Effect of harvesting time in the methane production on the anaerobic digestion of microalgae

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tent20 Environmental Technology ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tent20 Effect of harvesting time in the methane production on the anaerobic digestion of microalgae Fernando G. Fermoso, Catalina Hidalgo, Angeles Trujillo-Reyes, Juan CuberoCardoso & Antonio Serrano To cite this article: Fernando G. Fermoso, Catalina Hidalgo, Angeles Trujillo-Reyes, Juan Cubero-Cardoso & Antonio Serrano (2022): Effect of harvesting time in the methane production on the anaerobic digestion of microalgae, Environmental Technology, DOI: 10.1080/09593330.2022.2128893 To link to this article: https://doi.org/10.1080/09593330.2022.2128893 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 12 Oct 2022. Submit your article to this journal Article views: 122 View related articles View Crossmark data Effect of harvesting time in the methane production on the anaerobic digestion of microalgae Fernando G. Fermoso a , Catalina Hidalgo a , Angeles Trujillo-Reyes a , Juan Cubero-Cardoso a and Antonio Serrano b,c a Instituto de la Grasa (C.S.I.C.), Sevilla, Spain; b Institute of Water Research, University of Granada, Granada, Spain; c Department of Microbiology, Pharmacy Faculty, University of Granada, Granada, Spain ABSTRACT Microalgae are being proposed as excellent substrates for different biorefinery processes. Anaerobic digestion process of microalgae is one of these interesting processes but has some limitations in deleting cell walls. For this reason, many studies proposed different types of pre-treatments, entailing energy, operation, and investment costs. This work aims to optimize the anaerobic digestion of the microalgae Chlorella sorokiniana and Chlorella sorokiniana (strain S12/S13/S16) without any pre-treatment by selecting the optimal harvesting time. The greatest influence is seen at 5:00 PM in methane production for both microalgae. For Chlorella sorokiniana, it is the most optimal moment for anaerobic digestion, whereas Chlorella sorokiniana (strain S12/S13/S16) is the least optimal. In the other harvesting times, both microalgae present a similar methane production, i.e. 173 ± 12 mL CH 4 /g of total volatile solids. The highest methane production rate values were obtained during peak sunlight, 1:00 PM and 8:00 AM, respectively, and lower overnight. ARTICLE HISTORY Received 12 July 2022 Accepted 6 September 2022 KEYWORDS Biochemical potential test; carbohydrate; Chlorella sorokiniana; kinetic model; protein 1. Introduction Biorefinery based on microalgae cultures has gained a lot of attention in the last years due to the possibility of obtaining interesting compounds, such as amino acids, fine chemicals, or pigments, as well as the generation of fuels such as biogas, biodiesel, or biohydrogen [1,2]. The yield of these microalgae-based biorefineries is related to the composition of the microalgae cells, which depends on the stimulus received from surroundings such as carbon dioxide, water, nutrients deficit, light intensity, pH, and temperature [3,4]. For example, lipid content, fatty acid, and lipid class compositions of microalgae are strongly influenced by the culture age [5], light intensity, and photoperiod [6]. Other compounds such as photosynthetic pigments, proteins, and carbohydrates have been reported to be also influenced by light [7]. Microalgae have been widely proposed as a substrate for anaerobic digestion processes, allowing the conversion of the microalgae into biomethane for energy production. Species from the genus Chlorella and Scenedesmus have been the most widely studied as a substrate for anaerobic digestion processes [8]. Generally, the specific methane yield described for these processes ranged between 90 and 440 mL CH 4 /g VS (VS, total volatile solids) [8,9]. However, the biomethanization of microalgae can be limited due to the difficulty of degrading the cell walls, whose hydrolysis has been widely reported as one of the main challenges in the anaerobic digestion of microalgae [8,10]. Different pretreatments have been reported as a possible solution to overcome this challenge, such as thermal treatments, mechanical or the application of microwaves or ultrasounds [4,10]. These methods aim to break down the microalgae cell wall, allowing the solubilisation of the organic matter and, thus, facilitating the biological © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-ncnd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. CONTACT Juan Cubero-Cardoso [email protected] Instituto de la Grasa (C.S.I.C.), Campus Universidad Pablo de Olavide. Building 46. Ctra. De Utrera km. 1, Sevilla 41013, Spain ENVIRONMENTAL TECHNOLOGY https://doi.org/10.1080/09593330.2022.2128893 access of the anaerobic microorganisms to the organic matter to be converted into methane [8]. However, the energy consumption during the pre-treatment stage is not always compensated during the subsequent anaerobic digestion, especially for high-intensive pre-treatments [11]. For the pre-treatment of microalgae, the implementation of mechanical treatments is partly limited by the low solid concentration of the microalgae biomass, which results in negative energy balances [10, 12]. Likewise, the energy balances of the thermal treatments would be energetically unfeasible due to the high cost associated with heating the high water content in the microalgae biomass. Most of the studies about microalgae pre-treatments reviewed by Passos et al. [10] showed a ratio between the energy output from the anaerobic digestion and the energy input from the pre-treatments lower than one. On the contrary, Schwede et al. [13] reported a positive energetic balance in the anaerobic digestion of Nannochloropsis salina after a thermal pre-treatment of 100–120 °C for 2 h, relating their positive energy balance with the high concentration of the used microalgae feedstock. Another factor that would influence the methane yield of microalga is the organic matter composition, i.e. proteins, carbohydrates, or lipids content [10]. The composition of the microalgae can be related to the specific microalga species. For example, of the capability of accumulating polyunsaturated fatty acids, microalgae from the genus Bacillariophceae or Chlorophyceae tend to accumulate eicosapentaenoic acid. In contrast, other microalgae such as Crypthecodinium cohnii or species from the genus Schizochytrium have been reported to accrue docosahexaenoic acid [4]. Other authors have described that the anaerobic digestion of microalgae can also be influenced by different parameters such as cultivation conditions, the growth phase of the microalgae culture, or storage conditions due to variations in the microalgae composition [14]. The variation in the time of harvesting during the day would vary the microalgae composition due to variations in different interrelated factors, thus, affecting the anaerobic digestion of the microalgae biomass. This work aimed to determine the optimal moment of the one natural day to harvest the microalgae culture for enhancing methane production, through biochemical methane potential tests, as an alternative to usually proposed pre-treatments methods for enhancing the methane production. The microalgae Chlorella sorokiniana and Chlorella sorokiniana (strain S12/S13/S16) were cultured as model microalgae. The knowledge offered by this research relates the time of harvesting of the selected microalgae with the production of methane that leads to their biodegradation. Also, it was evaluated the substrate composition at each harvesting time as possible explanation of the differences in the methane production. These results could be applied not only to these microalgae, but also to other similar biomasses. 2. Materials and methods 2.1. Microalgae culture and harvesting conditions An initial culture of C. sorokiniana and C. sorokiniana (strain S12/S13/S16) were obtained from Algal Biotechnology group from CIDERTA-University of Huelva (Spain). The identification and description of C. sorokiniana (strain S12/S13/S16) can be found at Tapia et al. [15]. Both microalgae were cultured in a modification Chlamydomonas reinhardtii medium which contained [16]: 0.72 g KH 2 PO 4 ; 1.44 g K 2 HPO 4 ; 1 mL MgSO 4 ·7H 2 O (61% w/v); 1 mL CaCl 2 ·2H 2 O (20% w/v); 0.5 g NH 4 Cl; 0.95 g KNO 3 ; 0.0114 g H 3 BO 3 ; 0.0637 g Na 2 ·EDTA; 0.022 g ZnSO 4 ·H 2 O; 0.005 g MnCl 2 ·4H 2 O; 0.005 g FeSO 4 ·7H 2 O; 0.0016 g CoCl 2 ·6H 2 O; 0.0016 g CuSO 4 ·5H 2 O and 0.0011 g (NH 4 ) 6 Mo 7 O 24 · 4H 2 O per liter. Both microalgae were cultivated in outdoor batch photobioreactors at natural light, temperature, and light/dark period conditions. Each photobioreactor contained approximately 8-L of microalgae culture and was aerated and mixed with air to provide the needed CO 2 at 100 L/h. Stationary phase culture was harvested every 5 h in a 24-hour cycle, i.e. 8:00 AM, 1:00 PM, 5:00 PM, 10:00 PM, and 3:00 AM, by centrifuging at 5000 rpm for 5 min. The concentrate was immediately frozen in liquid nitrogen at –195.8°C for preservation by lyophilization in a lyophilizer Telstar LyoQuest (Spain). Lyophilized microalgae were stored at −20 °C for subsequent analysis. 