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Designing a CO2 supply strategy for microalgal biodiesel production under diurnal light in a cylindrical-membrane photobioreactor
Bioresource Technology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Designing a CO2 supply strategy for microalgal biodiesel production under diurnal light in a cylindrical-membrane photobioreactor Venkateswara R. Naira, Debasish Das, Soumen K. Maiti
⁎
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Cylindrical membrane photobioreactor Diurnal light FAME production Optimal CO2 supply Reversible photoinhibition Dark-phase growth
A cylindrical membrane photobioreactor with high CO2 mass transfer coefficient was designed and installed under customized unidirectional lighting. Combinatorial effect of light and CO2 on the growth of Chlorella sp. FC2 IITG was studied and an optimal CO2 supply without pH control strategy was developed under diurnal light similar to sunlight (17-2000-17 µE m−2 s−1). Unprecedentedly, broad range of saturated light levels (700–1500 µE m−2 s−1), reversible photoinhibition, no pH control requirement and dark-phase growth were noticed altogether in the strain. Under diurnal light, final biomass titer of 5.79 g L−1 and overall biomass productivity of 1.29 g L−1 day−1 were observed. The results were similar to optimal light (1130 µE m−2 s−1) and CO2 (2%) conditions. Subsequently, a highest FAME productivity of 265 mg L−1 day−1 was observed in last two days of lipid induction phase.
1. Introduction Microalgae remarked as an excellent autotrophic source for biodiesel production due to their high oil yielding capacities (Chisti, 2007). Additionally, microalgae mitigates the greenhouse effect by fixing CO2 and produces environmental friendly biodiesel compared to conventional diesel (Monyem et al., 2001). The microalgal culturing technology is mainly governed by light and CO2. At high light intensities, photoinhibition takes place due to production of reactive oxygen species (Murata et al., 2007). On other hand, concentration of dissolved CO2 (dCO2) in the culture medium has to be modulated to overcome limiting and inhibiting concentrations of CO2. However, overall CO2 mass transfer coefficient is the preliminary requirement to maintain desired dCO2 concentration. Above all, the demand of CO2 is depends on the light availability for the algal cells. Thus, the combinatorial effect of light and CO2 has to be considered for designing a cultivation technology (Pierobon et al., 2016). Apart from this, high concentration of dCO2 could inhibit the growth of algae due to two possible factors: lowered pH levels and usual substrate inhibition. Thus, it is very crucial to determine appropriate controlling pH before the evaluation of CO2 effect. In the present study, a cylindrical membrane photobioreactor (CMPBR) with a custom-made membrane sparger without agitation was designed. Since, algae cultivation under sunlight is the only feasible method (Wang et al., 2014), studies under diurnal lighting similar to outdoor sunlight is necessary. An LED lighting with computer-
⁎
dimmable technology was designed to mimic diurnal patterns similar to sunlight. In the CM-PBR, studies have mainly emphasized on pH control requirement and evaluation of inhibiting and limiting effects of both light and CO2 in a combinatorial manner for growth of microalgae. An optimal CO2 supply strategy was developed for maximizing biomass and lipid productivities grown under diurnal light cycle in the CM-PBR. 2. Materials and methods 2.1. Microorganism and culture medium Chlorella sp. FC2 IITG (referred as FC2), an indigenous novel microalgal isolate previously for biodiesel production (Muthuraj et al., 2014) was used in the studies. Culturing medium was modified BG11 medium with pH, 7.1 (Muthuraj et al., 2015). 2.2. Photobioreactor design, instrumentation and control Flanged cylindrical acrylic tube (O.D.: 75 mm, I.D.: 69 mm, H: 175 mm) with a low-cost membrane sparger was designed as CM-PBR (Fig. 1). A neoprene rubber sheet (thickness: 2 mm), made porous by 30 gauge fine needle was used as membrane sparger. Mixture of air and CO2 (2 bar pressure) was sparged from the bottom of CM-PBR. An LED (cool white) panel (3 × 50 W per reactor, 6500 K color temperature) was installed perpendicular to surface (one side) of reactor. Light (lux) was measured using a lux meter (Sigma Instruments, India). SNAP PAC
Corresponding author. E-mail address: [email protected] (S.K. Maiti).
https://doi.org/10.1016/j.biortech.2017.11.087 Received 18 October 2017; Received in revised form 25 November 2017; Accepted 27 November 2017 0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Naira, V.R., Bioresource Technology (2017), https://doi.org/10.1016/j.biortech.2017.11.087
Bioresource Technology xxx (xxxx) xxx–xxx
V.R. Naira et al.
Fig. 1. Schematic of the parallel cylindrical membrane photobioreactor (CM-PBR) system.
pH was not controlled) and ‘no control’ (pH was not controlled both in day and night) were tested at 10% CO2 level and constant light intensity. Thirdly, the growth of FC2 was investigated under constant supply of CO2 levels (% in air, v/v) ranging from atmospheric air to 15%. A constant light intensity of 1130 μE m−2 s−1 was used. Finally, FC2 growth characteristics and its FAME production were studied under simulated diurnal LED lighting that mimics outdoor sunlight (17–2000–17 μE m−2 s−1, light:dark cycle of 12:12 h). For lipid induction, FC2 cells were transferred to urea-free culture medium. A dCO2 concentration of 100 ± 10 mg L−1 was maintained throughout the culture. All studies were performed in batch and temperature was not controlled in any of the studies. During night periods, CO2 flow was stopped in all the studies (exception: ‘full control’ pH control strategy) and air flow remained same to ensure good mixing of algal cells without any settling.
