Bioresource Technology 174 (2014) 256–265
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Assessing microalgae biorefinery routes for the production of biofuels via hydrothermal liquefaction Diego López Barreiro a,⇑, Chiara Samorì b, Giuseppe Terranella c, Ursel Hornung c, Andrea Kruse c,d, Wolter Prins a a
Department of Biosystems Engineering, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium Centro Interdipartimentale di Ricerca Industriale (CIRI), University of Bologna, via S. Alberto 163, 48123 Ravenna, Italy Institute for Catalysis Research and Technology, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmoltz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany d Conversion Technology and Life Cycle Assessment of Renewable Resources (440f), Institute of Agricultural Engineering, University Hohenheim, Garbenstrasse 9, 70599 Stuttgart, Germany b c
h i g h l i g h t s HTL of two algae species was studied after extracting high value products. Extracting lipids has no beneficial effects in the biofuel quality and quantity. Extracting proteins increases the biofuel yields, reducing its nitrogen content. A high recovery of nutrients can be achieved in the aqueous by-product.
a r t i c l e
i n f o
Article history: Received 3 September 2014 Received in revised form 6 October 2014 Accepted 7 October 2014 Available online 14 October 2014 Keywords: Microalgae Hydrothermal liquefaction Biofuel production Biorefinery
a b s t r a c t The interest in third generation biofuels from microalgae has been rising during the past years. Meanwhile, it seems not economically feasible to grow algae just for biofuels. Co-products with a higher value should be produced by extracting a particular algae fraction to improve the economics of an algae biorefinery. The present study aims at analyzing the influence of two main microalgae components (lipids and proteins) on the composition and quantity of biocrude oil obtained via hydrothermal liquefaction of two strains (Nannochloropsis gaditana and Scenedesmus almeriensis). The algae were liquefied as raw biomass, after extracting lipids and after extracting proteins in microautoclave experiments at different temperatures (300–375 °C) for 5 and 15 min. The results indicate that extracting the proteins from the microalgae prior to HTL may be interesting to improve the economics of the process while at the same time reducing the nitrogen content of the biocrude oil. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The interest in microalgae as a source of third generation biofuels has increased during the last few years, due to the concerns related to the climate change and an increasing world population with higher energy demands. The advantages and disadvantages of microalgae have been highlighted in several papers (Patil et al., 2008; Malcata, 2011; Khoo et al., 2013; Liu et al., 2013), and the different techniques to convert them to biofuels widely reviewed (Brennan and Owende, 2010). In this context, hydrothermal liquefaction (HTL) has recently gained momentum as conversion technique, with a growing num⇑ Corresponding author. Tel.: +32 9 264 6190. E-mail address: [email protected] (D. López Barreiro). http://dx.doi.org/10.1016/j.biortech.2014.10.031 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
ber of publications (Zou et al., 2010; Yu et al., 2011; García Alba et al., 2012; Valdez et al., 2012). If an algae-based liquid fuel is pursued, HTL appears to be the best technology to achieve this, as it avoids the high energy costs of drying the algae, while converting all the algae fractions into biofuel by using the water naturally accompanying the feedstock. HTL benefits from the special properties of hot compressed water at near critical conditions (Peterson et al., 2008) that enhance the conversion of biomass to biofuel. Although much has been discussed about the optimal configuration for an HTL-based algal biorefinery, it is still not clear how it should look like. There are various approaches which can be divided into two main pathways: either directly using the whole microalgae biomass for HTL; or previously extracting valuable compounds from the algae cells and then converting the residual biomass into biofuel. A consensus seems to have been established
D. López Barreiro et al. / Bioresource Technology 174 (2014) 256–265
in support of the second pathway. Through this second approach it is possible to widen the portfolio of products that can be obtained in an algae biorefinery and improve its economics. VanthoorKoopmans et al. (2013) and Draaisma et al. (2013) have recently reviewed the possibilities of coupling the production of algae biofuels with the extraction of higher value compounds for food, feed and chemicals, and explored the market opportunities of these algae co-products. The extraction of lipids from algae has been extensively studied, due to the interest in obtaining algae biodiesel (Mata et al., 2010). Algal lipids can also be used for the production of omega 3 fatty acids for food (such as eicosapentanoic acid and docosahexaenoic acid) or building blocks for chemicals (Vanthoor-Koopmans et al., 2013). Some researchers have already investigated the performance under hydrothermal conditions of lipid-extracted algae (Vardon et al., 2012; Zhu et al., 2013). There is also some work available with regard to microalgal proteins. Huo et al. (2011) investigated the production of alcohol by using metabolic engineering of Escherichia coli to convert