2.2. Biomethane potential tests The potential of each microalgae sample taken at different times was evaluated in triplicate through biochemical methane potential (BMP) tests in reactors with a working volume of 120 mL at mesophilic conditions (35–37 °C) ,more details about the BMP system can be found in Serrano et al. [17]. The reactors were mechanically continuously stirred through magnetic bars at 300 rpm. The inoculum to substrate ratio was fixed in 2, based on VS. Initially, 120 µL of a micronutrient solution was added to supply all the required micronutrients to the microorganisms with the following composition: 2.28 g/L H 3 BO 3 , 2.747 g/L ZnSO 4 , 1.02 g/L MnCl 2 ·4H 2 O, FeSO 4 · 1.0 g/L 7H 2 O, 0.32 g/L CoCl 2 · 6H 2 O, 0.32 g/L CuSO 4 ·5H 2 O, and 0.22 g/L (NH 4 ) 6 Mo 7 O 24 2F. G. FERMOSO ET AL. ·4H 2 O[18]. 5 mL of a 65 g/L NaHCO 3 solution as an alkalinity source was added to each reactor to ensure the correct pH. Blanks were also used, in triplicate, to subtract the endogenous inoculum activity of the methane produced from the substrates. Methane production was measured daily by liquid displacement after CO 2 removal by bubbling in 2N NaOH. The methane volumes were expressed at standard temperature and pressure conditions for all measurements. 2.3. Chemical analyses The VS and the chemical oxygen demand (COD) determinations were carried out according to the protocols defined by the American Public Health et al. [19]. The determination of the concentration of ammonia was carried out by the colorimetric Nessler Method by Hannah kits. The Lowry method [20]wasusedto measure the protein content of the microalgae samples. Casein was used as a standard for the calibration curve, expressing the results of proteins in the samples as mg of casein equivalent. Total carbohydrates were estimated by the Dubois colorimetric method [21]. A calibration curve using glucose standards was used as a reference, expressing the results as mg of glucose equivalents. 2.4. Kinetic model Afirst-order kinetic model was used for evaluating the methane production for the calculation of the maximum methane yield coefficient (Equation 1) and the methane production rate (Equation 2), according to the following expressions [22]: G=Gmax [1 −exp (−k×t)] (1) where G(mL CH 4 /g VS) is the cumulative specific methane production, G max (mL CH 4 /g VS) is the ultimate specific methane production, k(d −1 )isthespecificrateconstant or apparent kinetic constant, and t(d) is the time. Rm=Gmax ×k(2) where R m (mL CH 4 /(g VS·d)) is the methane production rate. 3. Results and discussion 3.1. Relation between the harvesting time and microalgae composition The chemical characterization of the samples of both microalgae taken at different times is presented in Table 1. As it can be seen, both C. sorokiniana and C. sorokiniana (strain S12/S13/S16) showhed similar COD values, ranging from 655 ± 20 mg O 2 /L to 759 ± 11 mg O 2 /L (Table 1). Therefore, it was impossible to establish any relation between the harvesting time and the COD for both microalgae. Also, the almost constant organic matter concentration would indicate that the microalgae cultures were at a stationary growth phase at the different sampling times [23], ensuring that the obtained differences would not be a consequence of the growing process. The concentration of ammonia in the microalgae samples at each harvesting time is also shown in Table 1. As can be seen, the concentration remained almost constant at the different harvesting times, with mean values of 13.1 ± 0.4 mg/L and 9.2 ± 0.1 mg/L for C. sorokiniana and C. sorokiniana (strain S12/S13/S16), respectively, concentrations similar to those described by Rossi et al. [24]. The ammonium concentration is much lower than those shown to the anaerobic digestion process inhibition, i.e. higher than 1000–1500 mg / L [25]. The concentration of proteins in both C. sorokiniana and C. sorokiniana (strain S12/S13/S16) presented a similar trend, reaching a maximum concentration in the sample harvested at 10:00 PM. However, the proteins concentration in C. sorokiniana (strain S12/S13/S16) was markedly higher than in C. sorokiniana at all the harvesting times (Table 1). The maximum concentration of