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Gravimetric enrichment of high lipid and starch accumulating microalgae
Accepted Manuscript Gravimetric Enrichment of High Lipid and Starch Accumulating Microalgae Morteza Hassanpour, Mahsa Abbasabadi, Sirous Ebrahimi, Maryam Hosseini, Ahmad Sheikhbaglou PII: DOI: Reference:
S0960-8524(15)01008-1 http://dx.doi.org/10.1016/j.biortech.2015.07.046 BITE 15279
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
24 May 2015 14 July 2015 15 July 2015
Please cite this article as: Hassanpour, M., Abbasabadi, M., Ebrahimi, S., Hosseini, M., Sheikhbaglou, A., Gravimetric Enrichment of High Lipid and Starch Accumulating Microalgae, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.07.046
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gravimetric Enrichment of High Lipid and Starch Accumulating Microalgae Morteza Hassanpour, Mahsa Abbasabadi, Sirous Ebrahimi,*, Maryam Hosseini, Ahmad Sheikhbaglou Biotechnology Research Center, Faculty of Chemical Engineering, Sahand University of Technology, Tabriz, Iran,
*
Corresponding author: Sirous Ebrahimi
E-mail: [email protected] Tel: +98 41 33458180; Fax: +98 41 33444355
Abstract This study presents gravimetric enrichment of mixed culture to screen starch and lipid producing species separately in a sequencing batch reactor. In the enriched starch-producing mixed culture photobioreactor, the starch content at the end of steady state batch became 3.42 times the beginning of depletion. Whereas in the enriched lipid-producing photobioreactor, the lipid content at the end of steady state batch became 3 times the beginning of famine phase. The obtained results revealed that the gravimetric enrichment is a suitable screening method for specific production of storage compounds in none-sterile large-scaled condition. Keywords: Microalgae; Starch; Lipid; Biofuel; Gravimetric enrichment; Sequencing batch reactor 1. Introduction Increasing world population and fossil fuels depletion have increased global concerns about future. Greenhouse gas emissions and climate changes have induced attention to sustainable energy supplies like biofuels. Some characteristics such as high growth rate, arable land and potable water independency and storing of starch and triacylglycerol (TAG) have introduced microalgae as the most promising feedstock for future biofuel production (Chisti 2007 and Menetrez 2014). Starch can be used as fermentation feedstock for bioethanol production and TAG can be converted to biodiesel after an esterification step (Li et al. 2008 and Beetul et al. 2014). Selecting an appropriate species of microalgae is the first and most important step through the whole process of production of storage compounds (Mooij et al. 2013 and Talebi et al. 2014).
This selection is based on high storage capacity or high productivity. Many microalgae such as Chlorella species, Neochloris oleoabundans, Dunaliella species, Tetraselmis subcordiformis and Arthrospira platensis accumulate high levels of starch and TAG under nutrient-limiting conditions (Millán-Oropeza et al. 2015). For instance, in marine green microalga Tetraselmis subcordiformis, starch accumulated up to 62.1 and 54.3 % of dry weight (DW) under sulfur and nitrogen deprived conditions, respectively (Yao et al. 2012). The starch content of freshwater alga Chlorella vulgaris was reported to be 60, 55 and 38% DW in sulfur, phosphorus and nitrogen limitations, respectively (Brányiková et al. 2011). Marine microalga eustigmatophyte Nannochloropsis sp. attained 60% DW lipid content when was cultivated under nitrogen deprivation in 0.6-L bubbled tubes (Rodolfi et al. 2009). Selection of a pure culture will increase system contamination likelihood to invading species that can lead to lower production efficiency. Cultivation of microalgae in pure culture will require an aseptic process condition resulting in a high investment and operating cost. Such preparations present serious barriers to large scale application (Mooij et al. 2013). In addition, even in specific environmental conditions such as high salinity for Dunaliella Salina cultivation, where contamination likelihood is low, strain evolution remains as a great threat, since mutated strains with higher growth rate are able to outcompete the desired strain leading to a drop in productivity (Mooij et al. 2015). As an alternative to pure culture, the application of a mixed-culture-based process would be a technically attractive and an economically feasible solution (Reis et al. 2003). Screening mixed culture based on ecological role of product, would be a good solution to overcome cultivation contamination risk (Mooij et al. 2013). Exploring microbial diversity and operation in various conditions are the main advantages of using mixed culture under selective environment (Johnson
et al. 2009, Kleerebezem and van Loosdrecht 2007). Research in a semi-continuous process based on ecological role of accumulated fixed carbon resulted in the enrichment of microalgae with high levels of internal storage compounds. In that study, a sequencing batch mode of operation was adopted with cycles of 24 hours with a light and dark period in which nitrogen source was supplied only during the dark period of cycles. Therefore, only those species survived that were able to take up CO2 in light periods; store fixed carbon as internal storage compounds; and use it to uptake nitrogen source during the dark period (Mooij et al. 2013). Despite encouraging results, the applied approach does not provide a lipid/starch selective accumulation. Therefore, production of specific storage compound such as starch or TAG still remains a major challenge. Storage starch and TAG have density of approximately 1.5 and 0.95 g/mL, respectively (SalesCruz et al. 2010). The difference between starch and TAG densities imposes a selective environment based on buoyancy to screen starch and TAG accumulating species in mixed culture. Therefore, the aim of this study was to investigate screening starch and TAG accumulating species in a mixed culture in sequencing batch reactor (SBR) systems directing more photosynthetic carbon partition to either starch or lipid. It was expected that high starchstoring species can settle rapidly to the bottom and high TAG storing species can stay in supernatant of the cell suspension regarded to their cell density. Therefore, in the presented study for the first time gravimetric enrichment strategy was applied to screen starch and TAG accumulating species. 2. Materials and methods 2.1.