proteins into C4 and C5 alcohols. García-Moscoso et al. (2013) investigated the flash hydrolysis of Scenedesmus sp. to extract proteins and produce under mild hydrothermal conditions a biofuel intermediate with low nitrogen content (the presence of nitrogen in the biofuel is undesired as it would lead to NOx emissions). Proteins can be used for the production of fertilizers and antioxidants, and also for human and animal nutrition (Romero García et al., 2012; Vanthoor-Koopmans et al., 2013; Draaisma et al., 2013). Moreover, the use of amino acid based fertilizers has been indicated as beneficial for metabolic energy savings, since the nitrogen from amino acids does not need to be reduced in order to be uptaken by the biomass like in the case of nitrates (Huo et al., 2011; Romero García et al., 2012). Although the extraction of valuable co-products would positively impact the economics of the process, it is not clear what effect does the extraction of a certain fraction of the algae have on the biofuel yield and quality. Previous work carried in our group on a set of eight strains (López Barreiro et al., 2013) showed that via HTL the marine species Nannochloropsis gaditana and the freshwater species Scenedesmus almeriensis produced biocrude oil with a low amount of nitrogen. These two strains were selected for this study. A comparison has been made while considering the hydrothermal conversion of three different algae materials: in their raw state, after extracting lipids and after extracting proteins. Temperatures from 300 to 375 °C and reaction times of 5 and 15 min were tested in microautoclave experiments, with the objective of investigating the role of the presence/absence of lipids or proteins in the yield and quality of the biocrude oil. Currently many studies are being carried out analyzing the potential of HTL for several algae strains. But to the best of our knowledge, this paper is the first one reporting a systematic comparative study of the influence on the yields of HTL and the quality of the biocrude oil of the extraction of two distinct algae fractions (lipids and proteins) to obtain valuable co-products from marine and freshwater strains.
2. Methods 2.1. Chemicals All solvents and chemicals used in this study were obtained from Sigma–Aldrich and Merck (purities P98%) and used without any further purification.
257
2.2. Raw microalgae (RA) N. gaditana (CCAP 849/5) and S. almeriensis (CCAP 276/24) were obtained in a dry state from the cultivation facility in Las Palmerillas (University of Almería, Spain). They will be called NG and SA respectively from now onwards. The lipid content was determined gravimetrically by the Bligh & Dyer method (Bligh and Dyer, 1959), and the protein content was determined following González López et al. (2010). 2.3. Lipid-extracted algae (LEA) The lipid-extracted algae were obtained by subjecting 5 g of the dry raw algae to a Soxhlet extraction with n-hexane (VWRÒ, >99% purity) for 5 h, according to the DIN EN ISO 734–1 Norm (2006). The extraction was done in triplicate to check the reproducibility of the data. 2.4. Protein-extracted algae (PEA) The protein-extracted algae were obtained by subjecting approximately 200 g of the dry raw algae to the procedure developed by Romero García et al. (2012). This method consists of an enzymatic hydrolysis that yields amino acid concentrates. The algae cells were first disrupted via high-pressure homogenization, and then subjected to enzymatic hydrolysis by using the endoprotease Alcalase 2.5 L and the exoprotease Flavourzyme 1000 L to obtain the amino acids. Viscozyme L was added to the medium as well to reduce the viscosity of the mixture in order to enhance the mass transfer and thus improve the yield of the enzymatic hydrolysis. The success in the production of amino acid concentrates is measured by the degree of hydrolysis, which represents the amount of free amino acids hydrolyzed, compared to the total amount of proteins available for hydrolysis. For further details about the extraction method, the reader is referred to Romero García et al. (2012). 2.5. Algae paste characterization The dry weight of the algae feedstock was determined by subjecting the sample to 105 °C overnight, and the ash content was obtained at 550 °C for 5 h. The elemental composition (in weight percentage) was measured by a CHNS Analyzer Flash 2000 (Thermo Scientific) and used in Boie’s formula (Annamalai et al., 1987) (Eq. (1)) to calculate the Higher Heating Value (HHV) of the biocrude oil. 1
HHVBoie ðMJ kg Þ ¼ 0:3516 C þ 1:16225 H 0:1109 O þ 0:0628 N
ð1Þ
The composition of the inorganic matter was investigated by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) on an Agilent 7025 instrument. The salts from the cultivation medium need to be removed from the algae pellet before determining its organic content to avoid overestimations caused due to the presence of hydrated forms of these salts in the dry mass of the algae pastes (Zhu and Lee, 1997). The organic content was determined in a different way for each type of feedstock: for RA an algae pellet was centrifuged two times (10,000 rpm, 10 min) with 40 mL of de-ionized water, so that the salts in the slurry could dissolve and be removed with the supernatant after centrifugation. The remaining pellet was then dried overnight at 105 °C to determine the bio-dry weight (free of salts). The ash content of the pellet was further analyzed by subjecting the sample to 550 °C for 5 h in a muffle furnace.