proteins was 96.6 ± 12.4 mg casein eq/g VS and 129.2 ± 5.8 mg casein eq/g VS for C. sorokiniana and C. sorokiniana (strain S12/S13/S16), respectively. These values mean an increment of around 20% to the lowest concentration of protein for each microalga (Table 1). These protein values are lower than the percentages described for Chlorella sp. by other authors, which varied between 35 and 60% in dry weight [26, 27]. Similarly, the concentration of carbohydrates was also higher in C. sorokiniana (strain S12/S13/S16) than in C. sorokiniana (Table 1). Both microalgae presented a reasonable constant carbohydrate concentration regardless of the harvesting time, except for abnormal data for each microalga (8:00 AM for C. sorokiniana and 10:00 PM for C. sorokiniana (strain S12/S13/S16)). Concretely, the mean values for the concentration of carbohydrates were 12.2 ± 2.8 mg glucose eq./g VS and 15.2 ± 2.4 mg glucose eq./g VS for C. sorokiniana and C. sorokiniana (strain S12/S13/S16), respectively (Table 1). The increase in the protein concentration in the samples harvested at 10:00 PM would be intriguing due to these compounds’high theoretical methane potential, i.e. 0.851 L CH 4 /g VS, compared to the carbohydrates, whose theoretical methane yield is 0.415 L/g VS [28]. ENVIRONMENTAL TECHNOLOGY 3 3.2. Relation between the harvesting time and the biomethane production The variation of the methane yield coefficient throughout the experimental time for the anaerobic digestion of each microalga at each harvesting time is shown in Figure 1a and b. As it can be seen, the methane yield coefficient rapidly increased during the first 4-days, without any lag phase for both C. sorokiniana (Figure 1a) and C. sorokiniana (strain S12/S13/S16) (Figure 1b). The absence of a lag phase indicated that the hydrolysis was not a rate-limiting stage during the microalgae degradation due to readily degradable components in the substrate [29,30]. The decrease in the methane yield coefficient observed for some samples was a consequence of the activity of the microorganisms in the blanks, which still produced a small amount of biogas when the biogas production in the samples was the exhaust. The first-order kinetic model was implemented to calculate the methane production rate and facilitate the comparison of the methane yield coefficient with other authors [31]. Figure 2 shows the ultimate specific methane production for each microalga harvested at each time. As it can be seen, the optimal harvesting time for C. sorokiniana was at 5:00 PM, with a G max value of 214 ± 6 mL CH 4 /g VS (Figure 2). This value was 42% higher than the value of G max obtained for the same microalgae at 10:00 PM. The highest content of proteins would explain this difference in the samples of C. sorokiniana harvested at 10:00 PM (Table 1). The anaerobic degradation of proteins can increase the concentration of volatile fatty acids, ammonia, or, to a lesser extent, hydrogen and sulphur compounds which can inhibit the anaerobic digestion process at specific concentrations [32]. Samples of C. sorokiniana (strain S12/ S13/S16) presented quite similar values of G max to those obtained for C. sorokiniana (Figure 2), except for the sample harvested at 5:00 PM, which showed the lowest values of G max , i.e. only 132 ± 6 mL CH 4 /g VS. This value was 30% lower than the highest value of G max obtained for C. sorokiniana (strain S12/S13/S16) at 3:00 AM (Figure 2). Despite the lower value obtained at 5:00 PM, the values of G max for C. sorokiniana (strain S12/S13/S16) remained almost constant, with a mean value of 173 ± 12 mL CH 4 /g VS. Usually, the variation in the composition has been seen to influence the biodegrading of biomass [33,34]. However, the variation in the composition of the biomasses at the different harvesting time was not very marked, being not possible to establish a clear relation with the variations in the methane production, which would be related to other factors. For example, differences in the cell wall microalga morphology and thickness, which has been reported for different growth phases, could explain variations in the anaerobic biodegradability [35]. Therefore, it is possible to conclude that the harvesting time has a strong influence in the methane yield coefficient obtained from the microalgae, although was not possible to establish a clear relationship this variation and the analysed characteristics of the microalgae. Figure 3 shows the values of R m for each microalga harvested