control and data acquisition system (OPTO 22, USA) was used to control pH via online pH analyzer (Zest Engg., India) and LED light intensity via pulse width modulation technology (Mouser electronics, USA) (Fig. 1). The dCO2 concentration was measured using online dCO2 analyzer (Cole-Parmer). 2.3. Estimation of probe response time (τp) and overall mass transfer coefficient (KL a,CO2 ) Considering the probe response as first order process (Eq. (1)) (Philichi & Stenstrom, 1989), simple dynamic method in cell-free media (Eq. (2)) was used to estimate ‘KL a,CO2 ’ of membrane sparger and conventional L-sparger (Applikon Biotechnology B.V., Netherlands).
dCp dt
= kp ∗ (CL−Cp)
dCL = (KL a,CO2) ∗ (CL∗−CL) dt
(1)
2.5. Analytical techniques
(2)
2.5.1. Cell density, phosphate, urea and chlorophyll The biomass density was quantified by measuring optical density (OD) at 690 nm in a visible spectrophotometer (ESICO, India) “1 O.D = 0.4537 g dry cells l−1 (R2 = 0.99)”. The phosphate and urea in supernatant were estimated by ascorbic acid method (John, 1970) and diacetyl monoxime method (Momose et al., 1965) respectively. The chlorophyll was measured by methanol extraction (Pruvost et al., 2011). Detailed methodology is given in supplementary material.
where ‘Cp’ is probe reading at any time ‘t’, ‘CL’ is actual dCO2 concentration in bulk liquid and ‘kp’ is inverse of sensor time constant or response time (τp). After substituting the integrated form of Eq. (2) in Eq. (1), Eq. (1) was solved with initial and final conditions of ‘Cp’ as ‘Cpo’ and ‘CL∗’ respectively. The resulted expression (Eq. (3)) was used for estimation of ‘KL a,CO2 ’. Detailed experimental methodology is given in supplementary material.
KL a,CO2 ∗e−kp ∗ t kp ∗e−KL a,CO2 ∗t ⎫ − Cp = (CL∗) ∗ ⎧ ⎨ kp−KL a,CO2 ⎬ ⎭ ⎩ kp−KL aCO2 − ∗ k t p kp ∗e−KL a,CO2 ∗t ⎫ KL a,CO2 ∗e ⎧ − −(Cpo) ∗ ⎨ kp−KL a,CO2 kp−KL a,CO2 ⎬ ⎭ ⎩
2.5.2. Fatty acids extraction and estimation Intracellular fatty acids of FC2 culture were analyzed in terms of fatty acid methyl esters (FAMEs) using GC. The FAME extraction was carried out in hexane after direct transesterification of FC2 biomass using methanol (Kumar et al., 2014; Muthuraj et al., 2015). Detailed FAME extraction procedure and GC methodology were given in supplementary material.
(3)
2.4. Experiments 3. Results and discussions An optimum inoculum density of 0.07 g L−1 (see supplementary material) was used in all the conducted studies. Firstly, effect of constant light intensities were evaluated in the range of 106–2000 μE m−2 s−1 (basis: outdoor sunlight data). Secondly, three pH control strategies namely ‘full control’ (day: NaOH and night: CO2), ‘day control’ (day: NaOH and night:
3.1. Determination of KL a,CO2 for membrane sparger and conventional Lsparger The KL a,CO2 of membrane sparger and L-sparger were calculated. 2
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The mass transfer efficiency of membrane sparger (KL a,CO2 = 0.174 ± 0.006 ) was found 4.1 times of conventional L-sparger (KL a,CO2 = 0.0042 ± 0.0004 ). Note that the membrane sparger is disc type (dia: 69 mm) with thickness of 2 mm and L-sparger is around 70 mm with multiple holes of size around 0.5 mm. 3.2. Effect of constant light intensity on the growth of FC2 The saturated light intensity for the growth of FC2 was observed in a broad range of 700–1500 µE m−2 s−1 (Fig. 2B). Based on the specific growth rate of 1st day (light period), a clear photoinhibition was seen for 2000 µE m−2 s−1 light intensity. The photoinhibition was noticed reversible for the strain from 2nd day onwards and hence, an unusual and highest specific growth rate of ‘3.69 ± 0.009 day−1’ was observed (Fig. 2B). This could be due to available average light per cell in the PBR would have reached near to optimum value. In addition to the above reason, a special photo-protective mechanism (Tikkanen et al., 2014) could also be responsible for sudden appearance of an abnormally high specific growth rate in photoinhibited FC2 cells compared to the other light intensities. Total chlorophyll content was also satisfying the range of saturated photosynthesis, 700–1500 µE m−2 s−1 (Fig. 2A). Since there was no evidence of photoinhibition at 1130 µE m−2 s−1, this light intensity was chosen optimum for further studies. Moreover, highest final biomass titer (Fig. 2C) of 4.06 g L−1 was achieved at 1130 µE m−2 s−1. Note that this light intensity is close to the average daylight intensity range and much higher than typical optimum value, 200–300 µE m−2 s−1 (Dechatiwongse et al., 2014; Singh & Singh, 2015). 3.3. Analysis of pH control requirement for cultivation of FC2 In order to evaluate an appropriate pH control strategy, three pH control strategies were applied on FC2 culture. Since, HCl was unable to control alkaline pH in the night periods of culturing (data not shown), it was replaced with CO2 addition. The pH in full control and day control was maintained at 7.2 ± 0.1. In comparison to day control, full control strategy exhibited more biomass titer of 7.5 g L−1 (Fig. 3C). However, similar growth patterns were observed in both ‘full control’ and ‘no
Fig. 2. Biomass growth under various LED light intensities. A) Total chlorophyll B) Day wise (10 h) specific growth rates, and C) Final biomass titers and overall biomass productivities. CO2: 1% (v/v in air) and light:dark cycle: 10:14 h.
Fig. 3. Growth analysis of FC2 at various pH control strategies A) Biomass concentration profiles B) pH profile of ‘no control’ strategy C) Biomass concentration profiles at various pH setpoints using ‘full control strategy’. D) Urea and phosphate profiles of ‘no control’ strategy. Light intensity: 1130 µE m−2 s−1, CO2: 10% (v/v in air), light:dark cycle: 10:14 h.
