Process optimization for enhanced biodiesel production by Neochloris

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Bioengineering

Process optimization for enhanced biodiesel production by Neochloris oleoabundans UTEX 1185 with concomitant CO sequestration 2

Srijoni Banerjee, Harshita Singh, Debabrata Das, and Arnab Atta Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b05730 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Industrial & Engineering Chemistry Research

Process optimization for enhanced biodiesel production by Neochloris oleoabundans UTEX 1185 with concomitant CO2 sequestration Srijoni Banerjee1, Harshita Singh1, Debabrata Das*2,1, Arnab Atta*3,1 Advance Technology Development Center1 Department of Biotechnology2 Department of Chemical Engineering3 Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India

*Corresponding authors E-mail address: [email protected] (Arnab Atta), [email protected] (Debabrata Das)

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Abstract Algae are considered as a potential feedstock for biodiesel production. In this study, fresh water grown Neochloris oleoabundans UTEX 1185 is used as an impending source for lipid production in various configuration of photobioreactors (PBRs) namely, airlift, bubble column and flat panel. Different physico-chemical parameters viz. initial pH, cultivation temperature and nitrate concentrations are optimized using single and multi-parameter optimization techniques, which reveal that temperature is the most influential parameter for the optimum production of biomass and lipid. Higher production yields are experienced in bubble column PBR with ring sparger at the optimized condition. CFD study is carried out to understand the hydrodynamics of the bubble column PBR. Stoichiometric analysis shows that 1 g of algal biomass fixed 1.503 g of CO2 from air and released 1.506 g of O2. FAME analysis and fuel properties ascertain the suitability of algal lipid for biodiesel production.

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Introduction The demand of renewable energy sources is increasing day by day mainly due to the huge consumption of fossil fuels, which have limited reserve.1,2 Development of energy sustainability is essential for overcoming the energy crisis of the world.3 The alarming increase of the greenhouse gases in the atmosphere is significantly affecting global warming problem, which has drawn the attention around the globe. Biomass based biofuels as a substitute of fossil fuels is progressively receiving considerable importance.4,5 Microalgal biomass is a promising source of biodiesel, which is considered as third generation biofuel.6 Microalgae have several advantages for biofuel production such as large amount of biomass can be produced in lesser time, can be cultivated in smaller areas, and have significant photosynthetic efficiency.7,8 Biodiesel production from microalgae depends on the biomass concentration and lipid content of the microalgal biomass. Biomass productivity and lipid biosynthesis are influenced by different physico-chemical parameters like initial pH of the media, cultivation temperature, nitrate concentration, CO2 concentration etc. It has been reported that lipid accumulation is increased in nitrate stress condition.9 The lipid content in Chlorella vulgaris, Chlorella minutissima, Chlorella MJ 11/11, Scenedesmus obliquus was enhanced under both in nitrogen starvation and in deprived condition.9 Optimization of different parameters can be achieved through various statistical tools to find out the appropriate condition for maximization of biomass production and lipid synthesis. The most influential parameter for the growth of the microalgae can be also determined with the help of the statistical methods. Taguchi model10 is one of such statistical methods to identify the pivotal parameter for the growth of the microalgal biomass and lipid production. Furthermore, algal lipid is to be transesterified for the formation of fatty 3



acid methyl esters (FAME), which defines the diesel quality. It has been observed that presence of unsaturated fatty acid in FAME increases the storability of biodiesel and neutral lipid mainly tri-acyl glycerol (TAG) is preferred for biodiesel production.11,12 Hitherto, numerous studies13-15 had discretely delineated the optimal growth condition for N. oleoabundans, media selection and lipid enhancement. To the best of our knowledge, any study on the biomass and lipid production maximization from Neochloris oleoabundans UTEX 1185 by concomitant CO2 sequestration is completely absent in the literature. However, to make the process more sustainable and ecofriendlier, concomitant CO2 sequestration study is essential. Moreover, a complete FAME profile with respect to relative percent of essential fatty acids for biodiesel production in the total lipid and characterization of biodiesel extracted from Neochloris oleoabundans UTEX 1185 were never reported earlier. Even though, Beal et al.16 analyzed lipid profile of Neochloris oleoabundans by NMR spectroscopy and reported the functional groups from the profile in terms of peak intensities; their relative percent of concentration in the total lipid, and fatty acid (saturated and unsaturated) concentrations were not revealed, which are of paramount importance to assess the suitability of the lipid for biodiesel production and also to evaluate its quality. In the present work, six different algal strains17 were initially considered, and based on the lipid content N. oleoabundans UTEX 1185 was selected as a promising alga for biodiesel production. Gouveia et al.13 also showed that under suitable condition lipid content of N. oleoabundans can be increased to approximately 50 %w/w of the dry cell weight. Subsequently, key physico-chemical parameters are optimized by both single and multi-parameter optimization studies for maximization of biomass as well as lipid production. To determine the most influencing parameter and appropriate 4