at each time. For both microalgae, the highest R m values were obtained during daylight hours (from 8:00 AM to 5:00 PM), while the lowest R m values corresponded to samples harvested at night (Figure 2). It is worth noting that this trend was more marked for C. sorokiniana than for C. sorokiniana (strain S12/S13/ S16). Concretely, the values of R m for C. sorokiniana varied from only 41 mL CH 4 /(g VS·d) at 3:00 AM to 80 mL CH 4 /(g VS·d) at 5:00 PM, i.e. an increment of around 100%. These methane production rate values were in the same range as those reported by Beltrán et al. [36], which reported a methane production rate of 76.6 mL CH 4 /(g VS·d) during the batch anaerobic digestion of C. sorokiniana at mesophilic temperature. Table 1. Characterization of the microalgae samples harvested at each different time, where COD, chemical oxygen demand, VS, total volatile solids. Chlorella sorokiniana Harvesting time COD (mg O 2 /L) Proteins (mg casein eq./g VS) Carbohydrates (mg glucose eq./g VS) Ammonia (mg/L) 3:00 AM 707 ± 8 87.9 ± 2.5 12.8 ± 0.6 13.7 ± 0.4 8:00 AM 658 ± 8 86.0 ± 3.5 7.2 ± 0.6 12.9 ± 0.5 1:00 PM 681 ± 8 80.9 ± 5.5 13.6 ± 0.6 13.0 ± 0.3 5:00 PM 655 ± 20 89.6 ± 6.2 13.5 ± 0.5 13.1 ± 0.3 10:00 PM 759 ± 11 96.6 ± 12.4 13.9 ± 0.4 12.8 ± 0.1 Chlorella sorokiniana (strain S12/S13/S16) Harvesting time COD (mg O 2 /L) Proteins (mg casein eq./g VS) Carbohydrates (mg glucose eq./g VS) Ammonia (mg/L) 3:00 AM 682 ± 7 113.9 ± 2.7 15.5 ± 0.2 9.2 ± 0.3 8:00 AM 733 ± 66 109.6 ± 0.9 12.7 ± 0.2 9.0 ± 0.5 1:00 PM 693 ± 35 120.7 ± 2.0 14.1 ± 0.2 9.2 ± 0.4 5:00 PM 682 ± 22 108.3 ± 1.1 15.1 ± 0.4 9.3 ± 04 10:00 PM 665 ± 7 129.2 ± 5.8 19.1 ± 0.5 9.2 ± 0.3 4F. G. FERMOSO ET AL. Figure 1. (A) Variation of the methane production with time for C. sorokiniana harvested at different times, and (B) Variation of the methane production with time for C. sorokiniana (strain S12/S13/S16) harvested at different times, where VS, total volatile solids. ENVIRONMENTAL TECHNOLOGY 5 The enhancement in the values of R m achieved at varying the harvesting time was similar to one described by Córdova et al. [37], which reported a variation from 11.56 ± 0.15 mL CH 4 /(g VS·d) to values between 18 and 28 mL CH 4 /(g VS·d) at applying different physical pretreatment methods before the anaerobic digestion of Figure 2. Variation of the ultimate specific methane production (G max ) for C. sorokiniana and C. sorokiniana (strain S12/S13/S16) harvested at different times, where VS, total volatile solids. Figure 3. Variation of the methane production rate (R m ) for C. sorokiniana and C. sorokiniana (strain S12/S13/S16) harvested at different times, where VS, total volatile solids. 6F. G. FERMOSO ET AL. C. sorokiniana. Therefore, it was impossible to determine a clear relation between the microalgae composition and the anaerobic digestion behaviour at the different harvesting times. However, the harvesting time strongly influenced R m , resulting in variations like the obtained by other authors through the application of pre-treatments to the microalgae [33]. 4. Conclusions The anaerobic digestion process evaluated the methane production and methane production rate from C. sorokiniana than for C. sorokiniana (strain S12/S13/ S16). The microalgae have been harvested at one natural time every 5 h and evaluated through a biochemical methane potential test. The influence of sunlight in harvesting both microalgae has been demonstrated. In the other harvesting times, both microalgae present a similar methane production, i.e. 173 ± 12 mL CH 4 / g VS. The methane production rates have been the most optimal at 1:00 PM and 8:00 AM for C. sorokiniana and C. sorokiniana (strain S12/S13/ S16), respectively. Acknowledgement Antonio Serrano is grateful to the Economic Transformation, Industry, Knowledge and Universities Department of the Andalusia Autonomous Government for his Emergia fellowship (Reference: EMERGIA20_00114). Disclosure statement No potential conflict of interest was reported by the author(s). Funding The author(s) reported there is no funding associated with the work featured in this article. ORCID Fernando G. Fermoso http://orcid.org/0000-0002-2586-007X Angeles Trujillo-Reyes http://orcid.org/0000-0003-1064-5083 Juan Cubero-Cardoso http://orcid.org/0000-0002-4455-5417 Antonio Serrano http://orcid.org/0000-0002-4615-5038 References [1] Chew KW, Yap JY, Show PL, et al. Microalgae biorefinery: high value products perspectives. 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