3
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3.4. Evaluation of combinatorial effect of light and CO2 at various CO2 levels (%) In all the tested CO2 levels ranging from air (0.035%) to 15% (v/v), ∗ = 100 mg L−1) exhibited highest biomass cells grown at 2% CO2 (CCO 2 titer and overall biomass productivity of 4.33 g L−1 (Fig. 4A) and 1.08 g L−1 day−1 respectively. At 2% CO2, maximum specific growth rate (µmax) of 2.95 day−1 was observed on day 1. This was highest among all the individual µmax of other CO2 levels. On day 2, specific growth rates of 5% and 10% CO2, were observed higher than 2% CO2 (Fig. 4C). This could be either light limitation in 2% CO2 grown cells due to higher biomass or limitation of dCO2 concentration. Using BeerLambert’s light attenuation coefficient (Ka = 200 m2 kg−1) of Chlorella sp. light becomes almost zero (< 1% of incident light) after 35 mm (radius of PBR) when cell concentration reaches about 0.67 g L−1 (Ogbonna et al., 1995). However, continuously increasing cell density can also demand more CO2 at high light intensities. Due to this reason, CO2 levels (% in air) need to be elevated to maintain dCO2 as per increasing demand due to high light. At 1% CO2, dCO2 was decreased with time during the light periods, which implied that CO2 uptake rate for photosynthesis was more than the CO2 supply rate. In the case of 2% CO2, the dCO2 reached to steady state after certain hours of culturing (Fig. 4B). Where as in case of 5 and 10% CO2 levels, dCO2 was decreased during first 2 days (light period) and 1 day (light period) respectively. In the later stages of cultivation an increased dCO2 was observed mainly due to the domination of CO2 supply over decreasing total CO2 uptake rates. Cells grown at air and 15% CO2 were clearly exhibiting limited and inhibitive respectively. At 2% CO2 grown cells, steady state dCO2 was maintained at 83 mg L−1 consistently until 4.33 g L−1 biomass titer for 4 days. The steady state dCO2 concentration of 2% CO2 grown cells, 83 mg L−1 could drop to ∗ = 60 mg L−1) lesser value in large-scale cultivation. Since 1% CO2 (CCO 2 shown limiting to FC2 growth, the maintenance of constant dCO2 concentration at 100 mg/L was chosen as optimum CO2. 3.5. Growth and FAME analysis of FC2 culture under diurnal lighting pattern Automatic diurnal lighting and the resulted temperatures were given in Fig. 5C. To maintain dCO2 concentration at 100 ± 10 mg L−1, CO2 levels were adjusted in a discrete manner. The final biomass titer was observed as 5.79 g L−1 (Fig. 5B) with a biomass productivity of 1.29 g L−1 day−1, similar to 2% CO2 cultured cells at optimum light intensity 1130 µE m−2 s−1. The CO2 levels were increased (up to 3%) or decreased (up to 0.8%) depending on the light intensity (Fig. 5A and C). The reason would be an obvious increase/decrease in photosynthesis due to increase/decrease in light intensity. The low CO2 levels up to 0.8% were observed especially during low light intensities. The reason could be the release of CO2 along with regular consumption as equilibrium dCO2 con∗ centration (CCO ) of 100 mg L−1 corresponds to the 2% CO2 level. Since, 2 the final biomass titer was 5.79 g L−1, the growth of FC2 could not be limited by phosphate as it can grow until 7.4 g L−1 (Fig. 3D). Hence, it was assumed photorespiration in tandem with light limitation could be responsible for lesser biomass titer (Lloyd et al., 1977). For lipid induction, FC2 culture was transferred to urea-free culture medium. The final biomass titer was reduced to 5.08 g L−1 (explained in supplementary material). After 6 days of induction phase, total FAME content of 2.38 g L−1 (46.8% dry cell weight) was observed (Fig. 5D). The FAME productivities of 85 mg L−1 day−1 (for first 4 days) and 265 mg L−1 day−1 (for last 2 days) were noted. Thus, the total fatty acid production may increase furthermore if the study would be continued. The cetane number (CN) of the FAMEs was maintained in the range of 62–63 (see supplementary material). Apart from this, Fig. 5D was suggesting accumulation of key fatty acids (C16–C18) were only accounted (80.8 % of total FAME) for overall induction. Hence, the urea starvation improved both quality and quantity of biodiesel.
Fig. 4. Effect of CO2 levels (%) on growth of FC2 without pH control. A) Growth analysis of FC2 under various CO2 levels B) dCO2 concentrations profiles from 1 to 10 % CO2 levels C) Specific growth rates (10 h). Light intensity: 1130 µE m−2 s−1 and light:dark cycle: 10:14 h.
control’ strategies (Fig. 3A). This has suggested that FC2 might be capable of growing well in high range of acid-base environments, 4.6–8.6 pH (Fig. 3B and Fig. 3A). But for more clearance of above results, FC2 was also tested under three pH control set points, 5.9, 7.2 and 8.8 using ‘full control’ strategy. The pH of 7.2 was evaluated as optimum (Fig. 3B). Hence, the FC2 strain can decrease the cost of the process technology as it can grow without pH control. 4
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Fig. 5. Growth characteristics and FAME profiles of FC2 grown under diurnal lighting. A) CO2 level (%) profiles and dCO2 concentration. B) Biomass concentration and specific growth rates. C) Diurnal light intensity profile on the surface of CM-PBR and resulted temperatures. D) FAME analysis during the lipid induction phase. Light:dark cycle: 12:12 h. The pH was not controlled.
system was designed for FC2 growth under diurnal LED. The effect of light intensity, pH control strategies and CO2 levels were studied to develop a CO2 feeding strategy without pH control under diurnal lighting. The biomass and FAME production were 5.79 g L−1 in 4.5 days (growth-phase) and 2.38 g L−1 in 6 days (induction-phase) respectively. Being a qualitative biodiesel candidate, FC2 has also proved its novelty by showing high saturated light intensity, reversible photoinhibition, no pH control requirement and dark-phase growth.
