CET-vol 105 DOI: 10.3303/CET23105039 Paper Received: 21 February 2023; Revised: 22 April 2023; Accepted: 18 September 2023 Please cite this article as: Gurreri L., Calanni Rindina M., Luciano A., Falqui L., Mancini G., Fino D., 2023, Life Cycle Inventory Based on Primary Data of an Industrial Plant for the Cultivation of Chlorella Vulgaris, Chemical Engineering Transactions, 105, 229-234 DOI:10.3303/CET23105039 CHEMICAL ENGINEERING TRANSACTIONS VOL. 105, 2023 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: David Bogle, Flavio Manenti, Piero Salatino Copyright © 2023, AIDIC Servizi S.r.l. ISBN 979-12-81206-04-5; ISSN 2283-9216 Life Cycle Inventory Based on Primary Data of an Industrial Plant for the Cultivation of Chlorella Vulgaris Luigi Gurreria,*, Mirko Calanni Rindinaa, Antonella Lucianob, Luciano Falquic, Giuseppe Mancinia, Debora Finod a Department of Electrical, Electronic and Computer Engineering, University of Catania, Viale Andrea Doria 6, 95125, Catania, Italy b ENEA – Italian National Agency for the New Technologies, Energy and Sustainable Economic Development – Department for Sustainability, Casaccia Research Centre – Via Anguillarese 301, 00123, Rome, Italy c Plastica Alfa S.p.a., Zona Industriale, 95041, Caltagirone, Italy d Department of Applied Science and Technology (DISAT), Politecnico di Torino, Corso Duca Degli Abruzzi 24, 10129, Torino, Italy luigi.gurreri@unict.it Microalgae and cyanobacteria represent a valuable resource with great potential in the production of a large variety of high-added value products in the nutraceutical, cosmetic, pharmaceutical, and food sectors. However, the commercialization of microalgal systems and biorefineries is limited by environmental and economic sustainability concerns. Many life cycle assessments (LCAs) have been conducted so far, but they have the crucial limitation of using data extrapolated from lab-scale experiments or the literature, thus providing qualitative and uncertain projections. In this work, an industrial-scale plant's life cycle inventory (LCI) is compiled with primary data and analyzed. The plant is installed in Sicily (Italy) and has a 1200 kgDW year–1 capacity in the production of Chlorella vulgaris microalgae. The cultivation is performed in vertically stacked horizontal photobioreactors (PBRs) with a total volume of 40.4 m3. Dewatering is performed by centrifugation, producing a final algal suspension at ~200 gDW L–1. Energy, nutrients, water, chemicals, and infrastructure materials are inventoried to characterize the operation and construction. One kg of dry algae biomass is the functional unit chosen to report all elementary input/output flows. The results are compared with relevant LCIs from the literature, providing a picture of the current status of microalgae production in large-scale plants. 1. Introduction Microalgae have a huge potential for a multitude of production routes, including biofuels/bioenergy (Marangon et al., 2023), biomaterials (Nanda and Bharadvaja, 2022), and valuable biochemicals (Braun and Colla, 2022). However, the economic (Yadav et al., 2022) and environmental (Ubando et al., 2022) sustainability of microalgal production and biorefinery is uncertain and presents several challenges, hindering the competitiveness against established technologies. Many research efforts have been devoted to the life cycle assessment (LCA) of microalgal systems, especially in the last decade. Nevertheless, LCA studies have been conducted mostly with data extrapolated from lab-scale setups or even with literature data. In contrast, few studies have been based on primary data from large-scale plants (Gurreri et al., 2023). Moreover, LCAs of microalgal systems used different methodologies (e.g., functional units), and some of them show a lack of transparency and clarity, even in the life cycle inventory (LCI). It consists of collecting and analyzing all input/output flows (resource consumption, emissions, and wastes), representing a crucial step in the LCA. Industrial-scale plants evaluated by LCA studies are listed in Table 1 along with some examples of pilot-scale installations. Tubular