facilitates higher biomass growth through carbon utilization (Farooq et al., 2013; Venkata Mohan et al., 2013). The second phase (LIP) prevailing under autotrophic mode with variable salt concentrations showed positive influence on biomass growth compared to the natural stress (control) operation. Presence of salt-tolerant enzymes may also function over a wide range of salinities leading to increments in biomass growth of microalgae (Talukdar et al., 2012). Maximum biomass growth was observed in 1 g NaCl/l (6.12 g/l) operation which might be attributed to the enough salt concentration that supported growth and metabolic activities of microalgae (Britta and Kautsky, 2003). The prevalence of salt tolerant species in the culture might also have attributed for the growth. Lower biomass growth was observed with 2 g NaCl/l (5.55 g/l) operation which might be due to the hyper-saline conditions and the non-adaptability of the culture to high salinity (Vazquez and Arredondo, 1991). The salinity induced growth decrement might also be attributed to the accumulation of reactive oxygen species (Kalita et al., 2011). With 0.5 g NaCl/l operation, biomass growth of 5.35 g/l was observed which might be due to the hypo-saline conditions leading to the insufficient availability of salt concentrations that is required by microalgae for its growth and metabolic activities. LIP-NS operation with natural stress environment documented lower biomass growth (4.95 g/l) among the experimental variations studied. Chlorophyll pigment is considered as one of the indices for the biomass growth of microalgae. Total chlorophyll and its components estimated at the end of BGP and LIP operations showed some interesting observations pertaining to the relation among growth, concentrations and components of chlorophyll (Fig. 1b). Total chlorophyll concentration and biomass growth patterns during BGP documented linearity inferring the increments in chlorophyll concentration with increasing biomass growth. On the contrary, the total chlorophyll content estimated during LIP showed decrement with increasing biomass growth. The chlorophyll content noticed at the end of BGP (12.5 lg/mg) reduced after LIP (8.2 lg/mg (1 g NaCl/l); 5.5 lg/mg (2 g NaCl/l); 4.3 lg/mg (0.5 g NaCl/l); 4.1 lg/mg (LIP-NS)). Higher chlorophyll content at the end of BGP might be attributed to mixotrophic non-saline photosynthetic activity. Decrement in chlorophyll content after LIP might be attributed to saline stress caused due to limitations in photosynthetic electron transport and partial stomatal closure in saline environments (Asulabh et.al., 2012; Zhang et al., 2010). Chlorophyll components showed distinct variation with both the phase operation and as a function of salinity. Relatively higher Chl a (9.25 lg/mg) with lesser Chl b (3.25 lg/mg) was noticed at the end of BGP operation reflecting the increments in biomass growth. On the contrary, higher Chl b and lesser Chl a was observed
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stress operation depicting the effect of salinity on lipid composition. The lipid synthesis noticed with 2 g NaCl/l (TL/NL, 19.1/ 7.3%) operation indicates the possibility of microalgae adaptation to higher salt concentrations. BGP (TL/NL, 15.2/6%) also showed lipid synthesis along with biomass growth. The absence of nutrient and carbon during LIP-NS operation might have led to lesser lipid synthesis than BGP. Lipid productivity correlated well with the chlorophyll compositions where, LIP operations documented higher Chl b directing towards lipid accumulation and higher Chl a in BGP operations promoting biomass growth. The ratio of neutral to total lipids (N/T) illustrated some interesting observations. In spite of lower lipid productivity, the N/T value of BGP operation showed comparatively higher ratio equivalent to 1 g NaCl/l (0.39) operation followed by 2 g NaCl/l (0.38), 0.5 g NaCl/l (0.31) and LIP-NS (0.35). It is evident from the above results that the salt stress has definite influence on the lipid profile during synthesis.
Total Neutral
16 12 8 4 0 BGP
LIP-NS
LIP - 0.5 g NaCl/l
LIP - 1 g NaCl/l
LIP - 2 g NaCl/l
Experimental variation Fig. 2. Distinctions noticed in the (a) lipid content and (b) lipid productivity profiles during the study.
