Microsoft Word - 66iervolino.docx CHEMICAL ENGINEERINGTRANSACTIONS VOL. 65, 2018 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Eliseo Ranzi, Mario Costa Copyright © 2018, AIDIC Servizi S.r.l. ISBN978-88-95608- 62-4; ISSN 2283-9216 Investigation of Desmodesmus sp. Growth in Photobioreactor using Vinasse as a Carbon Source Gabriela F. Ferreiraa, Daniel S. Fernandesa, Luisa F. Ríos Pinto*a, Marija B. Tasicb, Rubens Maciel Filhoa a Faculty of Chemical Engineering, University of Campinas - 500 Albert Einstein Av, 13083-852, Campinas/SP, Brazil b Faculty of Technology, University of Niš, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia luisa.rpinto@yahoo.com Renewable source biofuels are a promising alternative to fossil fuel and can be economic and environmentally viable when associated with effluent gas and wastewater treatment. This work proposes the study of lipid accumulation and microalgae cultivation using vinasse which is a residue from sugarcane bioethanol production. Besides being a largely produced effluent that requires bioremediation, vinasse is a carbon and nutrient rich source that could be advantageous as microalgae cultivation medium. Therefore, the objective of this research is to cultivate the microalgae strain Desmodesmus sp. in different types of undiluted vinasse medium (raw, raw recycled and biodigested) in a photobioreactor system. Cultivation was performed under different conditions varying: I) bubble agitation; II) temperature; III) light incidence; IV) flux of atmospheric air supplemented with CO2. Biomass concentration was measured by cell density, lipid accumulation was measured gravimetrically after solvent extraction, and vinasse biochemical composition was determined using TOC-VCSN Shimadzu® analyzer. The levels of dissolved O2 and CO2 were monitored using O2 and CO2 electrodes, respectively. Raw vinasse with relative biomass concentration of 1.5 g L-1 showed to be preferred medium for high lipid accumulations and carbon consumption. The maximum lipid content of 24.48% was obtained under heterotrophic, aerobic (0.05 VVM) growth conditions, at a temperature of 26 ˚C, without magnetic agitation and CO2 presence. However, for effluent treatment, better results were obtained under mixotrophic, aerobic (0.05 VVM) and bubble agitated growth conditions, at 26 ˚C of temperature. Recorded carbon reduction was 46.35%. As vinasse consists of an organic carbon and nutrient source, cultivation cultures are susceptible to contamination by other microorganisms. Therefore, further study is necessary to the use of Desmodesmus sp. microalga for either biodiesel production or bioremediation. 1. Introduction Microalgae have been considered as a promising feedstock for biodiesel production due to several advantages comparing to conventional crops such as soybean and palm oil (Georgianna and Mayfield 2012). They have higher growth rate, can accumulate fuel precursor’s oils of good quality, do not require extensive areas and arable land for cultivation, are efficient CO2 sequesters, among others (Katiyar et al. 2017). In addition to bioenergy, microalgal biomass is also applicable in food and pharmaceutical industries once it allows to obtain proteins, carbohydrates, and lipids that may be feedstock for such industries. To produce large-scale sustainable microalgae products, however, it is still necessary to develop technologies that guarantee its economic viability (Moheimani et al. 2015). Because growth systems require large quantities of water, one alternative for sustainable microalgae cultivation is to use an industrial or domestic residue as a culture media, such as wastewater. Several microalgae species have been efficiently applied in wastewater and effluent treatment, once they assimilate nutrients from the medium (Samorì et al. 2013). A potential effluent that is both a source of nutrients (nitrogen, phosphorous, and potassium) and organic matter is vinasse. It is a residue generated after the fractional distillation of the wine from sugarcane juice fermentation, in the sugar and ethanol industry. For each litter of ethanol, 10 to 15 litters of vinasse are produced (Cortes-Rodríguez et al. 2018), which is mainly applied to fertirrigation of sugarcane crops. However, studies suggest that this application poses adverse impacts due to possible soil and subterranean 721 DOI: 10.3303/CET1865121 Please cite this article as: Ferreira G., Fernandes D., Rios Pinto L., Tasic M., Maciel Filho R., 2018, Investigation of desmodesmus sp. growth