Table 1 Dark phase growth of Chlorella sp. FC2 IITG in various studies. Experimental study
Variation of light intensities (µE m−2 s−1), 1% CO2, 4 days 106 700 1130 1500 2000
Overall night Biomass gain (g L−1)
0.14 0.80 0.93 0.70 0
Acknowledgement
Variation of CO2 levels (% in air) at constant light 1130 µE m−2 s−1, 4 days AIR 0.34 1 0.89 2 1.23 5 0.70 10 0.50 15 0.19 Diurnal LED lighting (100 ppm CO2, 0.8–3%), 4.5 days 0.88 0.82 Natural sunlight, 1% CO2 (3 days, September 2016, mixture of clear, cloudy and rainy daylights)
Authors would like to thanks to IIT Guwahati for supporting the fund for this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2017.11.087. References Chisholm, S.W., 1981. Temporal patterns of cell-division in unicellular algae. Can. Bull. Fish. Aquat. Sci. 210, 150–181. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Dechatiwongse, P., Srisamai, S., Maitland, G., Hellgardt, K., 2014. Effects of light and temperature on the photoautotrophic growth and photoinhibition of nitrogen-fixing cyanobacterium Cyanothece sp ATCC 51142. Algal Res. 5, 103–111. John, M.K., 1970. Colorimetric determination of phosphorus in soil and plant materials with ascorbic acid. Soil Sci. 109 (4), 214–220. Kumar, V., Muthuraj, M., Palabhanvi, B., Ghoshal, A.K., Das, D., 2014. Evaluation and optimization of two stage sequential in situ transesterification process for fatty acid methyl ester quantification from microalgae. Renewable Energy 68, 560–569. Lloyd, N.D.H., Canvin, D.T., Culver, D.A., 1977. Photosynthesis and Photorespiration in Algae. Plant Physiol. 59 (5), 936–940. Momose, T., Ohkura, Y., Tomita, J., 1965. Determination of urea in blood and urine with diacetyl monoxime-glucuronolactone reagent. Clin. Chem. 11 (2), 113–121. Monyem, A., Van Gerpen, J.H., Canakci, M., 2001. The effect of timing and oxidation on emissions from biodiesel-fueled engines. Trans. ASAE 44 (1), 35–42. Murata, N., Takahashi, S., Nishiyama, Y., Allakhverdiev, S.I., 2007. Photoinhibition of photosystem II under environmental stress. Biochim. Biophys. Acta -Bioenerg. 1767 (6), 414–421. Muthuraj, M., Chandra, N., Palabhanvi, B., Kumar, V., Das, D., 2015. Process engineering for high-cell-density cultivation of lipid rich microalgal biomass of Chlorella sp FC2 IITG. Bioenergy Res. 8 (2), 726–739. Muthuraj, M., Kumar, V., Palabhanvi, B., Das, D., 2014. Evaluation of indigenous microalgal isolate Chlorella sp FC2 IITG as a cell factory for biodiesel production and scale up in outdoor conditions. J. Ind. Microbiol. Biotechnol. 41 (3), 499–511. Ogbonna, J.C., Yada, H., Tanaka, H., 1995. Light supply coefficient – a new engineering parameter for photobioreactor design. J. Ferment. Bioeng. 80 (4), 369–376. Philichi, T.L., Stenstrom, M.K., 1989. Effects of dissolved-oxygen probe lag on oxygen-
3.6. Growth analysis in dark side of culturing Night biomass loss due to respiration remained as most common problem among the recent algal researchers. Present studies shown a fascinating night growth phenomenon during the dark-phase (Table. 1). Excluding the study of FC2 growth at highest light intensity (2000 µE m−2 s−1), night growth was observed in all the studies. However, better night growths were observed at suboptimal and optimal light levels. Notably, 80–90% of dark-phase growth was observed in the first 5 h of the 14 h dark period. As the saturated light intensity was very much high and broad range of 700–1500 µE m−2 s−1, the light energy above 700 µE m−2 s−1 may be responsible for generation of extra energy pools and carrying to dark phase, resulting growth for few hours (Chisholm, 1981). Thus, night growth may be dominating the loss due to respiration. Most of the species reported in the literature showing light saturation at very low and narrow range (200–300 µE m−2 s−1) compared to FC2. Hence, the scope of extra energy pools may be reduced and loss may be more dominating. 4. Conclusions An efficient cylindrical photobioreactor with membrane-sparging 5
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species: a review. Renewable Sustainable Energy Rev. 50, 431–444. Tikkanen, M., Mekala, N.R., Aro, E.M., 2014. Photosystem II photoinhibition-repair cycle protects Photosystem I from irreversible damage. Biochim. Biophys. Acta Bioenerg. 1837 (1), 210–215. Wang, S.K., Stiles, A.R., Guo, C., Liu, C.Z., 2014. Microalgae cultivation in photobioreactors: An overview of light characteristics. Eng. Life Sci. 14 (6), 550–559.