at the end of LIP (0.5 g NaCl/l (Chl a, 1.15 lg/mg; Chl b, 3.15 lg/ mg); 1 g NaCl/l (Chl a, 3.1 lg/mg; Chl b, 5.1 lg/mg); 2 g NaCl/l (Chl a, 2.25 lg/mg; Chl b, 3.25 lg/mg); LIP-NS (Chl a, 1.05 lg/mg; Chl b, 3.05 lg/mg)) showing specific increase of Chl b with salinity. Replacement of the methyl groups in chlorophyll with formyl groups under stress conditions might also be attributed to the increased Chl b content (Venkata Mohan and Devi, 2012). In spite of the lesser Chl a content, LIP operations showed biomass growth depicting the influence of salinity on growth rather than on chlorophyll. 3.2. Salinity vs. algal-lipid Experimental variations illustrated significant influence on the lipid content and lipid productivity of microalgae (Fig. 2a and b). Stress induced by changes in salt content has been shown to affect the total and neutral lipid fractions within microalgal cells (Rawat et al., 2013; Kalpesh et al., 2012). Experimental variations with induced salinity stress depicted higher lipid synthesis compared to the BGP and LIP-NS operations. Lipid composition showed significant variations in terms of total lipid (TL) and neutral lipid (NL), owing to the phenomenon, that microalgae growing under varying salinities undergo changes in lipid composition (Kalpesh et al., 2012). Maximum lipid synthesis was observed with 1 g NaCl/l (TL/NL, 23.4/9.2%) operation which might be due to optimum availability of salt concentration that facilitated efficient lipid synthesis. The presence of NaCl leads to the oxidative stress causing increment in the triacylglycerol content (Takagi, 2006; Asulabh et.al., 2012; Kan et al., 2012). The lipid synthesis observed at 0.5 g NaCl/l (TL/NL, 17.5/5.5%) was higher than the natural stress condition studied (LIP-NS; TL/NL, 11.8/4.2%) which again supports the hypothesis that salinity induces lipid synthesis. Increment in neutral lipid fraction was specifically observed with LIP-saline
3.2.1. Carbohydrates conversion Cellular carbohydrate (CHO) concentration was measured at the end of BGP and LIP to understand the role of carbohydrates conversion on lipid production. Carbohydrate concentrations were in accordance with lipid productivity profiles. The concentration of carbohydrate increased as a function of biomass growth and reached 320.5 mg/g of biomass from 80.3 mg/g of biomass by the end of BGP. The observed pattern indicates the gradual increment of CHO concentration that was well supported by the increasing biomass concentrations during BGP. After LIP, all the operations documented a decrement in CHO concentration indicating the conversion of accumulated carbohydrates to lipids. The pattern was well visualised by the lipid productivities noticed after LIP operation. Maximum CHO concentration observed at the end of LIP was higher with non-saline operation (LIP-NS, 275.5 mg/g of biomass) followed by 0.5 g NaCl/l (255.2 mg/g of biomass), 2 g NaCl/l (210.1 mg/g of biomass) and 1 g NaCl/l (132.4 mg/g of biomass). Maximum CHO conversion efficiency was noticed with 1 g NaCl/l (58.68%) followed by 2 g NaCl/l (34.48%), 0.5 g NaCl/l (20.37%) and LIP-NS (14.04%). 3.2.2. Total fatty acid composition Fatty acid composition (after transesterification) was studied at the end of BGP and LIP operations to enumerate the variation in the distribution of fatty acids (Fig. 3a). Supplementation of salt concentrations not only increased the lipid productivity but also caused changes in the fatty acid profile. Microalgal lipids are generally characterized by both saturated and unsaturated fatty acids. Fatty acids derived at all the operations showed higher SFAs than USFAs indicating less risk of combustion characteristics and ignition delay (Benjumea et al., 2011). Among the conditions, 1 g NaCl/l operation showed higher saturation profile (SFAs, 10; USFAs, 3) accounting for the highest saturation fraction of 3.3 while 2 g NaCl/l operation depicted the presence of 9 SFAs and 5 USFAs with a saturation fraction of 1.8. The 0.5 g NaCl/l operation with 8 SFAs and 3 USFAs showed a saturation fraction of 2.6 while BGP showed the presence of 7 SFAs and 5 USFAs with saturation fraction of 1.4. The LIP-NS operation documented 8 SFAs and 4 USFAs accounting for a saturation fraction of 2. Salinity showed marked improvement in the saturation fraction besides causing changes in the distribution of fatty acids. Ratio of SFAs and total number of fatty acids was higher with 1 g NaCl/l operation (0.58) followed by 2 g NaCl/l (0.52), 0.5 g NaCl/ l and LIP-NS (0.47) and BGP (0.41). The conditions with highest lipid productivity showed maximum ratio except with the BGP and LIP-NS operations. Results suggested the positive influence of salinity on increasing the saturation fraction of algal-oil and enhancing its fuel properties. Exposure of photosynthetic organisms to salt stress resulted in increment in the proportion of saturated fatty acids (Kan et al., 2012).
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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S. Venkata Mohan, M.P. Devi / Bioresource Technology xxx (2014) xxx–xxx
BGP LIP - NS LIP - 0.5 g NaCl/l LIP - 1 g NaCl/l LIP - 2 g NaCl/l
a 40
(%)
Fatty acids based on weight
50
30 20 10 0
Fatty acids Fatty acids (% on weight basis)
40
Palmitic acid Stearic acid Elaidic acid
b 30
20
10
0 BGP
LIP - NS
LIP - 0.5 g NaCl/l
LIP - 1 g NaCl/l
LIP - 2 g NaCl/l
Experimental Variation Fig. 3. (a) Total fatty acid profile; (b) weight based percentage of palmitic, stearic and elaidic acids detected during the study.