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D. López Barreiro et al. / Bioresource Technology 174 (2014) 256–265
The difference in weight between the bio-dry weight and the ash was taken as the organic content of the biomass. For LEA the determination of the organic content was done by subtracting the mass of lipids extracted from the initial organic content of the raw cells. The mass of lipids extracted was determined by weighting the dry algae feedstock before and after the Soxhlet extraction. For PEA the organic content was determined as for RA. The algae cells were broken after the enzymatic extraction, which could lead to a loss of organic matter in the supernatant during the centrifugation. However, the enzymatic extraction process already includes a centrifugation step, so all the possible losses of watersoluble organic matter would already take place at this stage. Therefore, we assume this method to be valid also for this feedstock, which is also confirmed by the good closure of the mass balances (above 90 wt% in most of the cases). 2.6. Hydrothermal liquefaction (HTL), product separation and analysis The HTL experiments have been carried out for RA, LEA and PEA in microautoclaves with a volume of 10 mL and made of stainless steel 1.4571. They were filled up to 70% of their volume with an algae–water mixture (mass ratio 1:10), and flushed with nitrogen to remove any air present inside. Following this, 20 bar of nitrogen were loaded to the reactor before closing it. The reaction was carried out by introducing the autoclaves in a GC-oven, which allowed an easy control of the temperature. A first set of experiments were carried out for RA from both strains at 350 °C to identify the best reaction time (5 or 15 min). Subsequently, four different temperatures (300, 325, 350 and 375 °C) have been applied for 15 min. The heating process took, depending on the selected reaction temperature, between 12 and 19 min, which corresponded to a heating rate of 19–23 °C min1. Once the reaction temperature was achieved, it was maintained constant during the desired reaction time. Subsequently, the autoclaves were submerged in an ice bath for fast quenching. After cooling down, the autoclaves were opened. The inner pressure of the autoclave was recorded with a manometer (Swagelok), and a gas sample was taken for analysis in a gas chromatograph (HP 6890). The other products (biocrude oil, aqueous phase and solid residue) were withdrawn from the reactor and filtered using a Whatman nylon membrane (47 mm, 0.45 lm pore size). Most of the biocrude oil produced remained stuck to the microautoclave walls, requiring the use of an organic solvent to complete its collection. 25 mL of dichloromethane were used, in stepwise additions of 5 mL. The solid products were recovered through vacuum filtration. To ensure that no biocrude remained in the filter cake, 10 mL of dichloromethane were additionally used to wash it. The filtrate consisted of a bi-phase mixture (water with organic matter dissolved, and biocrude oil dissolved in dichloromethane), which was centrifuged (7500 rpm, 10 min) to enable the separation between phases. The aqueous phase was recovered with a syringe. The biocrude oil, dissolved in dichloromethane, was flushed for 24 h with nitrogen to remove the solvent and any water that could still be present in the sample. The yields of the different product phases i (biocrude oil, aqueous phase, solid residue and gas) were calculated on an organic basis as the ratio of the weight of the recovered organic mass (mi) of each product i and the mass of microalgae (dry, ash free) initially loaded to the reactor, according to Eq. (2):
Yieldi ðwt%Þ ¼
mi mmicroalgaeðdafÞ
100
ð2Þ
For statistical reasons, the experiments were repeated four times for the RA and LEA, and three times for the PEA. Data is
reported with their corresponding standard deviations. The product yields were also subjected to a one-way analysis of variance (ANOVA) with regard to the temperature at a significance level of a = 0.05. The organic matter present in the aqueous phase was determined by drying aliquots of 1.5 mL at 60 °C for 24 h, and then treating the dry residue at 550 °C for 5 h to determine the ash content. Total carbon, total organic carbon and total nitrogen in the aqueous phase were measured by a DimatecÒ2000 instrument. Ion chromatography was applied for the determination of anions/ cations (Metrohm device) and organic acids (Merck Hitachi device). The mass of gas was calculated with the ideal gas law, considering the pressure of the gas after the HTL experiment and its composition (measured by GC), and it was then used to calculate the gas yield (Eq. (2)). The filtrated solids were quantified by drying them at 105 °C overnight to remove any residual water and dichloromethane after the filtration. Their ash content was determined by treating them at 550 °C for 5 h in a muffle furnace. The elemental composition of the biocrude oil was analyzed via a CHNS Analyzer Flash 2000 (Thermo Scientific) and the oxygen content was determined by difference (100-C-H-N-S). The values were used to calculate the HHV in Eq. (1).