Experimental set-up and operation
In order to screen the lightest and densest species, two 2 L none-sterile glass bioreactors (Takbiotech, Iran) were operated in SBR mode with 0.8 L working volume. The operating conditions are as follows: Cycle length, 6 day, 250 lux constant light provided by LED lamps, aeration rate of 1 vvm with 5% CO2 and stirrer speed 200 rpm. The pH of the bioreactor was controlled at 7.5 ± 0.1 by addition of 1 M HCl or 1 M NaHCO3, using a pH transmitter (2500, Mettler Toledo, Switzerland). Temperature was kept at 30 ± 1°C by using a water bath circulator with a heating system (UC4500, Sahandazar, Iran). A modified Combo-medium was used as nutrients (Kilham et al. 1998). No KCl, NaHCO3, vitamins and animal trace elements were added. Nitrogen source was 14 mg/L and other nutrients were supplied by a factor of three. The bioreactor was inoculated with 100 mL of combined samples of ponds and springs water which was cultivated in a glass bottle with the medium described above. In order to screen starch and TAG accumulating species, after 3 min settling, 30% of the bottom and supernatant of the bioreactor content was transferred to the next two batches as inoculums, respectively. The samples were taken regularly every week from both photobioreactors to observe the changes in microalgal population using a Leica DMLS light microscope (Leica Microsystems, Wetzlar GmbH, Germany). 2.2.
Biomass determination
It was assumed that cells contain four general classes of macromolecules: protein, polysaccharides, lipid, and nucleic acids. Total dry weight was determined by centrifugation of 40 mL of cell suspension at 5000 g for 5 min, decanting supernatant, washing the residues with distilled water, and drying at 105 °C for 24 h at the end of feast phase. Ash content was determined by incineration of dry biomass at 550 °C for 1 h. Volatile suspended solids amount (VSS) was calculated by difference between ash content and total dry weight. The difference
between the sum of protein, starch, lipid, and VSS at the end of feast phase was considered as nucleic acids. There was no cell division in famine phase. Thus, the amount of nucleic acids was constant, and the VSS was calculated as sum of starch, lipid, proteins, and nucleic acids theoretically in the other batch times. 2.3.
Starch determination
A modified method of Mc Cready et al. was used for starch determination (McCready et al. 1950). One mL of the cell suspension was centrifuged at 5000 g for 5 min. The pellets were collected. 6 mL of 96% ethanol was added and heated at 80 °C for 10 min to remove pigments. The extracts were centrifuged and supernatant was decanted. The retained pellets were dried over water bath. One mL of 50% perchloric acid was added to the pellets and incubated for 30 min at 25 °C in incubator shaking at 150 rpm. Four mL of distilled water was added to the sample, centrifuged at 3000 g for 5 min, pipetted out 1 mL of supernatant to Teflon-covered glass vials and was cooled to 0 °C. Then, 4 mL anthrone solution [2 g of anthrone in 1 L of 96% (v/v) H2SO4] was added, vortexed well and heated at 100 °C for 8 min. The sample was cooled to room temperature rapidly and absorbance was measured at 630 nm using UV/vis spectrophotometer (Pharo 300, MERCK, Germany). Glucose content in the samples was obtained by previously prepared standard curve. Starch content was calculated by multiplying factor 0.9. 2.4.
Lipid and protein determination
Lipid and protein determination procedures were the same as described by Mishra, Sanjiv K., et al. and Jakob H. Waterborg, respectively (Mishra et al. 2014, Waterborg 2009). 2.5.
Nitrate determination
A modified method of Cataldo et al. was used for determination of nitrate concentration (Cataldo et al. 1975). Briefly, 3 mL of culture sample was filtered using 0.45 µm cellulose nitrate filter (Sartorius Stedim). 0.25 mL of filtered sample was pipetted out to Teflon-covered glass vial, added 0.8 mL of salicylic acid solution (5% w/v salicylic acid in concentrated H2SO4) and vortexed well. After 20 min, 7.6 mL of 5 M NaOH was added, vortexed well and cooled to room temperature. The absorbance of solution at 410 nm was measured by spectrophotometer. Nitrate concentration was determined by previously prepared standard curve. 3. Results and discussion In order to enrich starch and TAG accumulating species, two photobioreactors were operated with six-day cycles for three months. Several surface waters were combined as inoculation. In constant light condition, after nitrogen source depletion, a large number of microalgae species accumulate fixed carbon in the form of starch and lipid. At the end of every cycle, cell suspension was allowed to settle for three minutes. High starch-storing species can fall rapidly to the bottom and high TAG-storing species can stay in supernatant of the cell suspension due to the differences in cell density. Transfer of the settled and supernatant microalgae species to the next batch as inoculation was the basis of enrichment of the starch and lipid-accumulating species. 30% of the bottom and supernatant of cell suspension was transferred to the starch and lipid photobioreactors for subsequent cultures, respectively. The samples were taken daily and analyzed for biomass, starch, lipid, protein, and nitrogen concentrations. After 8 weeks, steady state, in which initial and final starch to protein ratio and lipid to protein ratio in three sequence batch were constant for both photobioreactors, was reached. Starch, lipid, protein, and nitrogen profiles were comparable in every steady state cycle.
At the beginning of every steady state batch, at the presence of all nutrients, cells divided. Protein concentration was increased, while no increase in starch and lipid concentrations was observed. 3.1.