transfer parameter-estimation. J. Water Pollut. Control Fed. 61 (1), 83–86. Pierobon, S.C., Riordon, J., Nguyen, B., Sinton, D., 2016. Breathable waveguides for combined light and CO2 delivery to microalgae. Bioresour. Technol. 209, 391–396. Pruvost, J., Van Vooren, G., Le Gouic, B., Couzinet-Mossion, A., Legrand, J., 2011. Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application. Bioresour. Technol. 102 (1), 150–158. Singh, S.P., Singh, P., 2015. Effect of temperature and light on the growth of algae
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Designing a CO2 supply strategy for microalgal biodiesel production under diurnal light in a cylindrical-membrane photobioreactor Venkateswara R. Naira, Debasish Das, Soumen K. Maiti
⁎
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Cylindrical membrane photobioreactor Diurnal light FAME production Optimal CO2 supply Reversible photoinhibition Dark-phase growth
A cylindrical membrane photobioreactor with high CO2 mass transfer coefficient was designed and installed under customized unidirectional lighting. Combinatorial effect of light and CO2 on the growth of Chlorella sp. FC2 IITG was studied and an optimal CO2 supply without pH control strategy was developed under diurnal light similar to sunlight (17-2000-17 µE m−2 s−1). Unprecedentedly, broad range of saturated light levels (700–1500 µE m−2 s−1), reversible photoinhibition, no pH control requirement and dark-phase growth were noticed altogether in the strain. Under diurnal light, final biomass titer of 5.79 g L−1 and overall biomass productivity of 1.29 g L−1 day−1 were observed. The results were similar to optimal light (1130 µE m−2 s−1) and CO2 (2%) conditions. Subsequently, a highest FAME productivity of 265 mg L−1 day−1 was observed in last two days of lipid induction phase.
1. Introduction Microalgae remarked as an excellent autotrophic source for biodiesel production due to their high oil yielding capacities (Chisti, 2007). Additionally, microalgae mitigates the greenhouse effect by fixing CO2 and produces environmental friendly biodiesel compared to conventional diesel (Monyem et al., 2001). The microalgal culturing technology is mainly governed by light and CO2. At high light intensities, photoinhibition takes place due to production of reactive oxygen species (Murata et al., 2007). On other hand, concentration of dissolved CO2 (dCO2) in the culture medium has to be modulated to overcome limiting and inhibiting concentrations of CO2. However, overall CO2 mass transfer coefficient is the preliminary requirement to maintain desired dCO2 concentration. Above all, the demand of CO2 is depends on the light availability for the algal cells. Thus, the combinatorial effect of light and CO2 has to be considered for designing a cultivation technology (Pierobon et al., 2016). Apart from this, high concentration of dCO2 could inhibit the growth of algae due to two possible factors: lowered pH levels and usual substrate inhibition. Thus, it is very crucial to determine appropriate controlling pH before the evaluation of CO2 effect. In the present study, a cylindrical membrane photobioreactor (CMPBR) with a custom-made membrane sparger without agitation was designed. Since, algae cultivation under sunlight is the only feasible method (Wang et al., 2014), studies under diurnal lighting similar to outdoor sunlight is necessary. An LED lighting with computer-
⁎
dimmable technology was designed to mimic diurnal patterns similar to sunlight. In the CM-PBR, studies have mainly emphasized on pH control requirement and evaluation of inhibiting and limiting effects of both light and CO2 in a combinatorial manner for growth of microalgae. An optimal CO2 supply strategy was developed for maximizing biomass and lipid productivities grown under diurnal light cycle in the CM-PBR. 2. Materials and methods 2.1. Microorganism and culture medium Chlorella sp. FC2 IITG (referred as FC2), an indigenous novel microalgal isolate previously for biodiesel production (Muthuraj et al., 2014) was used in the studies. Culturing medium was modified BG11 medium with pH, 7.1 (Muthuraj et al., 2015). 2.2. Photobioreactor design, instrumentation and control Flanged cylindrical acrylic tube (O.D.: 75 mm, I.D.: 69 mm, H: 175 mm) with a low-cost membrane sparger was designed as CM-PBR (Fig. 1). A neoprene rubber sheet (thickness: 2 mm), made porous by 30 gauge fine needle was used as membrane sparger. Mixture of air and CO2 (2 bar pressure) was sparged from the bottom of CM-PBR. An LED (cool white) panel (3 × 50 W per reactor, 6500 K color temperature) was installed perpendicular to surface (one side) of reactor. Light (lux) was measured using a lux meter (Sigma Instruments, India). SNAP PAC
Corresponding author. E-mail address: [email protected] (S.K. Maiti).
https://doi.org/10.1016/j.biortech.2017.11.087 Received 18 October 2017; Received in revised form 25 November 2017; Accepted 27 November 2017 0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Naira, V.R., Bioresource Technology (2017), https://doi.org/10.1016/j.biortech.2017.11.087
Bioresource Technology xxx (xxxx) xxx–xxx
V.R. Naira et al.
Fig. 1. Schematic of the parallel cylindrical membrane photobioreactor (CM-PBR) system.
pH was not controlled) and ‘no control’ (pH was not controlled both in day and night) were tested at 10% CO2 level and constant light intensity. Thirdly, the growth of FC2 was investigated under constant supply of CO2 levels (% in air, v/v) ranging from atmospheric air to 15%. A constant light intensity of 1130 μE m−2 s−1 was used. Finally, FC2 growth characteristics and its FAME production were studied under simulated diurnal LED lighting that mimics outdoor sunlight (17–2000–17 μE m−2 s−1, light:dark cycle of 12:12 h). For lipid induction, FC2 cells were transferred to urea-free culture medium. A dCO2 concentration of 100 ± 10 mg L−1 was maintained throughout the culture. All studies were performed in batch and temperature was not controlled in any of the studies. During night periods, CO2 flow was stopped in all the studies (exception: ‘full control’ pH control strategy) and air flow remained same to ensure good mixing of algal cells without any settling.