The proportion of palmitic (C16:0), stearic (C18:0) and elaidic acids (C18:1n9t) varied with the experimental variations showing a significantly higher ratio under salinity stress (Fig. 3b). Palmitic acid was observed to have two to three folds higher concentration over stearic and elaidic acids. 1 g NaCl/l operation documented
relatively higher concentration of palmitic acid (38.4%) followed by 0.5 g NaCl/l (34.8%), BGP (20.3%), 2 g NaCl/l (18.5%) and LIP-NS (17.5%). Subsequent to palmitic acid, higher quantity of stearic acid was noticed in 0.5 g NaCl/l (15.8%) operation followed by 1 g NaCl/l (13.5%), 2 g NaCl/l (11%), LIP-NS (7.2%) and BGP (5.05%). Significant amounts of elaidic acid was observed in BGP operation (8.4%)
100 VFA
Nitrates
Phosphates
BGP
80
LIP - NS
LIP
LIP - 0.5 g NaCl/l
8.9
LIP - 1 g NaCl/l LIP - 2 g NaCl/l
60 8.4
pH
Removal efficiency (%)
9.4 COD
40
7.9 20 7.4 0 24
48
72
96
120
144
168
Cultivation time (h)
6.9 0
24 48 72 96 120 144 168 192 216 240 264 288 312 336 360
Cultivation time (h) Fig. 4. COD, VFA, nitrates and phosphate removal patterns noticed during the study.
Fig. 5. Distinctions in the pH profiles recorded at BGP and LIP of the study.
Please cite this article in press as: Venkata Mohan, S., Devi, M.P. Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.103
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followed by LIP-NS (6.5%), 1 g NaCl/l (3.3%), 2 g NaCl/l (3.06%) and 0.5 g NaCl/l (2.6%). The variations noticed at each operation might be attributed to the differences in the physiological state of the algae (Xin et al., 1997). Fatty acids derived during the study possess good applied value with diverse fuel characteristics that are desirable in the preparation of biofuels, formation of methyl esters and combustion fuels for engines. The SFAs and USFAs without biofuel properties can also be used as ingredients and excipients for several medicinal and industrial purposes (Venkata Mohan et al., 2011). 3.3. Wastewater treatment Wastewater treatment in terms of COD, volatile fatty acids (VFA), nitrates and phosphates removal was evaluated during BGP (Fig. 4). COD removal efficiency increased gradually and reached a maximum on the 8th day of mixotrophic operation (85.5%). Syntrophism assumes significantly when mixed cultures are used as biocatalyst. Algae along with many other living forms survive in the culture microenvironment developing a mutual dependence on each other and cause a reduction in the organic pollutants (Venkata Mohan et al., 2011; Devi et al., 2012). Similar to COD removal efficiency, VFA removal pattern during BGP was also found to increase till the end of BGP operation (58.1%). The removal pattern documented good agreement with the biomass growth profiles suggesting the fact that wastewater based carbon, forms main substrate for metabolism towards biomass growth as part of mixotrophic mode of cultivation. Nutrient removal monitored as nitrates and phosphates during the BGP documented increased removal patterns. The phosphorus and nitrogen gets utilized for the growth of microalgae. Among the nutrients evaluated, nitrates documented higher removal (75%) compared to phosphates (70%). In nitrification process, ammonia gets oxidized to nitrite and then to nitrate while in denitrification, the nitrate will be taken up by algae and either be converted to organic nitrogen in their cell tissues (algae assimilation) or will be reduced to elemental nitrogen (N2) and lost as a gas (Devi et al., 2013). The observed nutrients/carbon removal suggests that microalgae can effectively utilise nutrients/carbon for its growth with simultaneous reduction leading to the phenomenon of biological waste remediation. 3.4. Bioprocess monitoring Redox condition was regularly monitored during the BGP and LIP operations (Fig. 5). Initial pH for both BGP and LIP was adjusted to 8.2, which later showed slight variations during the experimental run and shifted towards basic (BGP, 8.9; LIP-NS, 8.7) and towards neutral (LIP, 2 g NaCl/l, 7.1; 1 g NaCl/l, 7.3; 0.5 g NaCl/l, 7.5). The shift towards basic microenvironment in BGP denotes the consumption of wastewater based carbon to form bicarbonates while in LIP-NS, the presence of accumulated bicarbonates in the first phase and the absence of salinity in the second phase might have led to the basic environment. Neutral pH patterns noticed during LIP saline stress operations might be due to the salt injury/osmotic stress caused due to the prevalence of saline environment (Rao et al., 2007). In spite of the neutral pH in LIP, biomass concentrations were found to increase, depicting the adaptation of the biocatalyst to saline environment. Oxidation-reduction potentials (ORP) recorded during the BGP and LIP showed good correlation with pH. BGP ( 165.1 mV) and LIP-NS ( 172.3 mV) operation showed negative values. In spite of the neutral pH, saline stress operations also registered negative ORP (2 g NaCl/l, 60.5 mV; 1 g NaCl/l, 62.5 mV; 0.5 g NaCl/l, 65.2 mV). The shift of ORP towards negative value supports the oxidation of carbon and its utilization in the metabolic pathway of microalgae which