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condition for maximization of biomass production, Taguchi model10 is utilized. To explore the impact of reactor configurations for enhanced biomass production, microalgae cultivation was carried out in three different reactors (viz. bubble column, flat panel, and air-lift photobioreactor) of identical working volume. As the biomass yield is found to be better in bubble column photobioreactor, a basic CFD model is developed to understand its hydrodynamics that eventually influences the mass transfer phenomena. A complete FAME profile, biodiesel characterization and its comparison with the conventional diesel fuel and ASTM standard diesel are subsequently described, which establish the potential of utilizing N. oleoabundans as a feedstock for biodiesel production. The optimization of both single and multi-parameters for the maximization of biomass and lipid production along with concomitant CO2 sequestration is the key novelty of this study, especially from the standpoint of biodiesel production through green and carbon neutral process. Materials and methods Selection of high oil containing algal strain Six different algal strains, viz. Chlorella sorokiniana (collected from IARI, Delhi), Scenedesmus obliqus 393 (University of Texas culture collection centre), Botrycococcus braunii (IARI Delhi), Neochloris oleoabundans UTEX 1185 (University of Texas culture collection centre), Anabeana sp. PCC 7120 (IARI Delhi) and Chlamydomonas reinhardtii UTEX 90 (University of Texas culture collection centre) were considered and cultivated for biomass as well as lipid production in the present study.

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Culture of microalgae and media composition The microalgal strain was selected on the basis of high lipid content. It has been reported in literature15 that media composition plays crucial role for the maximization of biomass and lipid production from the microalgae. Therefore, to screen the best strain for biodiesel production from microalgal biomass, six different strains were cultivated in the media that were reported as the most suitable media for their respective growth. Several studies18-21 reported that B. braunii grew faster in BG11 medium, and lipid content was also higher compared to results in CHU13. Accordingly, the growth characteristics of the selected microalgal strains were studied using different media like soil extract, TAP (-) acetate, BOLD 3N, modified BOLD 3N (MB3N), and BG11 media. MB3N is composed of 3 mL/ 100 mL NaNO3 (2.5 g/ 100 mL), 1 mL/ 100 mL of CaCl2 (0.25 g/ 100 mL), 1mL/ 100 mL MgSO4.7H2O (0.75 g/ 100 mL), 1 mL/ 100 mL K2HPO4 (0.75 g/ 100 mL), 1 mL/ 100 mL KH2PO4 (1.75 g/ 100 mL), 1 mL/ 100 mL NaCl (0.25 g/ 100 mL), and 4 mL/ 100 mL of soil extract. The major composition of the soil extract was analyzed by HPLC and atomic absorption spectroscopy (AAS), which showed the presence of 107 mg/ mL of humic acid (Fig. S1) and heavy metals like Cu (0.02 mg/L), Fe (0.12 mg/ L), Ni (0.02 mg / L), Ca (8 mg/ L), and Mg (3 mg/ L). Single parameter optimization From the literature,22 it was found that initial pH of the media, temperature and concentration of nitrate considerably influence the biomass production. Therefore, single parameter optimization experiments with initial pH of the media, cultivation temperature, and nitrate concentration in the media were carried out for the maximization of the biomass and lipid production. One parameter was varied at a time 6


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by keeping all the other parameters constant.23-26 The pH of the media was varied in the range of 6.0-9.0 with an interval of 0.5 for determining the optimal pH for biomass and lipid production. Temperature for the cultivation of microalgae was varied from 20-35 ºC with an interval of 5. Nitrate concentration in the range of 0.55-35.28 mM was maintained in the culture media for optimization of the suitable nitrate concentration. All experiments were conducted in 250 mL conical flasks with a working volume of 100 mL. For accuracy and repeatability, all the experiments were carried out in triplicates in the present work. Experimental set-up was placed in an illuminated shaker incubator (Innova, New Burnswick, NJ, USA). An artificial light from non-carbon source was used for photosynthesis. Compact fluorescent lamp (CFL) of 20 W was used as the light source. Following preliminary studies by developing a photoinhibition model (see supplementary information), the optimized light intensity was found to be 80 µmol/m2/s, which was measured by a quantum meter LI-250A of LI-COR, and rotational speed of 100 rpm was maintained in all subsequent experiments. Samples were drawn from the algal culture on regular interval time to study the growth kinetics. At the end of cultivation period, the cells were harvested for estimation of the lipid content. Multiple parameter optimization using Taguchi model Cumulative effect of the three physico-chemical parameters (initial pH of the media, cultivation temperature, and nitrate concentration) on the maximization of biomass and lipid production was realized by multiple parameters optimization study using Taguchi model.10 Three parameters were divided into different levels, and significance variance of different levels was set. Consequently, relative importance of the three factors on biomass and lipid production was determined by Taguchi model. L9 orthogonal array 7