photobioreactors (PBRs) or open raceway ponds (ORPs) are the most common cultivation systems of microalgae. Pérez-López et al. (2017) compared three pilot bioreactors, i.e., a horizontal PBR, a vertically stacked PBR, and an ORP, for the cultivation of Nannochloropsis sp. Data were collected during summer, fall, and winter in Wageningen, the Netherlands, and all systems were thermoregulated. The LCI showed large effects of seasonality and bioreactor configuration. The main inputs were ~0.4-9.0 kg kg–1 for 229 mailto:luigi.gurreri@unict.it construction materials (mainly aluminum, PMMA, and PP for the Hor, VSt, and ORP reactor, respectively), 128- 15984 L kgDW –1 for tap water (cleaning), 426-15628 L kgDW –1 for seawater (cultivation), ~0.9-6.6 kg kgDW –1 for NaNO3 (major nutrient), 559-~12.000 g kgDW –1 for cleaning chemicals, and ~280-5600 kWh kg–1 for energy consumption with major contributions due to heating/cooling and aeration. Onorato and Rösch (2020) compared three commercial-scale PBR plants (93 m3): an indoor Flat Panel Airlift (FPA) PBR, illuminated by LEDs and ventilated for temperature control; an outdoor Green Wall Panel (GWP) PBR (up-scaled data), illuminated by LEDs; an outdoor Unilayer Horizontal Tubular (UHT) PBR, cooled by freshwater spraying. The cultivation of Haematococcus pluvialis and the extraction of astaxanthin were studied. The FPA, GWP, and UHT PBR plants were located in Stuttgart (Germany), Montpellier (France), and Lisbon (Portugal), respectively. Their main inputs for microalgae cultivation were 346, 556, and 1574 L kgDW –1 for water, 50, 127, and 75 g kgDW –1 for nutrients, and 300, 318, and 25 kWh kgDW –1 for electricity, respectively. Herrera et al. (2021) assessed a 1000 m2 ORP installed in Almería (Spain) and producing biostimulants. Water consumption was either 540 or 2333 L kgDW –1 in the scenarios with or without recirculation from harvesting, respectively; N and P supply were 100 g kgDW –1 and 16 g kgDW –1, respectively, in the scenarios with added nutrients, while 100 L kgDW –1 for manure slurry was considered in other scenarios; the consumption of chemicals was 0.03, 0.08, and 20 g kgDW –1 for enzyme, NaOH, and flocculant, respectively; energy consumption was ~1 kWh kgDW –1 (optimized plant), while heat for hydrolysis was 1.8 MJ kgDW –1. Other seven bioreactors installed in the same facility of Almería were assessed by Pechsiri et al. (2023). Scenedesmus almeriensis was cultivated to extract biostimulants. Paddlewheel (~31 kg kg–1), LEDs (1.1-15.4 kg kg–1), PVC for electrocoagulation chamber (0.3 kg kg–1), and PMMA for reactor vessel (0.3 kg kg–1) dominated the construction materials in the ORP, indoor PBRs, TLC and VSt, respectively; water consumption was in the range 23-590 kg kg–1, most for cultivation or cleaning in the open or closed systems, respectively; nitrate and phosphate were 100 and 10 g kg–1, respectively; chemicals were ~10-30 g kg–1; electricity was ~11-85 kWh kg– 1, while heat for enzymatic hydrolysis was negligible. This study aims to compile the LCI of an industrial-scale PBR plant installed in Italy, thus taking a further step in the characterization of commercial implementations. The gathered primary data were elaborated, analyzed, and compared with LCIs from recent studies. Table 1: Industrial-scale microalgal plants providing primary data for previous LCA studies. Some examples of pilot plants are included. The references marked with “*” are used in this paper for LCI comparisons. Reference Reactor type Reactor size Passell et al. (2013) ORP 1000 m2 Beal et al. (2015) Horizontal serpentine airlift-driven PBR + ORP 25 + 60 m3 Silva et al. (2015) Vertically stacked PBR 12 m3 Pérez-López et al. (2017)* Horizontal PBR (Hor), vertically stacked PBR (VSt), ORP 0.56, 1.06, 4.73 m3 Onorato and Rösch (2020)* Flat Panel Airlift PBR (FPA), Green Wall Panel PBR (GWP), Unilayer Horizontal Tubular PBR (UHT) 93, 0.1, 93 m3 Herrera et al. (2021)* ORP 1000 m2 Pechsiri et al. (2023)* ORP, Thin Layer Cascade (TLC), vertically stacked PBR (VSt), Light Exchange Bubble column PBR (LEB), LEB mini reactors MR-1, MR-2, MR-3 20, 3, 4, 0.85, 0.36, 0.36, 