Enriched starch-producing mixed culture photobioreactor
The steady state profiles of nitrogen, starch, lipid, and protein in the enriched starch-producing mixed culture photobioreactor are illustrated in Fig. 1. Nitrogen source (NO3) was depleted after about 24 h. In the absence of nitrogen and presence of CO2, microalgae started to store fixed carbon, mainly in the form of starch, and reached to the value of 202.9 mg/L after 76 h. However, the increase in the lipid content was only 52 mg/L. At t=76 h, the VSS biomass concentration was 415.8 mg/L and the starch and lipid contents of biomass were 48.8 and 12.5 % of VSS, respectively. At the end of the batch, VSS was approximately 453 mg/L and the amount of starch and lipid reached to 217.4 and 75.0 mg/L or 48.0 and 16.6 % on the basis of VSS, respectively. The ratios of starch to protein and lipid to protein are depicted in Fig. 2. The starch content increased by a factor of 3.18 and 3.42 ( corresponding to 48.8 and 480.0% on the basis of VSS ) after 76 and 144 h in comparison with the onset of famine phase, respectively. The lipid content also increased by a factor of 1.7 and 2.4 (corresponding to 12.5 and 16.6% on the basis of VSS) after 76 and 144 h, respectively compared to the beginning of nitrate depletion. Fig. 1. Fig. 2. 3.2.
Enriched lipid-producing mixed culture photobioreactor
The steady state profiles of nitrogen, starch, lipid and protein in the enriched lipid-producing mixed culture photobioreactor are illustrated in Fig. 3. At the start of nitrogen depletion, VSS was 305 mg/L and the amount of the starch and lipid were approximately 81 and 51 mg/L, respectively. At t=76 h, the VSS was 503 mg/L and the amounts of the starch and lipid were increased to 215 and 110 mg/L or 42.8 and 21.9 % of VSS, respectively, while at the end of steady state batches, these values changed to 36 and 30% on the basis of VSS (Fig. 3). The ratios of starch and lipid to protein were also different, as shown in Fig. 4. Starch and lipid values increased by a factor of 2.55 and 2.07 after 76 h, and by the end of the batch increased up to a factor of 2.29 and 3 in comparison with the onset of famine. Fig. 3. Fig. 4. The obtained results in this study are consistent with the results of other researchers. The relative biological energy required for TAG synthesis is greater than starch synthesis per carbon. In addition, the energy return/carbon of fatty acid oxidation is higher than starch oxidation (Subramanian et al. 2013). Therefore, in the most studied species, microalgae start to store starch as primary form of energy storage compound in initial deal with famine phase. However, by extending period of food shortage, starch gradually degraded and lipid synthesis accelerated. The results of this study show in both starch and lipid photobioreactors, through gravimetric enrichment, species which accumulate starch as initial response to nitrogen depletion have been selected. In starch photobioreactor, after rapid accumulation of starch, starch productivity remained approximately constant and never decreased. While in lipid photobioreactor, in case of longer famine, starch partially degraded. After nitrogen depletion, lipid productivity in both
photobioreactors increased with constant rate. Lipid productivity in lipid photobioreactor was more than 2-fold the starch photobioreactor. Starch degradation in lipid photobioreactor may be due to spare more physical space to store lipid. The colors of the cell suspensions were different in photobioreactors. The photobioreactors content turned greenish and yellowish in enriched starch and lipid producing mixed culture photobioreactors, respectively. This color difference, together with the microscopic observation, reveals the different algal strains enrichment in two bioreactors. In addition, there was a larger microbial diversity in starch photobioreactor compared to lipid bioreactor. These visual results collectively can confirm that the gravimetric screening strategy has successfully resulted in two diverse micro-organism populations. This method comprised two phases: growth phase and depletion phase. Biomass reaches to maximum amount in growth phase and production of storage compound dramatically increased in depletion phase. It is strongly suggested that more distinguished results can be achieved by optimizing some parameters such as cycle length, settling time, volume exchange rate, light-dark regime, light intensity and different nitrogen sources and CO2 concentrations. For instance, by decreasing the batch time, species which have high productivity and store starch or lipid as initial response to depletion will be selected. Similarly, increasing the volume exchange rate imposes higher selective environment that can lead to an increase in starch or lipid accumulation. Prior to the present study, pure microalgal cultivation has been generally used to produce starch and lipid as internal storage compound. The only distinguished experience of using selective environment to screen energy-rich compound storing species in mixed culture led to starch accumulation. Emphasis on ecological role of product as an energy source cannot determine
whether starch or lipid is stored. Therefore, the novelty of this work is the enrichment of mixed cultures based on physical property of product leading to specific production of storage compounds in non-sterile condition. The results obtained in this investigation are comparable to pure culture cultivation, and optimization of involved parameter in this process can improve the obtained results. Ho et al. isolated three indigenous microalgae species from freshwater and examined their carbohydrate production ability. These three species, Chlorella vulgaris ESP-6, Chlorella vulgaris FSP-E, Chlamydomonas orbicularis Tai-04 accumulated 48.59, 54.13 and 47.35% VSS carbohydrate in eleven-day nitrogen starvation period respectively. Lipid content reached to 12.74, 11.61, 7.35% VSS, respectively, in the same period (Ho et al. 2013). In another study, Pruvost et al. investigated the ability of Neochloris oleoabundans to accumulate lipid under nitrogen starvation. Their results showed N. oleoabundans stored 37% DW lipid under nitrate starvation in an approximately six-day batch culture (Pruvost et al. 2009). Yoo et al. investigated biomass and lipid production of three microalgae species, Botryococcus braunii, Chlorella vulgaris, and Scenedesmus sp. under high level of carbon dioxide. In this study, the biomass and lipid productivity were 217.50 and 20.65 mg/L.day (9% of biomass) for Scenedesmus sp. and 26.55 and 5.51 mg/L.day (21% of biomass) for B. braunii respectively (Yoo et al. 2010). In another study, Yao et al. studied the accumulation of starch in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus deprivation with initial cell density of 1.5, 3, 6 and 3 × 106 cell/mL and figured out the starch content reached to 44.1 and 42.2 % of DW with initial cell density of 1.5 and 3 × 106 cell/mL (Yao et al. 2013). Gravimetric enrichment is a cheap, simple and flexible screening method that leads to screening starch and lipid producing microalgal species. This flexible method can be applied to various conditions such as marine environments to overcome obstacles relating to freshwater.