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was incorporated in the experimental design for organizing the parameters impacting biomass production and to vary those parameters into different levels. Taguchi model tests the suitable experimental combination to discern the most influential parameter for the product formation. Loss function (L(y)), as defined in Eq. 1,10 was used for assessing output of the performance with respect to the deviation from its target value. (1)

𝐿(𝑦) = 𝑘(𝑦 ― 𝑚)

where y denotes all the experimental value of each test. k is the proportionality constant and m denotes the target value. Loss function L(y) can also be represented by Eq. 2. 1

(2)

𝐿(𝑦) = 𝑘(𝑦2 ) Expected loss function can be defined as Eq. 3. 1

(3)

𝐸[𝐿(𝑦)] = 𝑘𝐸(𝑦2) where (1/y2) can be estimated from a sample of n and is described by Eq. 4.

( )=∑

𝐸

1

𝑦2

1 𝑛 ( )/𝑛 𝑖 = 1 𝑦2

(4)

Nine experiments with different combination in several levels were carried out. The statistical analysis with the experimental data was resolved using Minitab 15 (Minitab Inc., PA, USA) software package, where the signal-to-noise ratio (S/N) was calculated as follows (Eq. 5): 𝑆 𝑁

= 10 × 𝑙𝑜𝑔((𝑌2 ― 𝑠2 ÷ 𝑛) ÷ 𝑠2)

(5)

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where 𝑌 = mean, 𝑠 = standard deviation, and 𝑛 = number of the responses for the given factor level combination. The aforementioned equation helps to pick out the response and to form the S/N ratio on means and standard deviations. Determination of activation energy for the cell growth To determine the activation energy for growth of N. oleoabundans UTEX 1185, Arrhenius equation was considered (Eq. 6). 𝐸𝑎

(6)

µ𝑛𝑒𝑡 = 𝐴 exp ― 𝑅𝑇 which can be modified as Eq. 7. 𝐸𝑎

(7)

ln µ𝑛𝑒𝑡 = ln 𝐴 ― 𝑅𝑇

where µnet, A, Ea, and R indicate the net specific growth rate (d-1), Arrhenius constant, growth activation energy (kJ/mol), and universal gas constant, respectively. The value of R is 8.314 J/mol.K, and T is the absolute temperature (K). Growth kinetics of N. oleoabundans UTEX 1185 in various PBR configurations Reactor configuration has a great influence on microalgal biomass and lipid production maximization.27-30 Under the optimized cultivation conditions determined during the multi-parameter optimization studies, production yield of N. oleoabundans UTEX 1185 was studied in airlift reactor with ring sparger, bubble column reactor with ring sparger, and flat panel reactor (Fig. 1).

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Figure 1. (a) Schematic for microalgal cultivation in PBR, and (b) photograph of the experimental setup. The working volume of all the reactors was 1.4 L. Bubble column reactor was comprised of cylindrical tube with length of 45.1 cm, internal diameter 7.5 cm, and a ring sparger with 15 pores of 0.1 cm diameter. Alike bubble column, airlift reactor had similar cylindrical structure with ring sparger and an additional draft tube having length of 30 cm, internal diameter of 4 cm inside the outer cylinder. Flat panel reactor was also used in the present study (2L).28 All the reactors were fabricated in collaboration with Norwegian Institute for Agriculture and Environmental Research (Bioforsk), Norway at Indian Institute of Technology Kharagpur, India. CFD studies were performed on the bubble column reactor to understand its hydrodynamic behaviour, and to predict plausible optimal condition for sustainable operation. In this work, a 3D bubble column reactor is modeled using a commercial CFD solver, FLUENT 15.0 (ANSYS. Inc., USA). In line with the experimental design, air was purged through the fifteen openings 10


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each of 0.1 cm diameter available at the bottom of the column during simulations. Eulerian two-phase model was employed to predict volume fraction by solving NavierStokes and continuity equations for both phases. Prior to the calculation, entire computational domain was patched with media, and the outlet was set to atmospheric pressure condition. Effect of CO2 concentration on biomass and lipid production Effect of various CO2 concentrations on biomass and lipid production from N. oleoabundans UTEX 1185 was also investigated. These experiments were executed in bubble column with ring sparger, as mentioned earlier, and a mixture of CO2-air (%v/v) was sparged with a flow rate of 0.43 vvm, which was maintained using a rotameter and was considered as the prime source of carbon in the system. CO2-air mixtures were maintained to 0.03, 1.5, 2.5 and 3.5 %(v/v). The microalgae were cultivated in modified BOLD 3N media with the optimized parameters at a light intensity of 80 µmol/m2/s and the CO2 sequestration by microalgae was determined using the following equation Eq. 7.30 Chisti,5 demonstrated that 50 % of carbon in dry weight of microalgae which corresponds to 1.83 g of CO2 is required for the cultivation of 1 g of microalgae dry cell weight. 𝐶𝑂2𝑠𝑒𝑞𝑢𝑒𝑠𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑔/𝐿 ) = 1.83 × (𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛) (𝑔/𝐿)