is well associated with the increased biomass growth and increased substrate degradation noticed during BGP operation (Syrett et al., 1963). 4. Conclusions Experimental data documented the positive effect of salinity stress on lipid synthesis of microalgae. BGP showed effective biomass growth pattern with simultaneous substrate degradation and nutrient removal while LIP documented good lipid productivity associated with higher concentrations of Chl b supporting the biosynthesis of lipids. FAME profile related to fuel properties was found to be influenced by salinity. Integration of BGP with LIP (saline stress) illustrated the positive response of microalgae towards biodiesel production. The study also focussed on the flexibility of microalgae to utilize wastewater through mixotrophic mode for harnessing biofuels with simultaneous wastewater remediation. Acknowledgements Authors duly acknowledge the Director, CSIR-IICT for encouragement and support in carrying out this work. M.P.D. acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi for providing research fellowship. Research was supported by Council of Scientific and Industrial Research in the form of 12th Plan Task Force Project on ‘Photo-biological process to produce bioenergy through carbon sequestration and wastewater utilization-Biomass to Energy (BioEn, CSC-0116). References Alcantara, C., García-Encina, P.A., Munoz, R., 2013. Evaluation of mass and energy balances in the integrated microalgae growth-anaerobic digestion process. Chem. Eng. J. 221, 238–246. Alkayal, F., Albion, R.L., Tillett, R.L., Hathwaik, L.T., Lemos, M.S., Cushman, J.C., 2011. Expressed sequence tag (EST) profiling in hyper saline shocked Dunaliella salina reveals high expression of protein synthetic apparatus components. Plant Sci. 179, 437–449. Allakhverdiev, I.S., Sakamoto, A., Nishiyama, Y., Inaba, M., Murata, N., 2000. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp.. Plant Physiol. 123 (3), 1047–1056. American Public Health Association (APHA)American Water Works Association (AWWA), 1998. Standard Methods for the Examination of Water and Wastewater. Water Environment Federation, Washington, DC. Asulabh, K.S., Supriya, G., Ramachandra, T.V., 2012. Effect of salinity concentrations on growth rate and lipid concentration in Microcystis sp., Chlorococcum sp. and Chaetoceros sp. In: LAKE: National Conference on Conservation and Management of Wetland Ecosystems, pp. 27–32. Azachi, M., Sadka, A., Fisher, M., Goldshlag, P., Gokhman, I., Zamir, A., 2002. Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina. Plant Physiol. 129, 1320–1329. Benjumea, P., John, R.A., Andrés, F.A., 2011. Effect of the degree of unsaturation of biodiesel fuels on engine performance, combustion characteristics, and emissions. Energy Fuels 25, 77–85. Britta, E.T., Kautsky, L., 2003. Review on toxicity testing with marine macroalgae and the need for method standardization exemplified with copper and phenol. Mar. Pollut. Bull. 46, 171–181. Chandra, R., Rohit, M.V., Swamy, Y.V., Venkata Mohan, S., 2014. Regulatory function of organic carbon supplementation during growth and nutrient stress phases of mixotrophic microalgae cultivation on lipid synthesis. Bioresour. Technol. http://dx.doi.org/10.1016/j.biortech.2014.02.102. Devi, M.P., Swamy, Y.V., Venkata Mohan, S., 2013. Nutritional mode influences lipid accumulation in microalgae with the function of carbon sequestration and nutrient supplementation. Bioresour. Technol. 142, 278–286. Devi, M.P., Venkata Mohan, 2012. CO2 supplementation to domestic wastewater enhances microalgae lipid accumulation under mixotrophic microenvironment: effect of sparging period and interval. Bioresour. Technol. 112, 116–123. Devi, M.P., Subhash, G.V., Venkata Mohan, S., 2012. Heterotrophic cultivation of mixed microalgae for lipid accumulation and wastewater treatment during sequential growth and starvation phases. Effect of nutrient supplementation. J. Renew. Energy 43, 276–283. Farooq, W., Lee, Y.C., Ryu, B.G., Kim, B.H., Kim, H.S., Choi, Y.E., Yang, J.W., 2013. Twostage cultivation of two Chlorella sp. strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity. Bioresour. Technol. 132, 230–238.
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