control and data acquisition system (OPTO 22, USA) was used to control pH via online pH analyzer (Zest Engg., India) and LED light intensity via pulse width modulation technology (Mouser electronics, USA) (Fig. 1). The dCO2 concentration was measured using online dCO2 analyzer (Cole-Parmer). 2.3. Estimation of probe response time (τp) and overall mass transfer coefficient (KL a,CO2 ) Considering the probe response as first order process (Eq. (1)) (Philichi & Stenstrom, 1989), simple dynamic method in cell-free media (Eq. (2)) was used to estimate ‘KL a,CO2 ’ of membrane sparger and conventional L-sparger (Applikon Biotechnology B.V., Netherlands).
dCp dt
= kp ∗ (CL−Cp)
dCL = (KL a,CO2) ∗ (CL∗−CL) dt
(1)
2.5. Analytical techniques
(2)
2.5.1. Cell density, phosphate, urea and chlorophyll The biomass density was quantified by measuring optical density (OD) at 690 nm in a visible spectrophotometer (ESICO, India) “1 O.D = 0.4537 g dry cells l−1 (R2 = 0.99)”. The phosphate and urea in supernatant were estimated by ascorbic acid method (John, 1970) and diacetyl monoxime method (Momose et al., 1965) respectively. The chlorophyll was measured by methanol extraction (Pruvost et al., 2011). Detailed methodology is given in supplementary material.
where ‘Cp’ is probe reading at any time ‘t’, ‘CL’ is actual dCO2 concentration in bulk liquid and ‘kp’ is inverse of sensor time constant or response time (τp). After substituting the integrated form of Eq. (2) in Eq. (1), Eq. (1) was solved with initial and final conditions of ‘Cp’ as ‘Cpo’ and ‘CL∗’ respectively. The resulted expression (Eq. (3)) was used for estimation of ‘KL a,CO2 ’. Detailed experimental methodology is given in supplementary material.
KL a,CO2 ∗e−kp ∗ t kp ∗e−KL a,CO2 ∗t ⎫ − Cp = (CL∗) ∗ ⎧ ⎨ kp−KL a,CO2 ⎬ ⎭ ⎩ kp−KL aCO2 − ∗ k t p kp ∗e−KL a,CO2 ∗t ⎫ KL a,CO2 ∗e ⎧ − −(Cpo) ∗ ⎨ kp−KL a,CO2 kp−KL a,CO2 ⎬ ⎭ ⎩
2.5.2. Fatty acids extraction and estimation Intracellular fatty acids of FC2 culture were analyzed in terms of fatty acid methyl esters (FAMEs) using GC. The FAME extraction was carried out in hexane after direct transesterification of FC2 biomass using methanol (Kumar et al., 2014; Muthuraj et al., 2015). Detailed FAME extraction procedure and GC methodology were given in supplementary material.
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2.4. Experiments 3. Results and discussions An optimum inoculum density of 0.07 g L−1 (see supplementary material) was used in all the conducted studies. Firstly, effect of constant light intensities were evaluated in the range of 106–2000 μE m−2 s−1 (basis: outdoor sunlight data). Secondly, three pH control strategies namely ‘full control’ (day: NaOH and night: CO2), ‘day control’ (day: NaOH and night:
3.1. Determination of KL a,CO2 for membrane sparger and conventional Lsparger The KL a,CO2 of membrane sparger and L-sparger were calculated. 2
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The mass transfer efficiency of membrane sparger (KL a,CO2 = 0.174 ± 0.006 ) was found 4.1 times of conventional L-sparger (KL a,CO2 = 0.0042 ± 0.0004 ). Note that the membrane sparger is disc type (dia: 69 mm) with thickness of 2 mm and L-sparger is around 70 mm with multiple holes of size around 0.5 mm. 3.2. Effect of constant light intensity on the growth of FC2 The saturated light intensity for the growth of FC2 was observed in a broad range of 700–1500 µE m−2 s−1 (Fig. 2B). Based on the specific growth rate of 1st day (light period), a clear photoinhibition was seen for 2000 µE m−2 s−1 light intensity. The photoinhibition was noticed reversible for the strain from 2nd day onwards and hence, an unusual and highest specific growth rate of ‘3.69 ± 0.009 day−1’ was observed (Fig. 2B). This could be due to available average light per cell in the PBR would have reached near to optimum value. In addition to the above reason, a special photo-protective mechanism (Tikkanen et al., 2014) could also be responsible for sudden appearance of an abnormally high specific growth rate in photoinhibited FC2 cells compared to the other light intensities. Total chlorophyll content was also satisfying the range of saturated photosynthesis, 700–1500 µE m−2 s−1 (Fig. 2A). Since there was no evidence of photoinhibition at 1130 µE m−2 s−1, this light intensity was chosen optimum for further studies. Moreover, highest final biomass titer (Fig. 2C) of 4.06 g L−1 was achieved at 1130 µE m−2 s−1. Note that this light intensity is close to the average daylight intensity range and much higher than typical optimum value, 200–300 µE m−2 s−1 (Dechatiwongse et al., 2014; Singh & Singh, 2015). 3.3. Analysis of pH control requirement for cultivation of FC2 In order to evaluate an appropriate pH control strategy, three pH control strategies were applied on FC2 culture. Since, HCl was unable to control alkaline pH in the night periods of culturing (data not shown), it was replaced with CO2 addition. The pH in full control and day control was maintained at 7.2 ± 0.1. In comparison to day control, full control strategy exhibited more biomass titer of 7.5 g L−1 (Fig. 3C). However, similar growth patterns were observed in both ‘full control’ and ‘no
Fig. 2. Biomass growth under various LED light intensities. A) Total chlorophyll B) Day wise (10 h) specific growth rates, and C) Final biomass titers and overall biomass productivities. CO2: 1% (v/v in air) and light:dark cycle: 10:14 h.
Fig. 3. Growth analysis of FC2 at various pH control strategies A) Biomass concentration profiles B) pH profile of ‘no control’ strategy C) Biomass concentration profiles at various pH setpoints using ‘full control strategy’. D) Urea and phosphate profiles of ‘no control’ strategy. Light intensity: 1130 µE m−2 s−1, CO2: 10% (v/v in air), light:dark cycle: 10:14 h.