Characteristics such as elimination of sterilization and simple operation condition indicate that this screening method is practicable on large scale. 4. Conclusion A novel screening method of starch and lipid producing microalgae species in mixed culture was introduced based on their differences in cell density. Applying gravimetric enrichment in mixed culture can lead to explore microbial diversity of nature and identification of highly efficient microalgal starch and lipid production. At the end of steady state batches, starch and lipid contents increased up to 3.42 and 3 times the onset of nitrogen depletion in starch and lipid photobioreactors, respectively. Therefore, our findings demonstrate gravimetric enrichment as a simple, cheap, and flexible method to overcome specific storage compounds production obstacles in large scale. References 1. Beetul, K., Sadally, S. B., Hossenkhan, N., & Puchooa, D. (2014). An investigation of biodiesel production from microalgae found in mauritian waters. Biofuel Research Journal, 1(2), 58-64. 2. Brányiková, I., Maršálková, B., Doucha, J., Brányik, T., Bišová, K., Zachleder, V. and Vítová, M. (2011) Microalgae—novel highly efficient starch producers. Biotechnology and Bioengineering 108(4), 766-776. 3. Cataldo, D., Maroon, M., Schrader, L. and Youngs, V. (1975) Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid 1. Communications in Soil Science & Plant Analysis 6(1), 71-80. 4. Chisti, Y. (2007) Biodiesel from microalgae. Biotechnology advances 25(3), 294-306. 5. Ho, S.-H., Huang, S.-W., Chen, C.-Y., Hasunuma, T., Kondo, A. and Chang, J.-S. (2013) Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E. Bioresour Technol 135, 157-165.
6. Johnson, K., Jiang, Y., Kleerebezem, R., Muyzer, G. and Van Loosdrecht, M.C. (2009) Enrichment of a mixed bacterial culture with a high polyhydroxyalkanoate storage capacity. Biomacromolecules 10(4), 670-676. 7. Kilham, S.S., Kreeger, D.A., Lynn, S.G., Goulden, C.E. and Herrera, L. (1998) COMBO: a defined freshwater culture medium for algae and zooplankton. Hydrobiologia 377(1-3), 147-159. 8. Kleerebezem, R. and van Loosdrecht, M.C. (2007) Mixed culture biotechnology for bioenergy production. Current opinion in biotechnology 18(3), 207-212. 9. Li, Y., Horsman, M., Wu, N., Lan, C.Q. and Dubois‐Calero, N. (2008) Biofuels from microalgae. Biotechnology progress 24(4), 815-820. 10. McCready, R., Guggolz, J., Silviera, V. and Owens, H. (1950) Determination of starch and amylose in vegetables. Analytical chemistry 22(9), 1156-1158.
11. Menetrez, M. Y. (2014). Meeting the US renewable fuel standard: a comparison of biofuel pathways. Biofuel Research Journal, 1(4), 110-122. 12. Millán-Oropeza, A., Torres-Bustillos, L. G., & Fernández-Linares, L. (2015). Simultaneous effect of nitrate (NO3-) concentration, carbon dioxide (CO2) supply and nitrogen limitation on biomass, lipids, carbohydrates and proteins accumulation in Nannochloropsis oculata. Biofuel Research Journal, 2(1), 215-221. 13. Mishra, S.K., Suh, W.I., Farooq, W., Moon, M., Shrivastav, A., Park, M.S. and Yang, J.-W. (2014) Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method. Bioresour Technol 155, 330-333. 14. Mooij, P.R., Stouten, G.R., Tamis, J., van Loosdrecht, M.C. and Kleerebezem, R. (2013) Survival of the fattest. Energy Environ. Sci. 6(12), 3404-3406. 15. Mooij, P.R., Stouten, G.R., van Loosdrecht, M.C. and Kleerebezem, R. (2015) Ecology-based selective environments as solution to contamination in microalgal cultivation. Current opinion in biotechnology 33, 46-51. 16. Pruvost, J., Van Vooren, G., Cogne, G. and Legrand, J. (2009) Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor. Bioresour Technol 100(23), 59885995. 17. Reis, M., Serafim, L., Lemos, P., Ramos, A., Aguiar, F. and Van Loosdrecht, M. (2003) Production of polyhydroxyalkanoates by mixed microbial cultures. Bioprocess and Biosystems Engineering 25(6), 377-385. 18. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G. and Tredici, M.R. (2009) Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low‐cost photobioreactor. Biotechnology and Bioengineering 102(1), 100-112.
19. Sales-Cruz, M., Aca-Aca, G., Sánchez-Daza, O. and López-Arenas, T. (2010) Predicting critical properties, density and viscosity of fatty acids, triacylglycerols and methyl esters by group contribution methods.