3. Results and discussion 3.1. Extraction of lipids and proteins Prior to the extraction of lipids and proteins, the content of these two biochemical fractions in RA was determined. The amount of total lipids was 13.4% for NG and 13.1% for SA. The protein content was 32.2% for NG and 30.0% for SA. Typically, neutral lipids present in the form of triacylglycerides (TAGs) are preferred for high-value products derived from microalgae (Vanthoor-Koopmans et al., 2013). Therefore, n-hexane was the solvent of choice to produce lipid-extracted algae, due to its selectivity towards algae neutral lipids compared to other solvents (Shin et al., 2014). The amount of lipids extracted with n-hexane from each feedstock varied significantly among the two strains tested. For NG, the mass extracted with n-hexane was 11.1 ± 1.4 wt%, while for SA it was only 2.8 ± 0.5 wt%. This shows that in the case of NG a great fraction of the total lipids are neutral, but not in the case of SA. With regard to the extraction of proteins (done by enzymatic hydrolysis), again both species behaved differently. The degree of hydrolysis (percentage of the total proteins effectively extracted) was 44.3% for NG and 62.0% for SA, indicating that the enzymatic technique used was more effective towards the hydrolysis of the proteins from SA. It needs to be emphasized that the enzymatic extraction used to produce PEA leads to a significant loss of other algal fractions apart from proteins. For NG, 53.5% of the total mass from RA used for the extraction was lost. This loss was even higher for SA, reaching 80.9%. This shows that the enzymatic extraction removes not only proteins, but also significant amounts of the other biochemical fractions and inorganic matter.
3.2. Feedstock characterization A full characterization of the feedstock can be found in Table 1, which shows the high content in inorganic elements of the algae pastes. The high hygroscopicity of some salts present in water has been remarked in the Standard Methods for Examination of Water and Wastewater (APHA–AWWA–WPCF, 1998). These hydrated salts can lead to overestimations in the determination
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D. López Barreiro et al. / Bioresource Technology 174 (2014) 256–265 Table 1 Feedstock characterization: elemental composition, ash content, mineral elements (in wt%) and HHV (in MJ kg1).
*
Strain
Type
C
H
N
S
O*
Ash
HHV
Ca
Fe
K
Mg
Na
P
Si
Zn
Nannochloropsis gaditana
RA Without lipids (LEA) Without proteins (PEA)
47.6 43.9 52.8
7.5 7.1 8.3
6.9 7.4 6.5
0.5 1.1 1.0
25.1 26.0 19.0
12.4 14.5 12.4
23.1 21.3 26.5
0.50 0.62 0.68
0.05 0.05 0.05
1.30 1.56 3.12
0.27 0.32 0.23
3.02 3.64 1.49
1.43 1.72 1.30
50%, most of it as ammonia). Na and K exhibit an almost total recovery. The recoverability of P and S seems to be strongly affected by the other salts present in the feedstock. Acknowledgements The financial support by the Institute for the Promotion of Innovation by Science and Technology (IWT) from Belgium is acknowledged. Fundación Cajamar is also acknowledged for providing the algae biomass, and Franciso García Cuadra for the proteinextracted algae. Sonja Habbicht, Hermann Köhler and Armin Lautenbach are acknowledged for their assistance in the analyses. References Annamalai, K., Sweeten, J.M., Ramalingam, S.C., 1987. Estimation of gross heating values of biomass fuels. Trans. ASAE 30, 1205–1208. APHA–AWWA–WPCF, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. Washington (DC): American Public Health Association (APHA) American Water Works Association (AWWA) Water Pollution Control Federation (WPCF), Washington, DC. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae – a review of technologies for production, processing, and extractions of biofuels and co-products. Renewable Sustainable Energy Rev. 14, 557–577. Draaisma, R.B., Wijffels, R.H., Slegers, P.E., Brentner, L.B., Roy, A., Barbosa, M.J., 2013. Food commodities from microalgae. Curr. Opin. Biotechnol. 24, 169–177. European Standard UNE-EN ISO 734–1, 2006. Oilseeds Meals. Determination of oil Content. Part 1: Extraction Method with Hexane (or Light Petroleum), Brussels, Belgium. Garcia Alba, L., Torri, C., Samori, C., van der Spek, J., Fabbri, D., Kersten, S.R., Brilman, D.W.F., 2012. Hydrothermal treatment (HTT) of microalgae: evaluation of the process as conversion method in an algae biorefinery concept. Energy Fuels 26, 642–657. García Alba, L., Torri, C., Fabbri, D., Kersten, S.R., Brilman, D.W.F., 2013. Microalgae growth on the aqueous phase from hydrothermal liquefaction of the same microalgae. Chem. Eng. J. 228, 214–223. Garcia-Moscoso, J.L., Obeid, W., Kumar, S., Hatcher, P.G., 2013. Flash hydrolysis of microalgae (Scenedesmus sp.) for protein extraction and production of biofuels intermediates. J. Supercrit. Fluids 82, 183–190. González López, C.V., Cerón García, M.C., Acién Fernández, F.G., Segovia Bustos, C.R., Chisti, Y., Fernández Sevila, J.M., 2010. Protein measurements of microalgal and cyanobacterial biomass. Bioresour. Technol. 101, 7587–7591. Huo, Y.-X., Cho, K.M., Rivera, J.G.L., Monte, E., Shen, C.R., Yan, Y., Liao, J.C., 2011. Conversion of proteins into biofuels by engineering nitrogen flux. Nat. Biotechnol. 29, 346–351. Khoo, H., Koh, C., Shaik, M., Sharratt, P., 2013. Bioenergy co-products derived from microalgae biomass via thermochemical conversion – Life cycle energy balances and CO2 emissions. Bioresour. Technol. 143, 298–307.