(8)

Post experiment analytical methods Estimation of biomass and lipid content A sample of homogeneous algal cell suspension (1 mL) was collected from the photobioreactor. The biomass (pellet) was separated by centrifugation (6000 × g, 10 min, 4 C) and washed three times with 0.85 %w/v saline. This biomass was then dried 11



in a hot air oven at 80 ± 5 C in pre-weighed Aluminium (Al)-foil until a constant weight was obtained. The difference between the weights of the Al-foil with dried cells and the empty Al-foil was taken as the biomass concentration (g/L). Subsequently, optical density (OD) was determined in spectrophotometer (Chemito). Consequently, a calibration curve was plotted between dry cell weight of the biomass concentration vs OD to find out the correlation between them. As the algal cell drying is a timeconsuming process, further biomass concentration was determined from the correlation curve derived by the spectrophotometric method.31 Bligh and Dyer32 method was used for the extraction of lipid from the dried algal biomass. Total lipids were extracted from dry algal biomass by using chloroform: methanol (2:1) solvent mixture. Dried algae samples were homogenized by using pestle-motor in 5 mL of chloroform: methanol (2:1) for approximately 30 s. The biomass was then collected at the bottom of the falcon tube by centrifugation and the solvent was removed to a pre-weighed empty Supelco glass-vial. Extraction of biomass was repeated twice as described above. The organic extractions, pooled into a weighed vial, were dried by hot air oven for 12 h. The dried vials were again weighed to establish the total lipid content. Same methodology was used for higher amount of biomass but in Soxhlet apparatus. Dried algal biomass of 500 mg was taken and 150 mL of 2:1 ratio of chloroform : methanol solution was used in the soxhlet apparatus. As per the The Büchi B 811 (Switzerland) protocol of standard extraction using soxhlet, 1 g of algal biomass in 100 mL solvent is typically used during the lipid extraction from microalgal biomass. However, from our preliminary optimization study on the lipid extraction and transesterification process, enhanced lipid extraction was observed in case of using 500 mg of biomass in 150 mL solvent. The extracted solvent was then taken into 200 mL pre-weighed empty glass beakers and kept 12


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in oven for 12 h for drying. Then the dried beakers were again weighed to establish the total lipid content. However, the total lipid has to be transesterified to produce biodiesel. For the transesterification process, methanolic HCl was added to the total extracted lipid and was kept for 6 h in 65 ºC.33 Thereafter, hexane was added to the mixture, and equal amount of warm distilled water was also mixed. The mixture was then centrifuged at 6000 rpm for liquid-liquid phase separation. For better purification, the hexane layer containing FAME content was subsequently separated and washed twice with distilled water. This FAME was then analyzed by GC-technique to understand the suitability of the lipid to be used as biodiesel. Lipid content and biomass concentration were determined at every step in single and multiple parameter optimization studies, as well as during the operation of lab-scale PBRs and CO2 sequestration study in PBR. CHNS analysis Determination of elemental analysis (such as carbon, hydrogen, sulfur and nitrogen) of biomass (cultivated under different CO2-air mixture) was accomplished in a CHNS analyzer (Elementar, vario Macro cube), which had two chambers: oxidation, and reduction. Algal biomass was taken in a foil made up of tin (as it does not contain C, H, N, and S), and approximately 2-2.5 times of Tungsten oxide (WO3) (w/w) was added into the sample that was oxidized at 1150 ºC in the oxidation chamber in presence of oxygen. Oxidized gas was subsequently passed through reduction chamber containing copper fringe, where it was reduced. Reduced gas was then detected and quantified using a gas detector (TCD). Helium (He) was used as a carrier gas at a flow rate of 600 mL/min, and relative percent of oxygen was calculated by deducting the relative percent of C, H, N, S from overall content.

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Lipid accumulation study using Nile red staining and confocal microscopy A rapid estimation method of the intracellular lipid accumulation in microalgae using confocal microscopy can be done by staining the microalgal cell by Nile red. Nile red34 staining methods with dimethyl sulfoxide (DMSO) treatment were found to be effective for staining N. oleabundans UTEX 1185 cells. A mixture of 10 μL algal cells and 20 μL of DMSO was transferred into a 1 mL amber bottle and pretreated in microwave oven for 1 min, which is subsequently placed in a microwave oven for another 1 min after the addition of 935 μL of pure water and 10 μL of Nile red solution (100 μg/mL in acetone). Slides were prepared and observed under fluorescence microscope and confocal microscope for identification of the lipid droplets inside the algal cells. FAME analysis and fuel properties determination Lipid extracted from the dried algal biomass was transesterified by methanolic HCl at 65 ºC temperature for 6 h.33 Thereafter, biodiesel samples were analyzed in gas chromatography (Clarus 500, Perkin Elmer) using flame ionisation detector (FID) and Omegawax 250 capillary column of 30 m length, 0.25 µm film thickness, and 0.25 mm internal diameter (Sigma). The oven temperature and flow rate were 240 ºC and 1 mL/ min, respectively. Nitrogen was used as carrier gas. Standard FAME mixture (37 mix, Supelco Inc., Bellefonte, PA) was used for the calibration purpose. Extracted biodiesel from N. oleoabundans UTEX 1185 was characterized. Fuel properties such as kinematic viscosity, density, flash point, pour point, cetane number were analyzed, and subsequently were compared with ASTM (American Society for Testing and Material) standard and petroleum diesel.