0.35 m3 Bradley et al. (2023) Horizontal PBR + fermenters 60 + 3 m3 2. Materials and methods The inventoried plant is installed in Caltagirone, Sicily (Italy), within a facility occupying an area of ~1500 m2. Chlorella vulgaris, from which valuable products (e.g., biostimulants, lutein, and alternative proteins) can be extracted, is cultivated in vertically stacked (VSt) tubular horizontal PBRs with a total volume of 40.4 m3 (6 modules × 5.77 m3 + 2 modules × 2.88 m3, each module having either 4 or 2 loops of 1442 L) located in an 800 m2 greenhouse (Figure 1a). The plant capacity is 1200 kgDW year–1 and its average productivity is ~0.08 gDW L– 1 day–1. After cultivation, centrifugation harvesting increases the biomass concentration from 2 to 200 gDW L–1. The functional unit (FU) chosen to report all elementary input/output flows is 1 kg of dry-weight biomass (which is contained in 5 L of post-harvesting algal suspension). Energy, nutrients, water, chemicals, and infrastructure materials are inventoried to characterize the plant operation and construction. The LCI analysis was carried out by considering average flows representative of one year of industrial plant operation with 300 operating days per year and 10 years of PBR material lifespan. The cradle-to-gate approach was adopted, as depicted in Figure 1b, showing the main flows. The product system was divided into three subsystems, representing the process steps of reactor cleaning, cultivation (including inoculum), and harvesting. Cleaning and cultivation use 230 demineralized water produced by reverse osmosis (RO) of tap water (~700 mg/L TDS) and collected in storage tanks, each of 1500 L. The pre-treatments include quartzite filtration (100 μm), activated carbons, and cartridge microfiltration (10 and 5 μm). Eight RO modules (FILMTECTM BW30-4040, DuPont) are arranged in four parallel pressure vessels operated at 8-12 bar, producing ~1 m3 h–1 of permeate with ~90% water recovery. Waste streams (red arrows in Figure 1b) are discharged into the sewer. (a) (b) Figure 1: Cultivation of Chlorella vulgaris in the industrial-scale plant at the Plastica Alfa facility (Caltagirone, Italy): (a) Photo of the greenhouse with a portion of PBRs; (b) schematics of the analyzed process system. 2.1 Reactor cleaning The procedure for the PBRs cleaning and sterilization lasts 10 h and encompasses four sub-phases of flushing, each occurring in closed-loop: cleaning with an 80 mg/L sodium troclosene solution (6 h), washing with demi water (1 h), cleaning with a 3% citric acid solution (2 h), washing with demi water (1 h). The PBR modules are cleaned when needed, depending on the process conditions, to restore an optimal environment for microalgae growth. On average, it occurs roughly 5 times per year, thus the process is conducted with an average of 5 complete cycles per year. The exhausted washing solutions are discharged into the sewer. 2.2 Cultivation (including inoculum) The average growth rate is ~0.15 gDW L–1 d–1 in both inoculum and cultivation. The concentration target of all growth phases is 2 gDW L–1. The culture medium is prepared with demi water and added nutrients (~8 g/L as total concentration). The RO treatment provides an almost pure water that guarantees conditions suitable for the downstream extraction of high-added value compounds. CO2 is supplied from pressurized cylinders. The initial inoculum is performed in multiple steps, starting from growing the biomass in 1 L, then in 10 L, and culminating with an industrial-scale system consisting of 6 vertical airlift PBRs of 90 L each (540 L in total). The target concentration is achieved in ~20 days. UV light is used for sanitization of the fresh medium. The inoculum set-up is provided with (i) a monitoring and control system for the main process parameters, (ii) LED lamps, and (iii) a heat pump thermoregulation system. 