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Assessing microalgae biorefinery routes for the production of biofuels via hydrothermal liquefaction Diego López Barreiro a,⇑, Chiara Samorì b, Giuseppe Terranella c, Ursel Hornung c, Andrea Kruse c,d, Wolter Prins a a
Department of Biosystems Engineering, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium Centro Interdipartimentale di Ricerca Industriale (CIRI), University of Bologna, via S. Alberto 163, 48123 Ravenna, Italy Institute for Catalysis Research and Technology, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmoltz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany d Conversion Technology and Life Cycle Assessment of Renewable Resources (440f), Institute of Agricultural Engineering, University Hohenheim, Garbenstrasse 9, 70599 Stuttgart, Germany b c
h i g h l i g h t s HTL of two algae species was studied after extracting high value products. Extracting lipids has no beneficial effects in the biofuel quality and quantity. Extracting proteins increases the biofuel yields, reducing its nitrogen content. A high recovery of nutrients can be achieved in the aqueous by-product.
a r t i c l e
i n f o
Article history: Received 3 September 2014 Received in revised form 6 October 2014 Accepted 7 October 2014 Available online 14 October 2014 Keywords: Microalgae Hydrothermal liquefaction Biofuel production Biorefinery
a b s t r a c t The interest in third generation biofuels from microalgae has been rising during the past years. Meanwhile, it seems not economically feasible to grow algae just for biofuels. Co-products with a higher value should be produced by extracting a particular algae fraction to improve the economics of an algae biorefinery. The present study aims at analyzing the influence of two main microalgae components (lipids and proteins) on the composition and quantity of biocrude oil obtained via hydrothermal liquefaction of two strains (Nannochloropsis gaditana and Scenedesmus almeriensis). The algae were liquefied as raw biomass, after extracting lipids and after extracting proteins in microautoclave experiments at different temperatures (300–375 °C) for 5 and 15 min. The results indicate that extracting the proteins from the microalgae prior to HTL may be interesting to improve the economics of the process while at the same time reducing the nitrogen content of the biocrude oil. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The interest in microalgae as a source of third generation biofuels has increased during the last few years, due to the concerns related to the climate change and an increasing world population with higher energy demands. The advantages and disadvantages of microalgae have been highlighted in several papers (Patil et al., 2008; Malcata, 2011; Khoo et al., 2013; Liu et al., 2013), and the different techniques to convert them to biofuels widely reviewed (Brennan and Owende, 2010). In this context, hydrothermal liquefaction (HTL) has recently gained momentum as conversion technique, with a growing num⇑ Corresponding author. Tel.: +32 9 264 6190. E-mail address: [email protected] (D. López Barreiro). http://dx.doi.org/10.1016/j.biortech.2014.10.031 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
ber of publications (Zou et al., 2010; Yu et al., 2011; García Alba et al., 2012; Valdez et al., 2012). If an algae-based liquid fuel is pursued, HTL appears to be the best technology to achieve this, as it avoids the high energy costs of drying the algae, while converting all the algae fractions into biofuel by using the water naturally accompanying the feedstock. HTL benefits from the special properties of hot compressed water at near critical conditions (Peterson et al., 2008) that enhance the conversion of biomass to biofuel. Although much has been discussed about the optimal configuration for an HTL-based algal biorefinery, it is still not clear how it should look like. There are various approaches which can be divided into two main pathways: either directly using the whole microalgae biomass for HTL; or previously extracting valuable compounds from the algae cells and then converting the residual biomass into biofuel. A consensus seems to have been established
D. López Barreiro et al. / Bioresource Technology 174 (2014) 256–265
in support of the second pathway. Through this second approach it is possible to widen the portfolio of products that can be obtained in an algae biorefinery and improve its economics. VanthoorKoopmans et al. (2013) and Draaisma et al. (2013) have recently reviewed the possibilities of coupling the production of algae biofuels with the extraction of higher value compounds for food, feed and chemicals, and explored the market opportunities of these algae co-products. The extraction of lipids from algae has been extensively studied, due to the interest in obtaining algae biodiesel (Mata et al., 2010). Algal lipids can also be used for the production of omega 3 fatty acids for food (such as eicosapentanoic acid and docosahexaenoic acid) or building blocks for chemicals (Vanthoor-Koopmans et al., 2013). Some researchers have already investigated the performance under hydrothermal conditions of lipid-extracted algae (Vardon et al., 2012; Zhu et al., 2013). There is also some work available with regard to microalgal proteins. Huo et al. (2011) investigated the production of alcohol by using metabolic engineering of Escherichia coli to convert proteins into C4 and C5 alcohols. García-Moscoso