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Results and discussion Selection of suitable algal strain Initially, six different algal strains, viz. Chlorella sorokiniana, Scenedesmus obliquus UTEX 393, Botrycococcus braunii, Neochloris oleoabundans UTEX 1185, Anabeana sp. PCC7120 and Chlamydomonas reinhardtii UTEX 90 were chosen for biomass and ensuing lipid production. Their biochemical composition analysis (Table S1) revealed that the highest amount of lipid can be obtained from B. braunii. However, that lipid is mostly composed of large hydrocarbon molecules, which are ideal for liquid biofuel production by means of liquefaction or pyrolysis, and cannot be used for biodiesel production via transesterification.35 In few literature,36,37 B. braunii was also reported as a feedstock for biodiesel production, but its lipid productivity was lesser as compared to the present study. Moreover, the growth rate of N. oleoabundans UTEX 1185 is higher as compared to B. braunii. Therefore, in this study N. oleoabundans UTEX 1185 was chosen due to the higher lipid production as compared to other strains. Optimization of biomass and lipid production from N. oleoabundans UTEX 1185 Composition of media plays crucial role for the growth of microalgae. Algae require different types of nutrients for their growth. Media composition and concentration varies from species to species, which require different amount of nutrients for their growth and product formation. In this study, MB3N showed higher biomass yield as compared to soil extract, TAP (-) acetate, BOLD 3N, and BG11 media (Fig. 2).

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Figure 2. Growth characteristics of Neochloris oleoabundans UTEX 1185 in different growth medium. This observation resulted in further studies with modified BOLD 3N media (Table S2). It was also perceived that soil extract individually had less stimulating effect for the algal growth as compared to that of modified BOLD 3N medium. The soil extract present in the modified BOLD 3N media contained 107 mg/mL of humic acid (Fig. S1a, S1b). It also comprised trace amount of heavy metals like Fe 0.12 mg/L, Ca 7.73 mg/L, Mg 2.30 mg/L. Humic acid acts as a growth stimulant38 in algae and different types of plants. The heavy metals present in the soil extract can act as co-factors39 for various enzymes which regulate the growth of the microalgae as well as lipid production. Physico-chemical parameters like cultivation temperature, initial pH of the media and nitrate concentration in the media are the key parameters for the growth of microalgae. Therefore, single-parameter optimization technique in a batch process was considered to find out most suitable condition. Cellular enzymes are pH sensitive for the growth of the organism. Thus, the initial pH of the media significantly impacts suitable growth of the microalgae. Most of the microalgae favor neutral pH for their growth. Consequently, the initial pH of the media was varied in the range of 6.0-9.0 with an interval of 0.5 for 16


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determining the optimal pH for biomass and lipid production. Biomass production and lipid content of the biomass were 1.21 g/L and 30 %w/w, respectively, at pH 6.5 (Fig. 3a), which was comparatively higher than the other initial pH of the media.

Figure 3. The biomass production from Neochloris oleoabundans UTEX 1185 at different (a) initial pH of the media, (b) cultivation temperature, and (c) nitrate concentration (in mM). Temperature is one of the most influencing factors in all the biological activities in microalgae.11 Each of the microalgae has an optimum temperature for growth. Carbonic anhydrase activity is hampered due to small variation in temperature.11 Electron transport chain in cell can also be hampered due to temperature rise. Here, temperature for the cultivation of microalgae was varied from 20-35 ºC by keeping the other parameters unaltered. Maximum biomass concentration of 1.24 g/L was obtained at 25 ºC with 32 %w/w of lipid content (Fig. 3b). Microalgae also requires adequate amount of nitrogen source for production of biomass. For the growth of N. oleoabundans UTEX 1185, sodium nitrate was utilized as a nitrogen source in the cell growth media. 17



Biomass production also depends on the nitrate concentration in the media. The substrate affinity of nitrate uptake system varied from species to species. Therefore, nitrate concentration optimization study was carried out in the range of 0.55-35.28 mM. Maximum biomass concentration of 1.32 g/L was obtained at 8.82 mM nitrate concentration, and the lipid content in the biomass was 25 %w/w. However, the lipid content was observed to be higher in nitrate deprived concentration, which was obtained as 36 %w/w at 4.41 mM nitrate concentration with the biomass concentration of 0.897 g/L (Fig. 3c).