20. Subramanian, S., Barry, A.N., Pieris, S. and Sayre, R.T. (2013) Comparative energetics and kinetics of autotrophic lipid and starch metabolism in chlorophytic microalgae: implications for biomass and biofuel production. Biotechnol Biofuels 6(1), 150.
21. Talebi, A. F., Tabatabaei, M., & Chisti, Y. (2014). BiodieselAnalyzer: a user-friendly software for predicting the properties of prospective biodiesel. Biofuel Research Journal, 1(2), 55-57 22. Waterborg, J.H. (2009) The protein protocols handbook, pp. 7-10, Springer. 23. Yao, C.-H., Ai, J.-N., Cao, X.-P. and Xue, S. (2013) Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions. Applied microbiology and biotechnology 97(13), 6099-6110. 24. Yao, C., Ai, J., Cao, X., Xue, S. and Zhang, W. (2012) Enhancing starch production of a marine green microalga Tetraselmis subcordiformis through nutrient limitation. Bioresour Technol 118, 438-444. 25. Yoo, C., Jun, S.-Y., Lee, J.-Y., Ahn, C.-Y. and Oh, H.-M. (2010) Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol 101(1), S71-S74.
Figure captions Fig. 1. Nitrogen source, protein, starch and lipid concentrations in starch photobioreactor during steady state batch experiments Fig. 2. Starch and lipid content profiles in starch photobioreactor during steady state batch experiments Fig. 3. Nitrogen source, protein, starch and lipid concentrations in lipid photobioreactor during steady state batch experiments Fig. 4. Starch and lipid content profiles in lipid photobioreactor during steady state batch experiments
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HIGHLIGHTS: •4 •5 •6
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Introducing gravimetric enrichment to screen starch and lipid producing species Specific production of starch and lipid in mixed culture in semi continuous process Starch and lipid production in non-sterile conditions. 7
S0960-8524(15)01008-1 http://dx.doi.org/10.1016/j.biortech.2015.07.046 BITE 15279
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
24 May 2015 14 July 2015 15 July 2015
Please cite this article as: Hassanpour, M., Abbasabadi, M., Ebrahimi, S., Hosseini, M., Sheikhbaglou, A., Gravimetric Enrichment of High Lipid and Starch Accumulating Microalgae, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.07.046
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gravimetric Enrichment of High Lipid and Starch Accumulating Microalgae Morteza Hassanpour, Mahsa Abbasabadi, Sirous Ebrahimi,*, Maryam Hosseini, Ahmad Sheikhbaglou Biotechnology Research Center, Faculty of Chemical Engineering, Sahand University of Technology, Tabriz, Iran,
*
Corresponding author: Sirous Ebrahimi
E-mail: [email protected] Tel: +98 41 33458180; Fax: +98 41 33444355
Abstract This study presents gravimetric enrichment of mixed culture to screen starch and lipid producing species separately in a sequencing batch reactor. In the enriched starch-producing mixed culture photobioreactor, the starch content at the end of steady state batch became 3.42 times the beginning of depletion. Whereas in the enriched lipid-producing photobioreactor, the lipid content at the end of steady state batch became 3 times the beginning of famine phase. The obtained results revealed that the gravimetric enrichment is a suitable screening method for specific production of storage compounds in none-sterile large-scaled condition. Keywords: Microalgae; Starch; Lipid; Biofuel; Gravimetric enrichment; Sequencing batch reactor 1. Introduction Increasing world population and fossil fuels depletion have increased global concerns about future. Greenhouse gas emissions and climate changes have induced attention to sustainable energy supplies like biofuels. Some characteristics such as high growth rate, arable land and potable water independency and storing of starch and triacylglycerol (TAG) have introduced microalgae as the most promising feedstock for future biofuel production (Chisti 2007 and Menetrez 2014). Starch can be used as fermentation feedstock for bioethanol production and TAG can be converted to biodiesel after an esterification step (Li et al. 2008 and Beetul et al. 2014). Selecting an appropriate species of microalgae is the first and most important step through the whole process of production of storage compounds (Mooij et al. 2013 and Talebi et al. 2014).