et al. (2013) investigated the flash hydrolysis of Scenedesmus sp. to extract proteins and produce under mild hydrothermal conditions a biofuel intermediate with low nitrogen content (the presence of nitrogen in the biofuel is undesired as it would lead to NOx emissions). Proteins can be used for the production of fertilizers and antioxidants, and also for human and animal nutrition (Romero García et al., 2012; Vanthoor-Koopmans et al., 2013; Draaisma et al., 2013). Moreover, the use of amino acid based fertilizers has been indicated as beneficial for metabolic energy savings, since the nitrogen from amino acids does not need to be reduced in order to be uptaken by the biomass like in the case of nitrates (Huo et al., 2011; Romero García et al., 2012). Although the extraction of valuable co-products would positively impact the economics of the process, it is not clear what effect does the extraction of a certain fraction of the algae have on the biofuel yield and quality. Previous work carried in our group on a set of eight strains (López Barreiro et al., 2013) showed that via HTL the marine species Nannochloropsis gaditana and the freshwater species Scenedesmus almeriensis produced biocrude oil with a low amount of nitrogen. These two strains were selected for this study. A comparison has been made while considering the hydrothermal conversion of three different algae materials: in their raw state, after extracting lipids and after extracting proteins. Temperatures from 300 to 375 °C and reaction times of 5 and 15 min were tested in microautoclave experiments, with the objective of investigating the role of the presence/absence of lipids or proteins in the yield and quality of the biocrude oil. Currently many studies are being carried out analyzing the potential of HTL for several algae strains. But to the best of our knowledge, this paper is the first one reporting a systematic comparative study of the influence on the yields of HTL and the quality of the biocrude oil of the extraction of two distinct algae fractions (lipids and proteins) to obtain valuable co-products from marine and freshwater strains.
2. Methods 2.1. Chemicals All solvents and chemicals used in this study were obtained from Sigma–Aldrich and Merck (purities P98%) and used without any further purification.
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2.2. Raw microalgae (RA) N. gaditana (CCAP 849/5) and S. almeriensis (CCAP 276/24) were obtained in a dry state from the cultivation facility in Las Palmerillas (University of Almería, Spain). They will be called NG and SA respectively from now onwards. The lipid content was determined gravimetrically by the Bligh & Dyer method (Bligh and Dyer, 1959), and the protein content was determined following González López et al. (2010). 2.3. Lipid-extracted algae (LEA) The lipid-extracted algae were obtained by subjecting 5 g of the dry raw algae to a Soxhlet extraction with n-hexane (VWRÒ, >99% purity) for 5 h, according to the DIN EN ISO 734–1 Norm (2006). The extraction was done in triplicate to check the reproducibility of the data. 2.4. Protein-extracted algae (PEA) The protein-extracted algae were obtained by subjecting approximately 200 g of the dry raw algae to the procedure developed by Romero García et al. (2012). This method consists of an enzymatic hydrolysis that yields amino acid concentrates. The algae cells were first disrupted via high-pressure homogenization, and then subjected to enzymatic hydrolysis by using the endoprotease Alcalase 2.5 L and the exoprotease Flavourzyme 1000 L to obtain the amino acids. Viscozyme L was added to the medium as well to reduce the viscosity of the mixture in order to enhance the mass transfer and thus improve the yield of the enzymatic hydrolysis. The success in the production of amino acid concentrates is measured by the degree of hydrolysis, which represents the amount of free amino acids hydrolyzed, compared to the total amount of proteins available for hydrolysis. For further details about the extraction method, the reader is referred to Romero García et al. (2012). 2.5. Algae paste characterization The dry weight of the algae feedstock was determined by subjecting the sample to 105 °C overnight, and the ash content was obtained at 550 °C for 5 h. The elemental composition (in weight percentage) was measured by a CHNS Analyzer Flash 2000 (Thermo Scientific) and used in Boie’s formula (Annamalai et al., 1987) (Eq. (1)) to calculate the Higher Heating Value (HHV) of the biocrude oil. 1
HHVBoie ðMJ kg Þ ¼ 0:3516 C þ 1:16225 H 0:1109 O þ 0:0628 N
ð1Þ
The composition of the inorganic matter was investigated by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) on an Agilent 7025 instrument. The salts from the cultivation medium need to be removed from the algae pellet before determining its organic content to avoid overestimations caused due to the presence of hydrated forms of these salts in the dry mass of the algae pastes (Zhu and Lee, 1997). The organic content was determined in a different way for each type of feedstock: for RA an algae pellet was centrifuged two times (10,000 rpm, 10 min) with 40 mL of de-ionized water, so that the salts in the slurry could dissolve and be removed with the supernatant after centrifugation. The remaining pellet was then dried overnight at 105 °C to determine the bio-dry weight (free of salts). The ash content of the pellet was further analyzed by subjecting the sample to 550 °C for 5 h in a muffle furnace.