in photobioreactor using vinasse as a carbon source., Chemical Engineering Transactions, 65, 721-726 DOI: 10.3303/CET1865121 water pollution. Soil salinization was associated with vinasse probably due to high levels of total dissolved solids and conductivity (Fuess, Rodrigues, and Garcia 2017). Currently, the primary treatment for vinasse is biodigestion, a process that can aggregate value by producing biogas. Despite that, it does not eliminate the environmental impact obstacle, once the effluent still contains high quantity of inorganic nutrients (Candido and Lombardi, 2017). Therefore, microalgae are a possible and interesting solution as they could uptake nutrients and organic matter, potentially reducing oxygen biochemical and chemical demands, odor, conductivity, among other harmful parameters for soils and water environments. Different microalgae species have been tested for cultivation in vinasse, showing biomass production improvements. Santana et al. (2017) obtained higher biomass productivities using clarified vinasse (164.44 and 222.22 mg L-1 d-1) or 50% diluted vinasse (177.78 and 182.22 mg L-1 d-1) in comparison with Bold’s Basal Medium (BBM) (101.11 and 132.22 mg L-1 d-1), for Micractinium sp. and Chlamydomonas biconvexa, respectively, cultivated in 15 L airlift flat plate photobioreactors. Candido and Lombardi (2017) showed that Chlorella vulgaris could grow in 60% conventional and 80% biodigested vinasse solutions in 250-mL flasks, resulting in high specific growth rate (1.2 g day-1), competitively with the synthetic medium. Mattos and Bastos (2016) evaluated Desmodesmus sp. growth in sugarcane vinasse, obtaining 52.1% nitrogen removal and 36.2% chemical oxygen demand reduction. Other studies have approached microalgal growth in vinasse (Altenhofen da Silva et al. 2017; dos Santos et al. 2016; Olguín et al. 2015; Ramirez et al. 2014), thus confirming this is a potentially sustainable medium. Amongst those, Ramirez et al. (2014) were able to cultivate microalga Scenedesmus sp. at concentrations up to 40% vinasse in culture medium. Olguín et al. (2015) cultivated Neochloris oleoabundans in anaerobically digested vinasse (AEV), obtaining 62% cell density increase and 85.2% ammonium-nitrogen removal using 6% AEV and sodium bicarbonate, compared to BBM. In this context, this work proposes the use of vinasse (raw and biodigested) as a carbon concentration culture medium, for microalgae Desmodesmus sp. cultivation in a closed system, aiming lipid accumulation for biodiesel production. 2. Methodology 2.1 Microalgae inoculum Desmodesmus sp. strain was donated by the Aquatic Organisms Research Laboratory (LAPOA) from the Integrated Aquiculture and Ambiental Studies Group (GIA) at the Federal University of Paraná (UFPR) in Curitiba/PR. It was maintained in 100 mL tubes with BG-11 medium (Rippka et al., 1979), under continuous light (approximately 62 μmol m-2s-1). For the photobioreactor cultivation, the inoculum was previously transferred to a 250 mL Erlenmeyer and agitated through an orbital shaker (SOLAB SL-180/A) continuously operated for 2-3 weeks. All media for inoculum were sterilized in an autoclave (Phoenix Luferco® AV-50 Plus) and culture manipulation before inculcation in photobioreactor was performed in a laminar flux chamber (Pachane® Pa50) to avoid contamination. 2.2 Culture medium: vinasse Vinasse (both raw and biodigested) was donated by the Brazilian Bioethanol Science and Technology Laboratory (CTBE) and maintained refrigerated between 4 and 8 °C. The medium pretreatment consisted in to remove suspended solid particles by centrifugation (Eppendorf 5810R), for 20 min, 18 °C, and 5000 rpm. For all cultivations, pH was adjusted to 7.5 with a NaOH 6.5 M solution. When air and/or CO2 were added to the system, pH was placed between 7.5 and 8.0; and between 7.0 and 7.5 without CO2 addition to account for CO2 acidity in an aqueous medium. 2.3 Cultivation in flat-plate photobioreactor Cultivation was performed in a flat-plate vertical photobioreactor (FMT-150/1000 Photons Systems Instruments®), varying injection of CO2 and atmospheric air. For a total volume of 800 mL, cultures containing 20% inoculum in vinasse were added to the photobioreactor, with a headspace volume of 400 mL. All experiments (Table 1) were conducted from 4 to 11 days, varying parameters such as light, temperature, agitation, air injection, and vinasse type. During the nine cultivations, dissolved gases, temperature, pH, cell density, dry biomass, and nutrients (carbon and nitrogen) present in the substrate were monitored. A rotameter controlled the gas injection. Aliquots were taken daily to evaluate biomass growth, monitoring cell density, using an optical microscope (Olympus® CX21) and Neubauer chamber. Also, total carbon (TC) and total nitrogen (TN) concentrations were determined using a total organic carbon analyzer (TOC-VCSN Shimadzu®). After cultivation, total lipid was quantified by Bligh and Dyer (1959) method. 