500 L of inoculum are used in one loop of the VSt PBRs to start up the cultivation, while the remaining 40 L are used to reactivate the inoculum. The cultivation in the VSt PBRs is a semi-continuous process. Indeed, a start-up phase takes place through a three-stage dilution (with fresh medium) and growth process after cleaning. Overall, the start-up step lasts ~20 days and the PBRs are filled at ~70% with the liquid. Then, the cultivation batches “at regime” are performed by replacing 500 L of algal suspension per PBR loop with fresh medium, thus growing the microalgal biomass from ~1 to 2 gDW L–1 (6.13 days). The cultivation at regime is performed for ~39 batches for each loop per year. The liquid is circulated through the cultivation PBRs by seven pneumatic pumps (one pump for 4 PBR loops). Compressed air (produced by a compressor and stored in a vessel) is used to drive the pumps and to mix the liquid in the PBRs. Nutrients are dosed in a tank containing the fresh medium (~130 L) that is continuously recirculated during UV sterilization. The culture temperature is maintained at the optimal value of 24 °C with a thermoregulation system based on a heat pump and including a recirculation pump and shell and tube heat exchangers. During some dark hours (~8 h/day) the photosynthetic algal growth is maintained with the support of artificial light provided by LEDs (4 panels per each couple of PBR loops). 2.3 Harvesting The algal suspension (~1650 L/day) is concentrated via centrifugation. The centrifuge (Macfuge 325) works ~1 hour per day producing a dewatered suspension at 200 gDW L–1 (concentration factor equal to 100). Algal suspension 2 gDW L–1 Algal suspension 200 gDW L–1 Retentate Waste cleaning solution Electrical energy Tap water System boundary Electrical energy Cleaning agents CLEANING Nutrients Electrical energyCO2 Tap water Retentate Residual culture medium HARVESTING (centrifugation) Infrastructure materials Waste infrastructure materials CULTIVATION (including inoculum) 231 3. Results and discussion Table 2 reports the LCI results. Overall, more than 1.1 m3 kgDW –1 of tap water is consumed almost in equal portions in the two phases of cleaning and cultivation, producing an almost equal total amount of wastewater (mainly from cleaning and harvesting). The chemical consumption amounts to ~4 kg kgDW –1 for both cleaning and cultivation, with major contributions in the two phases due to citric acid and sodium bicarbonate, respectively. PMMA usage in the PBRs is ~0.8 kg kgDW –1. The total energy consumption of the plant amounts to ~376 kWh kgDW –1 and is dominated by thermoregulation (217 kWh kgDW –1) and pumping & agitation (115 kWh kgDW –1), followed by LED lighting (40 kWh kgDW –1), in the cultivation phase. In contrast, energy consumption plays a marginal role in cleaning and harvesting (centrifugation). The CO2 consumption was estimated from laboratory tests, assuming also a mild loss (20% of the input) limited by the dosage controlled by pH measurements. The photosynthetic oxygen was estimated as 1 kgO2 kgCO2 –1 assimilated. Data on possible residual chemicals in the wastewater are not available, but chemical analyses will be performed in the future. Table 2: Life Cycle Inventory of the analysed full-scale PBRs plant producing Chlorella vulgaris. All values are reported for the FU of 1 kg of DW biomass. Cleaning Cultivation Harvesting Input Unit Value Input Unit Value Input Unit Value Tap water L 563 Tap water L 556 Sodium troclosene g 10 Na2MoO4·2H2O g 33 Citric acid g 3797 CaCl2·2H2O g 167 Na2EDTA·2H2O g 83 FeSO4·7H2O g 167 K2HPO4 g 333 K2SO4 g 333 MgSO4·7H2O g 333 MnCl2·4H2O g 33 NaCl g 333 NaHCO3 g 1667 CuSO4·5H2O g 333 H3BO3 g 167 ZnSO4·7H2O g 167 Air for pumping&agitation m3 22 Air for pumping&agitation m3 2672 CO2, kg 2.5 PMMA for PBR infrastructure g 810 Pumping&Agitation kWh 2 Pumping&Agitation kWh 115 Centrifugation kWh 1.4 Lighting kWh 40 Thermoregulation kWh 217 Output Unit Value Output Unit Value Output Unit Value Wastewater L 563 Wastewater L 56 Wastewater L 495 Air m3 22 Air m3 2672 Residual chemicals g N.A. CO2 kg 0.5 Residual chemicals g N.A. O2 kg 2 Waste plastic mat. g 810 A comparative analysis of material and energy inputs was performed with LCI results from the literature (Figure 2). From Pérez-López et al. (2017), data referring to the fall season were selected by assuming that they are the most representative of the