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The difference in weight between the bio-dry weight and the ash was taken as the organic content of the biomass. For LEA the determination of the organic content was done by subtracting the mass of lipids extracted from the initial organic content of the raw cells. The mass of lipids extracted was determined by weighting the dry algae feedstock before and after the Soxhlet extraction. For PEA the organic content was determined as for RA. The algae cells were broken after the enzymatic extraction, which could lead to a loss of organic matter in the supernatant during the centrifugation. However, the enzymatic extraction process already includes a centrifugation step, so all the possible losses of watersoluble organic matter would already take place at this stage. Therefore, we assume this method to be valid also for this feedstock, which is also confirmed by the good closure of the mass balances (above 90 wt% in most of the cases). 2.6. Hydrothermal liquefaction (HTL), product separation and analysis The HTL experiments have been carried out for RA, LEA and PEA in microautoclaves with a volume of 10 mL and made of stainless steel 1.4571. They were filled up to 70% of their volume with an algae–water mixture (mass ratio 1:10), and flushed with nitrogen to remove any air present inside. Following this, 20 bar of nitrogen were loaded to the reactor before closing it. The reaction was carried out by introducing the autoclaves in a GC-oven, which allowed an easy control of the temperature. A first set of experiments were carried out for RA from both strains at 350 °C to identify the best reaction time (5 or 15 min). Subsequently, four different temperatures (300, 325, 350 and 375 °C) have been applied for 15 min. The heating process took, depending on the selected reaction temperature, between 12 and 19 min, which corresponded to a heating rate of 19–23 °C min1. Once the reaction temperature was achieved, it was maintained constant during the desired reaction time. Subsequently, the autoclaves were submerged in an ice bath for fast quenching. After cooling down, the autoclaves were opened. The inner pressure of the autoclave was recorded with a manometer (Swagelok), and a gas sample was taken for analysis in a gas chromatograph (HP 6890). The other products (biocrude oil, aqueous phase and solid residue) were withdrawn from the reactor and filtered using a Whatman nylon membrane (47 mm, 0.45 lm pore size). Most of the biocrude oil produced remained stuck to the microautoclave walls, requiring the use of an organic solvent to complete its collection. 25 mL of dichloromethane were used, in stepwise additions of 5 mL. The solid products were recovered through vacuum filtration. To ensure that no biocrude remained in the filter cake, 10 mL of dichloromethane were additionally used to wash it. The filtrate consisted of a bi-phase mixture (water with organic matter dissolved, and biocrude oil dissolved in dichloromethane), which was centrifuged (7500 rpm, 10 min) to enable the separation between phases. The aqueous phase was recovered with a syringe. The biocrude oil, dissolved in dichloromethane, was flushed for 24 h with nitrogen to remove the solvent and any water that could still be present in the sample. The yields of the different product phases i (biocrude oil, aqueous phase, solid residue and gas) were calculated on an organic basis as the ratio of the weight of the recovered organic mass (mi) of each product i and the mass of microalgae (dry, ash free) initially loaded to the reactor, according to Eq. (2):
Yieldi ðwt%Þ ¼
mi mmicroalgaeðdafÞ
100
ð2Þ
For statistical reasons, the experiments were repeated four times for the RA and LEA, and three times for the PEA. Data is
reported with their corresponding standard deviations. The product yields were also subjected to a one-way analysis of variance (ANOVA) with regard to the temperature at a significance level of a = 0.05. The organic matter present in the aqueous phase was determined by drying aliquots of 1.5 mL at 60 °C for 24 h, and then treating the dry residue at 550 °C for 5 h to determine the ash content. Total carbon, total organic carbon and total nitrogen in the aqueous phase were measured by a DimatecÒ2000 instrument. Ion chromatography was applied for the determination of anions/ cations (Metrohm device) and organic acids (Merck Hitachi device). The mass of gas was calculated with the ideal gas law, considering the pressure of the gas after the HTL experiment and its composition (measured by GC), and it was then used to calculate the gas yield (Eq. (2)). The filtrated solids were quantified by drying them at 105 °C overnight to remove any residual water and dichloromethane after the filtration. Their ash content was determined by treating them at 550 °C for 5 h in a muffle furnace. The elemental composition of the biocrude oil was analyzed via a CHNS Analyzer Flash 2000 (Thermo Scientific) and the oxygen content was determined by difference (100-C-H-N-S). The values were used to calculate the HHV in Eq. (1).
3. Results and discussion 3.1. Extraction of lipids and proteins Prior to the extraction of lipids and proteins, the content of these two biochemical fractions in RA was determined. The amount of total lipids was 13.4% for NG and 13.1% for SA. The protein content was 32.2% for NG and 30.0% for SA. Typically, neutral lipids present in the form of triacylglycerides (TAGs) are preferred for high-value products derived from microalgae (Vanthoor-Koopmans et al., 2013). Therefore, n-hexane was the solvent of choice to produce lipid-extracted algae, due to its selectivity towards algae neutral lipids compared to other solvents (Shin et al., 2014). The amount of lipids extracted with n-hexane from each feedstock varied significantly among the two strains tested. For NG, the mass extracted with n-hexane was 11.1 ± 1.4 wt%, while for SA it was only 2.8 ± 0.5 wt%. This shows that in the case of NG a great fraction of the total lipids are neutral, but not in the case of SA. With regard to the extraction of proteins (done by enzymatic hydrolysis), again both species behaved differently. The degree of hydrolysis (percentage of the total proteins effectively extracted) was 44.3% for NG and 62.0% for SA, indicating that the enzymatic technique used was more effective towards the hydrolysis of the proteins from SA. It needs to be emphasized that the enzymatic extraction used to produce PEA leads to a significant loss of other algal fractions apart from proteins. For NG, 53.5% of the total mass from RA used for the extraction was lost. This loss was even higher for SA, reaching 80.9%. This shows that the enzymatic extraction removes not only proteins, but also significant amounts of the other biochemical fractions and inorganic matter.