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3.4. Evaluation of combinatorial effect of light and CO2 at various CO2 levels (%) In all the tested CO2 levels ranging from air (0.035%) to 15% (v/v), ∗ = 100 mg L−1) exhibited highest biomass cells grown at 2% CO2 (CCO 2 titer and overall biomass productivity of 4.33 g L−1 (Fig. 4A) and 1.08 g L−1 day−1 respectively. At 2% CO2, maximum specific growth rate (µmax) of 2.95 day−1 was observed on day 1. This was highest among all the individual µmax of other CO2 levels. On day 2, specific growth rates of 5% and 10% CO2, were observed higher than 2% CO2 (Fig. 4C). This could be either light limitation in 2% CO2 grown cells due to higher biomass or limitation of dCO2 concentration. Using BeerLambert’s light attenuation coefficient (Ka = 200 m2 kg−1) of Chlorella sp. light becomes almost zero (< 1% of incident light) after 35 mm (radius of PBR) when cell concentration reaches about 0.67 g L−1 (Ogbonna et al., 1995). However, continuously increasing cell density can also demand more CO2 at high light intensities. Due to this reason, CO2 levels (% in air) need to be elevated to maintain dCO2 as per increasing demand due to high light. At 1% CO2, dCO2 was decreased with time during the light periods, which implied that CO2 uptake rate for photosynthesis was more than the CO2 supply rate. In the case of 2% CO2, the dCO2 reached to steady state after certain hours of culturing (Fig. 4B). Where as in case of 5 and 10% CO2 levels, dCO2 was decreased during first 2 days (light period) and 1 day (light period) respectively. In the later stages of cultivation an increased dCO2 was observed mainly due to the domination of CO2 supply over decreasing total CO2 uptake rates. Cells grown at air and 15% CO2 were clearly exhibiting limited and inhibitive respectively. At 2% CO2 grown cells, steady state dCO2 was maintained at 83 mg L−1 consistently until 4.33 g L−1 biomass titer for 4 days. The steady state dCO2 concentration of 2% CO2 grown cells, 83 mg L−1 could drop to ∗ = 60 mg L−1) lesser value in large-scale cultivation. Since 1% CO2 (CCO 2 shown limiting to FC2 growth, the maintenance of constant dCO2 concentration at 100 mg/L was chosen as optimum CO2. 3.5. Growth and FAME analysis of FC2 culture under diurnal lighting pattern Automatic diurnal lighting and the resulted temperatures were given in Fig. 5C. To maintain dCO2 concentration at 100 ± 10 mg L−1, CO2 levels were adjusted in a discrete manner. The final biomass titer was observed as 5.79 g L−1 (Fig. 5B) with a biomass productivity of 1.29 g L−1 day−1, similar to 2% CO2 cultured cells at optimum light intensity 1130 µE m−2 s−1. The CO2 levels were increased (up to 3%) or decreased (up to 0.8%) depending on the light intensity (Fig. 5A and C). The reason would be an obvious increase/decrease in photosynthesis due to increase/decrease in light intensity. The low CO2 levels up to 0.8% were observed especially during low light intensities. The reason could be the release of CO2 along with regular consumption as equilibrium dCO2 con∗ centration (CCO ) of 100 mg L−1 corresponds to the 2% CO2 level. Since, 2 the final biomass titer was 5.79 g L−1, the growth of FC2 could not be limited by phosphate as it can grow until 7.4 g L−1 (Fig. 3D). Hence, it was assumed photorespiration in tandem with light limitation could be responsible for lesser biomass titer (Lloyd et al., 1977). For lipid induction, FC2 culture was transferred to urea-free culture medium. The final biomass titer was reduced to 5.08 g L−1 (explained in supplementary material). After 6 days of induction phase, total FAME content of 2.38 g L−1 (46.8% dry cell weight) was observed (Fig. 5D). The FAME productivities of 85 mg L−1 day−1 (for first 4 days) and 265 mg L−1 day−1 (for last 2 days) were noted. Thus, the total fatty acid production may increase furthermore if the study would be continued. The cetane number (CN) of the FAMEs was maintained in the range of 62–63 (see supplementary material). Apart from this, Fig. 5D was suggesting accumulation of key fatty acids (C16–C18) were only accounted (80.8 % of total FAME) for overall induction. Hence, the urea starvation improved both quality and quantity of biodiesel.
Fig. 4. Effect of CO2 levels (%) on growth of FC2 without pH control. A) Growth analysis of FC2 under various CO2 levels B) dCO2 concentrations profiles from 1 to 10 % CO2 levels C) Specific growth rates (10 h). Light intensity: 1130 µE m−2 s−1 and light:dark cycle: 10:14 h.
control’ strategies (Fig. 3A). This has suggested that FC2 might be capable of growing well in high range of acid-base environments, 4.6–8.6 pH (Fig. 3B and Fig. 3A). But for more clearance of above results, FC2 was also tested under three pH control set points, 5.9, 7.2 and 8.8 using ‘full control’ strategy. The pH of 7.2 was evaluated as optimum (Fig. 3B). Hence, the FC2 strain can decrease the cost of the process technology as it can grow without pH control. 4
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Fig. 5. Growth characteristics and FAME profiles of FC2 grown under diurnal lighting. A) CO2 level (%) profiles and dCO2 concentration. B) Biomass concentration and specific growth rates. C) Diurnal light intensity profile on the surface of CM-PBR and resulted temperatures. D) FAME analysis during the lipid induction phase. Light:dark cycle: 12:12 h. The pH was not controlled.
system was designed for FC2 growth under diurnal LED. The effect of light intensity, pH control strategies and CO2 levels were studied to develop a CO2 feeding strategy without pH control under diurnal lighting. The biomass and FAME production were 5.79 g L−1 in 4.5 days (growth-phase) and 2.38 g L−1 in 6 days (induction-phase) respectively. Being a qualitative biodiesel candidate, FC2 has also proved its novelty by showing high saturated light intensity, reversible photoinhibition, no pH control requirement and dark-phase growth.