During photosynthesis, the formation of NADP+ (major electron

acceptor) results in higher biomass production due to the consumption of ATP and NADPH molecules.26 Fatty acids (mainly TAG) also boosted up consuming NADPH and replenish NADP+.26 Additionally, carbon available in the media producing (lipid) TAG rather than protein synthesis might be the possible reason for increment of lipid content in nitrate deprived condition. Multi-parameters optimization Multi-parameters optimization was performed by Taguchi model to find out the most influential parameter for biomass production (Table S3). From the results obtained from single parameter optimization, three levels (L1, L2 and L3) of each parameter were determined. Maximum biomass concentration of 1.55 g/L and lipid concentration of 0.558 g/L were achieved at the temperature of 25 °C, and 4.41 mM sodium nitrate concentration with initial pH of the media as 7.0. Fig. 4 shows the relative influence of each parameter on biomass production. Thereafter, parameters were ranked according to the respective degree of influence of each factor, denoted by the difference between L2 and L1, as mentioned in Table S4. Microalgae utilize light by limiting the electron transport chain at lower temperature. Photorespiration increases in cell due to rise in 18


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temperature. Therefore, it can be interpreted that nitrate accumulation and different enzymatic reaction inside the microalgal cell can be hampered as a consequence of temperature increment. Biomass concentration was found to be highest at 25 ºC.

Figure 4. Main effect plots for means from Taguchi design. Hence, temperature is ascertained as the most influential parameter for biomass and lipid production from N. oleoabundans UTEX 1185. Determination of activation energy for the growth of the cell Establishing the temperature as the dominant parameter, the growth activation energy of N. oleoabundans UTEX 1185 was determined using Arrhenius equation in the range of 20 ºC to 35 ºC. Fig. 5a demonstrates the variation of the net specific growth rate of N. oleoabundans UTEX 1185 with temperature. From the slope of natural logarithm of net specific growth rate vs. reciprocal of temperature plot, growth activation energy was estimated (Fig. 5b).

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Figure 5. (a) Specific growth profile, (b) plot of natural logarithm of net specific growth rate vs reciprocal of temperature. For the plot, only those temperatures were chosen in which the correlation coefficient was close to -1.0. This led to a lesser number of points involving only three temperatures, 20 ºC, 25 ºC and 35 ºC. In these temperatures, correlation coefficient was approximately -0.96. Fig. 5a shows that net specific growth rate (µnet) was highest at 25 ºC, which decreases sharply after 25 ºC. This can be ascribed to the increased substrate affinity of microalgae with increasing temperature and beyond 25 ºC, denaturation of cellular components may take place at high temperature. Hence, the cells become dead with disruption of cellular metabolism. The cell growth activation energy (Ea) of 23.40 kJ/mol was then obtained at 25 ºC, which helps in formulating a specific relation for estimating µnet at any temperature in the range of range of 20 ºC to 35 ºC, as described in Eq. 9. This value is comparable with Ea value of Chlorella vulgaris which is 21.46 kJ/mol.11 ln 𝜇𝑛𝑒𝑡 = 4.95 ―

23.4

(9)

𝑅𝑇

20


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Effect of different reactor configurations for biomass production Airlift, bubble column and flat panel reactors were operated by maintaining the optimized conditions (from single and multi-parameter optimization) for the improvement of biomass as well as lipid yield. Initial pH of the media was maintained at 7.0, cultivation temperature was maintained at 25 ºC, and nitrate concentration was maintained to 4.41 mM. Table S5 shows that maximum biomass concentration of 2.01 g/L was obtained in bubble column reactor with ring sparger, and the lipid content was also found to be highest (46 %w/w). This can be attributed to the larger cross-sectional area available for the sparger at the inlet of the bubble column reactor that facilitates better radial dispersion of air. CFD results for bubble column hydrodynamics endorsed this flow homogeneity that subdued dead zones inside the PBR during cultivation, which is desirable for growth of the microalgae. Fig. 6 qualitatively demonstrates the air distribution profiles at two different axial position (0.25 m and 0.15 m) from the inlet. Fig. 7 shows the transient velocity profiles at a plane above 0.25 m from the inlet that attained pseudo-steady state within 33 s for air flow rate of 0.43 vvm.