This selection is based on high storage capacity or high productivity. Many microalgae such as Chlorella species, Neochloris oleoabundans, Dunaliella species, Tetraselmis subcordiformis and Arthrospira platensis accumulate high levels of starch and TAG under nutrient-limiting conditions (Millán-Oropeza et al. 2015). For instance, in marine green microalga Tetraselmis subcordiformis, starch accumulated up to 62.1 and 54.3 % of dry weight (DW) under sulfur and nitrogen deprived conditions, respectively (Yao et al. 2012). The starch content of freshwater alga Chlorella vulgaris was reported to be 60, 55 and 38% DW in sulfur, phosphorus and nitrogen limitations, respectively (Brányiková et al. 2011). Marine microalga eustigmatophyte Nannochloropsis sp. attained 60% DW lipid content when was cultivated under nitrogen deprivation in 0.6-L bubbled tubes (Rodolfi et al. 2009). Selection of a pure culture will increase system contamination likelihood to invading species that can lead to lower production efficiency. Cultivation of microalgae in pure culture will require an aseptic process condition resulting in a high investment and operating cost. Such preparations present serious barriers to large scale application (Mooij et al. 2013). In addition, even in specific environmental conditions such as high salinity for Dunaliella Salina cultivation, where contamination likelihood is low, strain evolution remains as a great threat, since mutated strains with higher growth rate are able to outcompete the desired strain leading to a drop in productivity (Mooij et al. 2015). As an alternative to pure culture, the application of a mixed-culture-based process would be a technically attractive and an economically feasible solution (Reis et al. 2003). Screening mixed culture based on ecological role of product, would be a good solution to overcome cultivation contamination risk (Mooij et al. 2013). Exploring microbial diversity and operation in various conditions are the main advantages of using mixed culture under selective environment (Johnson
et al. 2009, Kleerebezem and van Loosdrecht 2007). Research in a semi-continuous process based on ecological role of accumulated fixed carbon resulted in the enrichment of microalgae with high levels of internal storage compounds. In that study, a sequencing batch mode of operation was adopted with cycles of 24 hours with a light and dark period in which nitrogen source was supplied only during the dark period of cycles. Therefore, only those species survived that were able to take up CO2 in light periods; store fixed carbon as internal storage compounds; and use it to uptake nitrogen source during the dark period (Mooij et al. 2013). Despite encouraging results, the applied approach does not provide a lipid/starch selective accumulation. Therefore, production of specific storage compound such as starch or TAG still remains a major challenge. Storage starch and TAG have density of approximately 1.5 and 0.95 g/mL, respectively (SalesCruz et al. 2010). The difference between starch and TAG densities imposes a selective environment based on buoyancy to screen starch and TAG accumulating species in mixed culture. Therefore, the aim of this study was to investigate screening starch and TAG accumulating species in a mixed culture in sequencing batch reactor (SBR) systems directing more photosynthetic carbon partition to either starch or lipid. It was expected that high starchstoring species can settle rapidly to the bottom and high TAG storing species can stay in supernatant of the cell suspension regarded to their cell density. Therefore, in the presented study for the first time gravimetric enrichment strategy was applied to screen starch and TAG accumulating species. 2. Materials and methods 2.1.
Experimental set-up and operation
In order to screen the lightest and densest species, two 2 L none-sterile glass bioreactors (Takbiotech, Iran) were operated in SBR mode with 0.8 L working volume. The operating conditions are as follows: Cycle length, 6 day, 250 lux constant light provided by LED lamps, aeration rate of 1 vvm with 5% CO2 and stirrer speed 200 rpm. The pH of the bioreactor was controlled at 7.5 ± 0.1 by addition of 1 M HCl or 1 M NaHCO3, using a pH transmitter (2500, Mettler Toledo, Switzerland). Temperature was kept at 30 ± 1°C by using a water bath circulator with a heating system (UC4500, Sahandazar, Iran). A modified Combo-medium was used as nutrients (Kilham et al. 1998). No KCl, NaHCO3, vitamins and animal trace elements were added. Nitrogen source was 14 mg/L and other nutrients were supplied by a factor of three. The bioreactor was inoculated with 100 mL of combined samples of ponds and springs water which was cultivated in a glass bottle with the medium described above. In order to screen starch and TAG accumulating species, after 3 min settling, 30% of the bottom and supernatant of the bioreactor content was transferred to the next two batches as inoculums, respectively. The samples were taken regularly every week from both photobioreactors to observe the changes in microalgal population using a Leica DMLS light microscope (Leica Microsystems, Wetzlar GmbH, Germany). 2.2.
Biomass determination
It was assumed that cells contain four general classes of macromolecules: protein, polysaccharides, lipid, and nucleic acids. Total dry weight was determined by centrifugation of 40 mL of cell suspension at 5000 g for 5 min, decanting supernatant, washing the residues with distilled water, and drying at 105 °C for 24 h at the end of feast phase. Ash content was determined by incineration of dry biomass at 550 °C for 1 h. Volatile suspended solids amount (VSS) was calculated by difference between ash content and total dry weight. The difference
between the sum of protein, starch, lipid, and VSS at the end of feast phase was considered as nucleic acids. There was no cell division in famine phase. Thus, the amount of nucleic acids was constant, and the VSS was calculated as sum of starch, lipid, proteins, and nucleic acids theoretically in the other batch times. 2.3.
Starch determination
A modified method of Mc Cready et al. was used for starch determination (McCready et al. 1950). One mL of the cell suspension was centrifuged at 5000 g for 5 min. The pellets were collected. 6 mL of 96% ethanol was added and heated at 80 °C for 10 min to remove pigments. The extracts were centrifuged and supernatant was decanted. The retained pellets were dried over water bath. One mL of 50% perchloric acid was added to the pellets and incubated for 30 min at 25 °C in incubator shaking at 150 rpm. Four mL of distilled water was added to the sample, centrifuged at 3000 g for 5 min, pipetted out 1 mL of supernatant to Teflon-covered glass vials and was cooled to 0 °C. Then, 4 mL anthrone solution [2 g of anthrone in 1 L of 96% (v/v) H2SO4] was added, vortexed well and heated at 100 °C for 8 min. The sample was cooled to room temperature rapidly and absorbance was measured at 630 nm using UV/vis spectrophotometer (Pharo 300, MERCK, Germany). Glucose content in the samples was obtained by previously prepared standard curve. Starch content was calculated by multiplying factor 0.9. 2.4.
Lipid and protein determination
Lipid and protein determination procedures were the same as described by Mishra, Sanjiv K., et al. and Jakob H. Waterborg, respectively (Mishra et al. 2014, Waterborg 2009). 2.5.