yearly average behaviour. Figure 2 shows that all inputs are scattered across at least two orders of magnitude and that the plant inventoried in this study has an intermediate performance. The low water consumption of several systems (FPA and GWP from Onorato and Rösch, 2020, all systems from Pechsiri et al. 2023) is due to recirculation from harvesting, while the high water consumption of the UHT system is due to PBR tubes cooling via water spraying. The minimum and maximum values of water consumption from Herrera et al. (2021) fall in the medium-high range, but they considered also a wastewater scenario, whose actual minimum consumption of freshwater or seawater is zero. Regarding the consumption of chemicals, the plant inventoried in the present study is characterized by high values in line with those reported by Pérez-López et al. (2017), while other studies show values reduced even to ~1 g kgDW –1 for cleaning and to zero for cultivation 232 (nutrients from wastewater). Moreover, the present plant exhibits a middle-high value of plastic mass for bioreactor construction, in line with other VSt systems. The total energy consumption of the present plant is quite high. Compared to the values reported by other studies (where applicable and available), its consumption for thermoregulation is the lowest one, while its consumption for lighting is the second lowest, likely due to the favorable climatic conditions. However, energy for pumping and aeration plays a major role. Figure 2: Main inputs (per kg of DW biomass) of pilot/industrial-scale plants for microalgae cultivation. 4. Conclusions An LCI analysis of an industrial-scale plant cultivating Chlorella vulgaris was performed by using primary data. The results revealed that the plant consumes ~1120 L kgDW –1 of tap water, 7960 g kgDW –1 of chemicals, 810 g kgDW –1 of plastic in the PBRs, and 376 kWh kgDW –1 of electricity. Water and chemicals are used in similar amounts between the cleaning and cultivation phases, while energy is almost fully consumed in the cultivation phase due to thermoregulation, pumping and aeration, and lighting (217, 115, and 40 kWh kgDW –1, respectively). Compared to other inventoried plants, the present one exhibits intermediate levels of resource consumption. Data from the literature are scattered across several orders of magnitude, affected by technical features and process performance (i.e., productivity), including factors related to the location. Moreover, some LCIs are incomplete and not fully transparent, thus weakening the comparison. However, the results highlight that strategies of water recycling (including the use of wastewater, depending on the final product) and energy optimization are crucial for the minimization of resource consumption. The collected primary data will be integrated with further details, including construction materials and transport of materials, thus providing a robust base to conduct LCA studies of great interest for the environmental analysis of commercial implementations. Abbreviations DW – dry weight FPA – flat panel airlift (PBR) FU – functional unit GWP – green wall panel (PBR) Hor – horizontal (PBR) 0.1 1 10 100 1000 10000 VSt Hor VSt ORP FPA GWP UHT ORPmin ORPmax ORP TLC VSt LEB MR-1 MR-2 MR-3 El e ct ri ci ty [ kW h ] Total Thermoregulation Lighting 1 10 100 1000 10000 C h e m ic al s [g ] Cleaning Cultivation 10 100 1000 10000 100000 W at e r [L ] 1 10 100 1000 10000 P la st ic in b io r. [ g] Pérez-López et al. (2017) Onorato and Rösch (2020) This work Herrera et al. (2021) Pechsiri et al. (2023) N.A. N.A. N.A. N.A. N.A.0 N.A. N. A. 0 N. A. 