Figure 6. Distribution of air velocity inside a bubble column PBR at different heights from the inlet (a) 0.25 m, (b) 0.15 m, and (c) inlet for an air flow rate of 0.43 vvm. 21



Elevated draft tube design for air inlet in the airlift reactor, and the pore distribution at the inlet for the flat panel reactor are plausibly unable to provide a homogeneous radial mixing of the liquid inside the system.40,41

Figure 7: Radial velocity distribution inside the bubble column PBR at a height of 0.25 m from the inlet and air flow rate of 0.43 vvm. Effect of CO2 concentration on biomass and lipid production The integration of biofuel production strategy with CO2 sequestration by microalgae would increase the feasibility of the process by reducing the greenhouse gas effect and increasing the product (biomass and lipid) yield. Different factors determine the CO2 sequestration capability in microalgae. PBR configuration and its mass transfer efficiency play a key role for algal CO2 sequestration. Kumar et al.30 revealed that the higher mass transfer of CO2 in water resulted in enhanced biomass and lipid production. Other factors like initial pH of the media and cultivation temperature also influence the CO2 sequestration in microalgae. Therefore, the optimized conditions for the cultivation of N. oleoabundans UTEX 1185 were maintained, and subsequent experiments were conducted in bubble column reactor with ring sparger as it provided maximum biomass concentration and the lipid content compared to other PBR configurations (Table S5). 22


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Modified BOLD 3N media was used for the cultivation of the organism. The microalgae were grown in the presence of air, 1.5, 2.5 and 3.5 %v/v CO2-air mixtures. The highest biomass concentration of 2.56 g/L with lipid content of 49 %w/w was found in the presence of 2.5 %v/v CO2-air mixture (Fig. 8).

Figure 8. Cell growth profiles of Neochloris oleoabundans UTEX 1185 in modified BOLD 3N media using different CO2-air (% v/v) gas mixture. Biomass concentrations of 1.33 g/L and 1 g/L were achieved using 1.5 %v/v CO2-air mixture and air, respectively. There was a sharp pH drop in the cultivation media for 3.5 %v/v CO2-air gas mixture. At 3.5% CO2-air (v/v) mixture, the final pH was found to be nearly 4.3. Therefore, it can be perceived that pH may be one of the critical factors influencing the growth of this strain. It was also reported in several literature6,11 that microalgae favorably grew in alkaline pH. As the pH of the culture dropped to 4.3, growth inhibitions took place for N. oleoabundans UTEX 1185. Chiu et al.42,43 reported that the growth of microalgae Chlorella. sp. and N. oculata were completely inhibited when the CO2 concentration was higher than 5%. Consequently, minimal growth of microalgae was observed in that condition. Higher CO2-air gas mixture was also studied but any perceptible growth was not observed due to further drop in pH. The presence of CO2 for microalgae growth may have caused the storage of lipids owing to metabolic 23



Page 24 of 39

flux shifting toward neutral lipids production pathway rather than forming phospholipids for membrane function and growth under stressed environment. CHNS analysis and determination of CO2 sequestration C, H, N, and S content of N. oleoabundans UTEX 1185 algal biomass were determined at different experimental conditions, as illustrated in Table S6. All the four elements C, H, N, and S of N. oleoabundans UTEX 1185 grown on 2.5 %v/v CO2-air gas mixture were significantly higher than that of the 1.5 %v/v CO2-air gas mixture and normal air. Molecular formula of the algal biomass of N. oleoabundans UTEX 1185 was determined based on the relative percent of C, H, N. Highest molecular weight of N. oleoabundans UTEX 1185 was 29.26 g when grown in 2.5 %v/v CO2-air gas mixture. Fig. 9 illustrates the stoichiometric analysis (Eqs. 10-12), which reveals that the synthesis of 1 g of algal biomass consumes 1.503 g of CO2 from air, and releases 1.506 g of O2. For 0.03 % v/v CO2 – air: 𝐶𝑂2 +0.99𝐻2𝑂 + 0.17𝑁𝑂3 ― →𝐶𝐻1.98𝑁0.17𝑂0.65 +1.42𝑂2

(10)

For 1.5 % v/v CO2 – air: 𝐶𝑂2 + 0.96𝐻2𝑂 + 0.11𝑁𝑂3 ― →𝐶𝐻1.92𝑁0.11𝑂0.74 + 1.28𝑂2

(11)

For 2.5 % v/v CO2 – air: 𝐶𝑂2 + 1.01𝐻2𝑂 + 0.18𝑁𝑂3 ― →𝐶𝐻2.02𝑁0.18𝑂0.79 + 1.38𝑂2

24


(12)

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Figure 9. Material balance of the PBR considered for the cultivation of microalgae. Regarding total CO2 sequestration, the results indicate that 2.5 %v/v CO2-air is the most efficient stream that sequestrated 3.91 g/L of CO2 in a batch of 8 d in bubble column reactor with ring sparger, which is higher than the total CO2 sequestration capability of airlift reactor (2.89 g/L) and flat panel reactor (2.09 g/L). Lipid accumulation, FAME analysis of N. oleoabundans UTEX 1185 and fuel properties of the biodiesel The improvement of staining technique with hydrophobic solvent DMSO and microwave pretreatment enable the visualization of the lipid droplet inside the green algal cells under fluorescence and confocal microscopes. This staining technique effectively penetrates the rigid cell wall of the microalgal cell wall and binds with the lipid droplets present inside the cell. Discernable neutral lipid droplets (Fig. 10) were found to increase with respect to cultivation time, and it was observed that lipid accumulation increased significantly after the 3rd day of cultivation.