Nitrate determination
A modified method of Cataldo et al. was used for determination of nitrate concentration (Cataldo et al. 1975). Briefly, 3 mL of culture sample was filtered using 0.45 µm cellulose nitrate filter (Sartorius Stedim). 0.25 mL of filtered sample was pipetted out to Teflon-covered glass vial, added 0.8 mL of salicylic acid solution (5% w/v salicylic acid in concentrated H2SO4) and vortexed well. After 20 min, 7.6 mL of 5 M NaOH was added, vortexed well and cooled to room temperature. The absorbance of solution at 410 nm was measured by spectrophotometer. Nitrate concentration was determined by previously prepared standard curve. 3. Results and discussion In order to enrich starch and TAG accumulating species, two photobioreactors were operated with six-day cycles for three months. Several surface waters were combined as inoculation. In constant light condition, after nitrogen source depletion, a large number of microalgae species accumulate fixed carbon in the form of starch and lipid. At the end of every cycle, cell suspension was allowed to settle for three minutes. High starch-storing species can fall rapidly to the bottom and high TAG-storing species can stay in supernatant of the cell suspension due to the differences in cell density. Transfer of the settled and supernatant microalgae species to the next batch as inoculation was the basis of enrichment of the starch and lipid-accumulating species. 30% of the bottom and supernatant of cell suspension was transferred to the starch and lipid photobioreactors for subsequent cultures, respectively. The samples were taken daily and analyzed for biomass, starch, lipid, protein, and nitrogen concentrations. After 8 weeks, steady state, in which initial and final starch to protein ratio and lipid to protein ratio in three sequence batch were constant for both photobioreactors, was reached. Starch, lipid, protein, and nitrogen profiles were comparable in every steady state cycle.
At the beginning of every steady state batch, at the presence of all nutrients, cells divided. Protein concentration was increased, while no increase in starch and lipid concentrations was observed. 3.1.
Enriched starch-producing mixed culture photobioreactor
The steady state profiles of nitrogen, starch, lipid, and protein in the enriched starch-producing mixed culture photobioreactor are illustrated in Fig. 1. Nitrogen source (NO3) was depleted after about 24 h. In the absence of nitrogen and presence of CO2, microalgae started to store fixed carbon, mainly in the form of starch, and reached to the value of 202.9 mg/L after 76 h. However, the increase in the lipid content was only 52 mg/L. At t=76 h, the VSS biomass concentration was 415.8 mg/L and the starch and lipid contents of biomass were 48.8 and 12.5 % of VSS, respectively. At the end of the batch, VSS was approximately 453 mg/L and the amount of starch and lipid reached to 217.4 and 75.0 mg/L or 48.0 and 16.6 % on the basis of VSS, respectively. The ratios of starch to protein and lipid to protein are depicted in Fig. 2. The starch content increased by a factor of 3.18 and 3.42 ( corresponding to 48.8 and 480.0% on the basis of VSS ) after 76 and 144 h in comparison with the onset of famine phase, respectively. The lipid content also increased by a factor of 1.7 and 2.4 (corresponding to 12.5 and 16.6% on the basis of VSS) after 76 and 144 h, respectively compared to the beginning of nitrate depletion. Fig. 1. Fig. 2. 3.2.
Enriched lipid-producing mixed culture photobioreactor
The steady state profiles of nitrogen, starch, lipid and protein in the enriched lipid-producing mixed culture photobioreactor are illustrated in Fig. 3. At the start of nitrogen depletion, VSS was 305 mg/L and the amount of the starch and lipid were approximately 81 and 51 mg/L, respectively. At t=76 h, the VSS was 503 mg/L and the amounts of the starch and lipid were increased to 215 and 110 mg/L or 42.8 and 21.9 % of VSS, respectively, while at the end of steady state batches, these values changed to 36 and 30% on the basis of VSS (Fig. 3). The ratios of starch and lipid to protein were also different, as shown in Fig. 4. Starch and lipid values increased by a factor of 2.55 and 2.07 after 76 h, and by the end of the batch increased up to a factor of 2.29 and 3 in comparison with the onset of famine. Fig. 3. Fig. 4. The obtained results in this study are consistent with the results of other researchers. The relative biological energy required for TAG synthesis is greater than starch synthesis per carbon. In addition, the energy return/carbon of fatty acid oxidation is higher than starch oxidation (Subramanian et al. 2013). Therefore, in the most studied species, microalgae start to store starch as primary form of energy storage compound in initial deal with famine phase. However, by extending period of food shortage, starch gradually degraded and lipid synthesis accelerated. The results of this study show in both starch and lipid photobioreactors, through gravimetric enrichment, species which accumulate starch as initial response to nitrogen depletion have been selected. In starch photobioreactor, after rapid accumulation of starch, starch productivity remained approximately constant and never decreased. While in lipid photobioreactor, in case of longer famine, starch partially degraded. After nitrogen depletion, lipid productivity in both
photobioreactors increased with constant rate. Lipid productivity in lipid photobioreactor was more than 2-fold the starch photobioreactor. Starch degradation in lipid photobioreactor may be due to spare more physical space to store lipid. The colors of the cell suspensions were different in photobioreactors. The photobioreactors content turned greenish and yellowish in enriched starch and lipid producing mixed culture photobioreactors, respectively. This color difference, together with the microscopic observation, reveals the different algal strains enrichment in two bioreactors. In addition, there was a larger microbial diversity in starch photobioreactor compared to lipid bioreactor. These visual results collectively can confirm that the gravimetric screening strategy has successfully resulted in two diverse micro-organism populations. This method comprised two phases: growth phase and depletion phase. Biomass reaches to maximum amount in growth phase and production of storage compound dramatically increased in depletion phase. It is strongly suggested that more distinguished results can be achieved by optimizing some parameters such as cycle length, settling time, volume exchange rate, light-dark regime, light intensity and different nitrogen sources and CO2 concentrations. For instance, by decreasing the batch time, species which have high productivity and store starch or lipid as initial response to depletion will be selected. Similarly, increasing the volume exchange rate imposes higher selective environment that can lead to an increase in starch or lipid accumulation. Prior to the present study, pure microalgal cultivation has been generally used to produce starch and lipid as internal storage compound. The only distinguished experience of using selective environment to screen energy-rich compound storing species in mixed culture led to starch accumulation. Emphasis on ecological role of product as an energy source cannot determine