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 233 LCA – life cycle analysis LCI – life cycle inventory LEB – light exchange bubble column (PBR) LED – light-emitting diode MR – (LEB) mini reactor N.A. – not available ORP – open raceway pond PBR – photobioreactor PMMA – polymethylmethacrylate PP – polypropylene PVC – polyvinylchloride RO – reverse osmosis TLC – thin layer cascade UHT – unilayer horizontal tubular (PBR) UV – ultraviolet VSt – vertically stacked (PBR) Acknowledgments This work was carried out with the co-funding of European Union, European Social Fund – REACT EU, PON Ricerca e Innovazione 2014-2020, Azione IV.4 “Dottorati e contratti di ricerca su tematiche dell'innovazione” and Azione IV.6 “Contratti di ricerca su tematiche Green” (DM 1062/2021). Part of this research was supported by Piano di incentivi per la ricerca di Ateneo 2020/2022 (Pia.ce.ri.) Linea 2D - University of Catania. This study was partially carried out within the Agritech National Research Center and received funding from the European Union Next-GenerationEU (Piano nazionale di ripresa e resilienza (PNRR) – Missione 4 Componente 2, Investimento 1.4 – D.D. 1032 17/06/2022, CN00000022). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them. References Beal C.M., Gerber L.N., Sills D.L., Huntley M.E., Machesky S.C., Walsh M.J., Tester J.W., Archibald I., Granados J., Greene C.H., 2015, Algal biofuel production for fuels and feed in a 100-ha facility: A comprehensive techno-economic analysis and life cycle assessment, Algal Research, 10, 266–279. Bradley T., Rajaeifar M.A., Kenny A., Hainsworth C., del Pino V., del Valle Inclán Y., Povoa I., Mendonça P., Brown L., Smallbone A., Roskilly A.P., Joyce S., Heidrich O., 2023, Life cycle assessment of microalgae- derived biodiesel, International Journal of Life Cycle Assessment, 28, 590–609. Braun J.C.A., Colla L.M., 2022, Use of Microalgae for the Development of Biofertilizers and Biostimulants, BioEnergy Research, 1, 3. Gurreri L., Calanni Rindina M., Luciano A., Lima S., Scargiali F., Fino D., Mancini G., 2023, Environmental sustainability of microalgae-based production systems: Roadmap and challenges towards the industrial implementation, Sustainable Chemistry and Pharmacy, 35, 101191. Herrera A., D’Imporzano G., Acién Fernandez F.G., Adani F., 2021. Sustainable production of microalgae in raceways: Nutrients and water management as key factors influencing environmental impacts, Journal of Cleaner Production, 287, 125005. Marangon B.B., Magalhães I.B., Pereira A.S.A.P., Silva T.A., Gama R.C.N., Ferreira J., Castro J.S., Assis L.R., Lorentz J.F., Calijuri M.L., 2023, Emerging microalgae-based biofuels: Technology, life-cycle and scale-up, Chemosphere, 326, 138447. Nanda N., Bharadvaja N., 2022, Algal bioplastics: current market trends and technical aspects, Clean Technologies and Environmental Policy, 24, 2659–2679. Onorato C., Rösch C., 2020, Comparative life cycle assessment of astaxanthin production with Haematococcus pluvialis in different photobioreactor technologies, Algal Research, 50, 102005. Passell H., Dhaliwal H., Reno M., Wu B., Ben Amotz A., Ivry E., Gay M., Czartoski T., Laurin L., Ayer N., 2013, Algae biodiesel life cycle assessment using current commercial data, Journal of Environmental Management, 129, 103–111. Pechsiri J.S., Thomas J.-B.E., El Bahraoui N., Fernandez F.G.A., Chaouki J., Chidami S., Tinoco R.R., Martin J.P., Gomez C., Combe M., Gröndahl F., 2023, Comparative life cycle assessment of conventional and novel microalgae production systems and environmental impact mitigation in urban-industrial symbiosis, Science of the Total Environment, 854, 158445. Pérez-López P., de Vree J.H., Feijoo G., Bosma R., Barbosa M.J., Moreira M.T., Wijffels R.H., van Boxtel A.J.B., Kleinegris D.M.M., 2017, Comparative life cycle assessment of real pilot reactors for microalgae cultivation in different seasons, Applied Energy, 205, 1151–1164. Silva A.G., Carter R., Merss F.L.M., Corrêa D.O., Vargas J.V.C., Mariano A.B., Ordonez J.C., Scherer M.D., 2015, Life cycle assessment of biomass production in microalgae compact photobioreactors, GCB Bioenergy, 7, 184–194. Ubando A.T., Ng E.A.S., Chen W.H., Culaba A.B., Kwon E.E., 2022, Life cycle assessment of microalgal biorefinery: A state-of-the-art review, Bioresource Technology, 360, 127615. Yadav K., Vasistha S., Nawkarkar P., Kumar S., Rai M.P., 2022, Algal biorefinery culminating multiple value- added products: recent advances, emerging trends, opportunities, and challenges, 3 Biotech, 12, 244. 234 58gurreri.pdf Life Cycle Inventory Based on Primary Data of an Industrial Plant for the Cultivation of Chlorella Vulgaris