Table 1 Dark phase growth of Chlorella sp. FC2 IITG in various studies. Experimental study
Variation of light intensities (µE m−2 s−1), 1% CO2, 4 days 106 700 1130 1500 2000
Overall night Biomass gain (g L−1)
0.14 0.80 0.93 0.70 0
Acknowledgement
Variation of CO2 levels (% in air) at constant light 1130 µE m−2 s−1, 4 days AIR 0.34 1 0.89 2 1.23 5 0.70 10 0.50 15 0.19 Diurnal LED lighting (100 ppm CO2, 0.8–3%), 4.5 days 0.88 0.82 Natural sunlight, 1% CO2 (3 days, September 2016, mixture of clear, cloudy and rainy daylights)
Authors would like to thanks to IIT Guwahati for supporting the fund for this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2017.11.087. References Chisholm, S.W., 1981. Temporal patterns of cell-division in unicellular algae. Can. Bull. Fish. Aquat. Sci. 210, 150–181. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Dechatiwongse, P., Srisamai, S., Maitland, G., Hellgardt, K., 2014. Effects of light and temperature on the photoautotrophic growth and photoinhibition of nitrogen-fixing cyanobacterium Cyanothece sp ATCC 51142. Algal Res. 5, 103–111. John, M.K., 1970. Colorimetric determination of phosphorus in soil and plant materials with ascorbic acid. Soil Sci. 109 (4), 214–220. Kumar, V., Muthuraj, M., Palabhanvi, B., Ghoshal, A.K., Das, D., 2014. Evaluation and optimization of two stage sequential in situ transesterification process for fatty acid methyl ester quantification from microalgae. Renewable Energy 68, 560–569. Lloyd, N.D.H., Canvin, D.T., Culver, D.A., 1977. Photosynthesis and Photorespiration in Algae. Plant Physiol. 59 (5), 936–940. Momose, T., Ohkura, Y., Tomita, J., 1965. Determination of urea in blood and urine with diacetyl monoxime-glucuronolactone reagent. Clin. Chem. 11 (2), 113–121. Monyem, A., Van Gerpen, J.H., Canakci, M., 2001. The effect of timing and oxidation on emissions from biodiesel-fueled engines. Trans. ASAE 44 (1), 35–42. Murata, N., Takahashi, S., Nishiyama, Y., Allakhverdiev, S.I., 2007. Photoinhibition of photosystem II under environmental stress. Biochim. Biophys. Acta -Bioenerg. 1767 (6), 414–421. Muthuraj, M., Chandra, N., Palabhanvi, B., Kumar, V., Das, D., 2015. Process engineering for high-cell-density cultivation of lipid rich microalgal biomass of Chlorella sp FC2 IITG. Bioenergy Res. 8 (2), 726–739. Muthuraj, M., Kumar, V., Palabhanvi, B., Das, D., 2014. Evaluation of indigenous microalgal isolate Chlorella sp FC2 IITG as a cell factory for biodiesel production and scale up in outdoor conditions. J. Ind. Microbiol. Biotechnol. 41 (3), 499–511. Ogbonna, J.C., Yada, H., Tanaka, H., 1995. Light supply coefficient – a new engineering parameter for photobioreactor design. J. Ferment. Bioeng. 80 (4), 369–376. Philichi, T.L., Stenstrom, M.K., 1989. Effects of dissolved-oxygen probe lag on oxygen-
3.6. Growth analysis in dark side of culturing Night biomass loss due to respiration remained as most common problem among the recent algal researchers. Present studies shown a fascinating night growth phenomenon during the dark-phase (Table. 1). Excluding the study of FC2 growth at highest light intensity (2000 µE m−2 s−1), night growth was observed in all the studies. However, better night growths were observed at suboptimal and optimal light levels. Notably, 80–90% of dark-phase growth was observed in the first 5 h of the 14 h dark period. As the saturated light intensity was very much high and broad range of 700–1500 µE m−2 s−1, the light energy above 700 µE m−2 s−1 may be responsible for generation of extra energy pools and carrying to dark phase, resulting growth for few hours (Chisholm, 1981). Thus, night growth may be dominating the loss due to respiration. Most of the species reported in the literature showing light saturation at very low and narrow range (200–300 µE m−2 s−1) compared to FC2. Hence, the scope of extra energy pools may be reduced and loss may be more dominating. 4. Conclusions An efficient cylindrical photobioreactor with membrane-sparging 5
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species: a review. Renewable Sustainable Energy Rev. 50, 431–444. Tikkanen, M., Mekala, N.R., Aro, E.M., 2014. Photosystem II photoinhibition-repair cycle protects Photosystem I from irreversible damage. Biochim. Biophys. Acta Bioenerg. 1837 (1), 210–215. Wang, S.K., Stiles, A.R., Guo, C., Liu, C.Z., 2014. Microalgae cultivation in photobioreactors: An overview of light characteristics. Eng. Life Sci. 14 (6), 550–559.
transfer parameter-estimation. J. Water Pollut. Control Fed. 61 (1), 83–86. Pierobon, S.C., Riordon, J., Nguyen, B., Sinton, D., 2016. Breathable waveguides for combined light and CO2 delivery to microalgae. Bioresour. Technol. 209, 391–396. Pruvost, J., Van Vooren, G., Le Gouic, B., Couzinet-Mossion, A., Legrand, J., 2011. Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application. Bioresour. Technol. 102 (1), 150–158. Singh, S.P., Singh, P., 2015. Effect of temperature and light on the growth of algae
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