whether starch or lipid is stored. Therefore, the novelty of this work is the enrichment of mixed cultures based on physical property of product leading to specific production of storage compounds in non-sterile condition. The results obtained in this investigation are comparable to pure culture cultivation, and optimization of involved parameter in this process can improve the obtained results. Ho et al. isolated three indigenous microalgae species from freshwater and examined their carbohydrate production ability. These three species, Chlorella vulgaris ESP-6, Chlorella vulgaris FSP-E, Chlamydomonas orbicularis Tai-04 accumulated 48.59, 54.13 and 47.35% VSS carbohydrate in eleven-day nitrogen starvation period respectively. Lipid content reached to 12.74, 11.61, 7.35% VSS, respectively, in the same period (Ho et al. 2013). In another study, Pruvost et al. investigated the ability of Neochloris oleoabundans to accumulate lipid under nitrogen starvation. Their results showed N. oleoabundans stored 37% DW lipid under nitrate starvation in an approximately six-day batch culture (Pruvost et al. 2009). Yoo et al. investigated biomass and lipid production of three microalgae species, Botryococcus braunii, Chlorella vulgaris, and Scenedesmus sp. under high level of carbon dioxide. In this study, the biomass and lipid productivity were 217.50 and 20.65 mg/L.day (9% of biomass) for Scenedesmus sp. and 26.55 and 5.51 mg/L.day (21% of biomass) for B. braunii respectively (Yoo et al. 2010). In another study, Yao et al. studied the accumulation of starch in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus deprivation with initial cell density of 1.5, 3, 6 and 3 × 106 cell/mL and figured out the starch content reached to 44.1 and 42.2 % of DW with initial cell density of 1.5 and 3 × 106 cell/mL (Yao et al. 2013). Gravimetric enrichment is a cheap, simple and flexible screening method that leads to screening starch and lipid producing microalgal species. This flexible method can be applied to various conditions such as marine environments to overcome obstacles relating to freshwater.
Characteristics such as elimination of sterilization and simple operation condition indicate that this screening method is practicable on large scale. 4. Conclusion A novel screening method of starch and lipid producing microalgae species in mixed culture was introduced based on their differences in cell density. Applying gravimetric enrichment in mixed culture can lead to explore microbial diversity of nature and identification of highly efficient microalgal starch and lipid production. At the end of steady state batches, starch and lipid contents increased up to 3.42 and 3 times the onset of nitrogen depletion in starch and lipid photobioreactors, respectively. Therefore, our findings demonstrate gravimetric enrichment as a simple, cheap, and flexible method to overcome specific storage compounds production obstacles in large scale. References 1. Beetul, K., Sadally, S. B., Hossenkhan, N., & Puchooa, D. (2014). An investigation of biodiesel production from microalgae found in mauritian waters. Biofuel Research Journal, 1(2), 58-64. 2. Brányiková, I., Maršálková, B., Doucha, J., Brányik, T., Bišová, K., Zachleder, V. and Vítová, M. (2011) Microalgae—novel highly efficient starch producers. Biotechnology and Bioengineering 108(4), 766-776. 3. Cataldo, D., Maroon, M., Schrader, L. and Youngs, V. (1975) Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid 1. Communications in Soil Science & Plant Analysis 6(1), 71-80. 4. Chisti, Y. (2007) Biodiesel from microalgae. Biotechnology advances 25(3), 294-306. 5. Ho, S.-H., Huang, S.-W., Chen, C.-Y., Hasunuma, T., Kondo, A. and Chang, J.-S. (2013) Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E. Bioresour Technol 135, 157-165.
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21. Talebi, A. F., Tabatabaei, M., & Chisti, Y. (2014). BiodieselAnalyzer: a user-friendly software for predicting the properties of prospective biodiesel. Biofuel Research Journal, 1(2), 55-57 22. Waterborg, J.H. (2009) The protein protocols handbook, pp. 7-10, Springer. 23. Yao, C.-H., Ai, J.-N., Cao, X.-P. and Xue, S. (2013) Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions. Applied microbiology and biotechnology 97(13), 6099-6110. 24. Yao, C., Ai, J., Cao, X., Xue, S. and Zhang, W. (2012) Enhancing starch production of a marine green microalga Tetraselmis subcordiformis through nutrient limitation. Bioresour Technol 118, 438-444. 25. Yoo, C., Jun, S.-Y., Lee, J.-Y., Ahn, C.-Y. and Oh, H.-M. (2010) Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol 101(1), S71-S74.
Figure captions Fig. 1. Nitrogen source, protein, starch and lipid concentrations in starch photobioreactor during steady state batch experiments Fig. 2. Starch and lipid content profiles in starch photobioreactor during steady state batch experiments Fig. 3. Nitrogen source, protein, starch and lipid concentrations in lipid photobioreactor during steady state batch experiments Fig. 4. Starch and lipid content profiles in lipid photobioreactor during steady state batch experiments
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HIGHLIGHTS: •4 •5 •6
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Introducing gravimetric enrichment to screen starch and lipid producing species Specific production of starch and lipid in mixed culture in semi continuous process Starch and lipid production in non-sterile conditions. 7