25



Figure 10. Lipid accumulation study by confocal microscopy on the (a) first day, (b) third day, and (c) fifth day of cultivation. (bright yellow spots are the lipid droplets inside microalgal cells). It was observed from the fluorescence and confocal microscopic analysis that lipid accumulation increased with the cultivation time and with increasing biomass concentration, which can be attributed to the nitrate stress condition. During photosynthesis in microalgae, NADP+ is generated, which is considered as a major electron acceptor in photosynthetic process. Generation of NADP+ yields higher biomass production by consumption of ATP and NADPH molecules.26 Under nitrogen starvation condition NADP+ is depleted, which might be a possible reason for TAG accumulation in microalgae. Table 1 demonstrates the composition of the transesterified fatty acid.

26


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Table 1. FAME analysis of the lipid extracted from N. oleoabundans UTEX 1185 Fatty acids

C14:0 C16:0 C16:1

Relative percent of fatty acid composition (0.03 %v/v CO2-air) 3.42 ± 0.3 21.63 ± 1.0 24.31± 1.1

Relative percent of fatty acid composition (1.5 %v/v CO2-air) 2.32 ± 0.2 20.52 ± 1.1 24.31 ± 0.8

Relative percent of fatty acid composition (2.5 %v/v CO2-air) 2.78 ± 0.2 18.16 ± 0.9 28.05 ± 1.0

C16:2

1.07 ± 0.2

1.25 ± 0.4

1.95 ± 0.2

C18:0

1.81 ± 0.2

1.28 ± 0.1

1.62 ± 0.2

C18:1

41.16 ± 0.8

41.37 ± 1.7

38.56 ± 1.8

C18:2

3.98 ± 0.3

3.25 ± 0.3

4.50 ± 0.4

C20:0

1.06 ± 0.3

4.17 ± 0.5

1.69 ± 0.2

Unsaturated 69.88 ± 0.8 71.68 ± 1.0 77.75 ± 1.2 fatty acids Saturated 30.12 ± 0.5 28.32 ± 0.7 22.25 ± 0.8 fatty acids Major fatty acids found in N. oleoabundans UTEX 1185 were palmitic (16:0), palmitoleic (16:1), oleic acid (18:1), and linoleic (18:2). Arachidic (20:0) acids were also found as minor components (Fig S3). Interestingly, a complete FAME profile with biodiesel characterization was not reported in the literature. Du et al.44 reported a partial FAME analysis of the biodiesel extracted by N-ethylbutylamine (EBA) from N. oleoabundans that comprised of C18:1 (oleic acid 17%), C18:2 (linoleic acid 21%) and C16:2 (hexadecadienoic acid 2%). In the present study, an increase in the concentration of unsaturated fatty acids was observed with increased CO2 levels (Table 1). The level of unsaturated fatty acids determines the quality of the biodiesel and the presence of higher level of saturated fatty acids indicates the good oxidative stability of the biodiesel. The fatty acids obtained has a carbon chain length of C14–C20 in significant amount and is higher than the reported data,45,46 which justifies the use of N. oleoabundans UTEX 1185 as feedstock for biodiesel production. Furthermore, fuel properties of the biodiesel produced from N. oleoabundans UTEX 1185 were compared with ASTM standard and 27



petroleum diesel. The kinematic viscosity and density of the fuel influence the engine performance and its emission characteristics. Consequently, kinematic viscosity (4.7 mm2/s) and density (882 kg/m3) of the biodiesel were determined, which were found to be comparable with petroleum diesel (Table 2). Table 2. Comparison of N. oleoabundans UTEX 1185 biodiesel with ASTM standard and petroleum diesel Fuel properties ASTM Diesel Biodiesel -2 -1 Kinematic viscosity at 40 ºC (mm s ) 1.9-6 4.1 4.7 -3 Density (kg m ) 825 882 -1 Calorific value (MJ kg ) 45 40 -1 Acid value (mg KOH g ) < 0.8 0.4 0.6 -1 Iodine (g I2 100 g ) 79.4 80 Flash point (ºC) 130 115 120 Pour point (ºC) -15 -25 -18 Cetane number >= 47 49 53 The acid number of fuels signifies the corrosive potential of the fuel. In this case, the presence of acid moieties in the biodiesel (0.6 mg KOH/g) is in the comparable limit with the petroleum diesel (0.4 mg KOH/g) and ASTM standard (

Process optimization for enhanced biodiesel production by Neochloris

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