722 Table 1: Growth conditions of Desmodesmus sp. cultivation in a flat-plate photobioreactor. Parameters/ Cultivation Period (days) Vinasse Light Temperature (°C) Agitation Contamination Air flow (vvm) CO2 flow (%/vvm) A 11 Raw No 26 No Bacteria 0.5 0 B 10 Raw Yes 25 Yes (bubbling) Fungi 0.5 0 C 4 Recicled (B) No 25 Yes (bubbling) Bacteria 0.5 0 D 7 Recicled (C) No 25 Yes (bubbling) Bacteria 0.5 0 E 7 Recicled (D) 50% diluted Yes 25 No Fungi 0.5 0 F 7 Raw No 20 No Bacteria 0.5 0 G 10 Raw Yes 21 Yes (bubbling) Fungi 0.25 10 H 9 Recicled (F) + 1.28 g K2HPO4 Yes 12:12h light:dark 21 Yes (bubbling) Bacteria 1 5 I 10 Biodigested No 21 Yes (bubbling) Fungi 0.5 0 3. Results and discussion During all cultivations the pH showed no significant changes, remaining at approximately 8.0 and between 6.0 and 8.0 without and with the CO2 injection, respectively. These values indicate the buffer characteristic of vinasse, which interfered in the dissolution of CO2 since HCO3 ions are generated (Nguyen and Rittmann, 2016). In turn, this was not observed during the injection of air, once the dissolution is not governed by a chemical reaction, but by mass transfer and oxygen solubility in water (Lee, 2017). The Desmodesmus sp. strain was cultivated in various vinasse types under different conditions (Table 1) to evaluate the effect of cultivation conditions upon microalgae growth (Figure 1), nutrients consumption (Figure 2) and lipid content (Figure 3). As can be seen from Table 1 cultivations were performed under mixotrophic (B, E, G, and H) or heterotrophic (A, C, D, F, and I) conditions. At this point is worthwhile mentioning that microalgae growth, presented as algal cell density, was periodically measured up to some interference such as contamination appeared. It was observed the presence of fungi and high concentration of bacteria, which compromised the further analysis of the growth curves. Comparing to heterotrophic, mixotrophic conditions using raw vinasse show to be a more favorable way of Desmodesmus sp. cultivation (Figure 1). While heterotrophic cultures (A and F) showed practically constant cell density throughout the cultivations, mixotrophic cultures (B and G) presented an increase in cell density. Further, increasing temperature and air injection did not lead to significant accumulation of biomass (cell density of B cultures was higher compared to G). This means that low temperature kept increase dissolving of air-CO2 thus favoring the photosynthesis. However, carbon consumption in culture B was higher probably because: a) presence of contaminations or; b) of the higher carbon decomposition rate caused by higher temperature), as depicted in Figure 2. The same trend was observed in heterotrophic cultures (A and F). Figure 1: Cell density of Desmodesmus sp. growth in vinasse, monitored using an optical microscope, for cultivations: A (■), B (□), C (●), D (○), E (▲), F (∆), G (▼), H (∇), and I (✕). 723 Figure 2: Percent reduction of total organic carbon ( ), inorganic carbon ( ), total carbon ( ), and total nitrogen ( ) in vinasse medium. Due to the low nutrient consumption, cultures with raw vinasse recycled once (C and H), twice (D) and three times (E) were used for cultivation of Desmodesmus sp. Due to the depletion of nutrients, the recycling was limited at twice (as can be concluded from the results of cultivation E) and recycling gave better results with add-in nutrients, such as of K2HPO4 (culture H). Heterotrophic conditions (C and D cultivations) favored higher cell density using raw recycled vinasse, but the highest cell density value was obtained for mixotrophic (H) culture because of phosphate supplementation (Figure 1). According to Prokop, Bajpa, and Zappi (2014), phosphate is an essential nutrient for cell growth, and consequently, carbon consumption. Although higher cell density was obtained under heterotrophic conditions, carbon reduction was more drastic in the mixotrophic cultures (Figure 2). Which is due to initially higher cell density (culture C) and presence of phosphate supplementation (culture H). In cultivation with biodigested vinasse (I), there was a decrease in the final cell density (three times less than in the initial) and an increase in the content of carbon. The decrease in cell density was the consequence of cell death and lysis due to the presence of the harmful components in the biodigested vinasse, mainly relative to the concentration of phenolic compounds (Candido and Lombardi, 2017) and presence of contaminats which resulted in nutrient shortages. On the other hand, cell death and lysis lead in an increase in carbon content (Kan and Pan, 2010). Further studies aimed to test the potential of Desmodesmus sp, cultivated on various types of vinasse, as an alternative source of oil (lipids). Also, biomass’s results were explained concerning algal biomass productivity rather than cell density or biomass concentration, since it is of commercial importance. As lipid and biomass data are interconnected when it comes to economic viability, it is plausible to analyze it concomitantly (Figure 3). As observed in Figure 3, for raw vinasse cultures the highest lipid content (approximately 24%, cultures A, and F) and highest biomass productivity (culture F) were obtained using heterotrophic conditions. Among nutrients, nitrogen is of most importance for biomass production (Prokop, Bajpa and Zappi, 2014) and lipid content (Rios et al., 2015). Obtained highest lipid content was due to lowest TN reduction. As observed in Figure 2, there was a low consumption of nitrogen in all raw vinasse cultures, leading the accumulation of lipids. In the analysis of recycled raw vinasse, the highest lipid content and biomass production were also obtained for heterotrophic conditions in once recycled vinasse (culture C). Although high biomass production and lipid content could also be noticed in mixotrophic cultivation (culture H), these increases were due to the addition of phosphate and increased CO2 flux. Also, recycled cultivation results indicate better total carbon and nitrogen contents reduction (up to 46%). Despite the decrease in cell density, biodigested vinasse showed to be preferable for biomass production rather than lipid content. However, the most significant difference comparing to other cultivations was the increase of both TC and TN content. In this case, TN increasing had no relation with lipid accumulation, but preferably with cell death and lysis. 724 Figure 3: Biomass productivity ( ) and lipid content (■) for Desmodesmus sp. cultivated in different types of vinasse. The lack of studies involving Desmodesmus sp. cultivation integrated to effluent treatment makes it difficult to compare with other’s results. Santana et al. (2017) obtained lower biomass productivities than in this work, however, using different microalgae species, cultivated in 100% clarified and 50% diluted vinasse. Also, lower TOC reduction (around 8%) in comparison to the 14% obtained on average. Mattos and Bastos (2016) achieved higher nitrogen consumption in a 30-h batch cultivation of Desmodesmus sp in vinasse., but an initial biomass concentration of 1 g L-1. Moreover, bacteria growth was also observed, with an approximate exponential behavior. Therefore, microalgae cultivation in vinasse is dependent of various experimental conditions and can be strongly influenced by contamination. 4. Conclusions and final remarks Microalgae growth in a sustainable medium still faces challenges for scale-up and, mainly, to avoid contamination with other microorganisms. The presented study evaluated the possibility of growing microalga Desmodesmus sp. in a closed system, using vinasse as carbon and nutrient source. It was investigated the use of raw, recycled and biodigested vinasses. Results showed that under heterotrophic conditions, recycled raw vinasse and raw vinasse were the most suitable media for biomass production and lipid content, respectively. However, for effluent treatment, mixotrophic culture in once recycled raw vinasse with nutrient supplementation (phosphate) was more suitable. Due to contamination with other microorganisms (fungi and bacteria), it was not possible to conduct all the experiments during the same period. Despite that, promising results aiming microalgae biomass production were obtained, and bioremediation analyzed through carbon and nitrogen reductions. Recycling raw vinasse was proved to be a potential step, and biodigested vinasse presents a harsh environment for this microalga growth. 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