3.2. Feedstock characterization A full characterization of the feedstock can be found in Table 1, which shows the high content in inorganic elements of the algae pastes. The high hygroscopicity of some salts present in water has been remarked in the Standard Methods for Examination of Water and Wastewater (APHA–AWWA–WPCF, 1998). These hydrated salts can lead to overestimations in the determination
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D. López Barreiro et al. / Bioresource Technology 174 (2014) 256–265 Table 1 Feedstock characterization: elemental composition, ash content, mineral elements (in wt%) and HHV (in MJ kg1).
*
Strain
Type
C
H
N
S
O*
Ash
HHV
Ca
Fe
K
Mg
Na
P
Si
Zn
Nannochloropsis gaditana
RA Without lipids (LEA) Without proteins (PEA)
47.6 43.9 52.8
7.5 7.1 8.3
6.9 7.4 6.5
0.5 1.1 1.0
25.1 26.0 19.0
12.4 14.5 12.4
23.1 21.3 26.5
0.50 0.62 0.68
0.05 0.05 0.05
1.30 1.56 3.12
0.27 0.32 0.23
3.02 3.64 1.49
1.43 1.72 1.30
50%, most of it as ammonia). Na and K exhibit an almost total recovery. The recoverability of P and S seems to be strongly affected by the other salts present in the feedstock. Acknowledgements The financial support by the Institute for the Promotion of Innovation by Science and Technology (IWT) from Belgium is acknowledged. Fundación Cajamar is also acknowledged for providing the algae biomass, and Franciso García Cuadra for the proteinextracted algae. Sonja Habbicht, Hermann Köhler and Armin Lautenbach are acknowledged for their assistance in the analyses. References Annamalai, K., Sweeten, J.M., Ramalingam, S.C., 1987. Estimation of gross heating values of biomass fuels. Trans. ASAE 30, 1205–1208. APHA–AWWA–WPCF, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. Washington (DC): American Public Health Association (APHA) American Water Works Association (AWWA) Water Pollution Control Federation (WPCF), Washington, DC. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae – a review of technologies for production, processing, and extractions of biofuels and co-products. Renewable Sustainable Energy Rev. 14, 557–577. Draaisma, R.B., Wijffels, R.H., Slegers, P.E., Brentner, L.B., Roy, A., Barbosa, M.J., 2013. Food commodities from microalgae. Curr. Opin. Biotechnol. 24, 169–177. European Standard UNE-EN ISO 734–1, 2006. Oilseeds Meals. Determination of oil Content. Part 1: Extraction Method with Hexane (or Light Petroleum), Brussels, Belgium. Garcia Alba, L., Torri, C., Samori, C., van der Spek, J., Fabbri, D., Kersten, S.R., Brilman, D.W.F., 2012. Hydrothermal treatment (HTT) of microalgae: evaluation of the process as conversion method in an algae biorefinery concept. Energy Fuels 26, 642–657. García Alba, L., Torri, C., Fabbri, D., Kersten, S.R., Brilman, D.W.F., 2013. Microalgae growth on the aqueous phase from hydrothermal liquefaction of the same microalgae. Chem. Eng. J. 228, 214–223. Garcia-Moscoso, J.L., Obeid, W., Kumar, S., Hatcher, P.G., 2013. Flash hydrolysis of microalgae (Scenedesmus sp.) for protein extraction and production of biofuels intermediates. J. Supercrit. Fluids 82, 183–190. González López, C.V., Cerón García, M.C., Acién Fernández, F.G., Segovia Bustos, C.R., Chisti, Y., Fernández Sevila, J.M., 2010. Protein measurements of microalgal and cyanobacterial biomass. Bioresour. Technol. 101, 7587–7591. Huo, Y.-X., Cho, K.M., Rivera, J.G.L., Monte, E., Shen, C.R., Yan, Y., Liao, J.C., 2011. Conversion of proteins into biofuels by engineering nitrogen flux. Nat. Biotechnol. 29, 346–351. Khoo, H., Koh, C., Shaik, M., Sharratt, P., 2013. Bioenergy co-products derived from microalgae biomass via thermochemical conversion – Life cycle energy balances and CO2 emissions. Bioresour. Technol. 143, 298–307.
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