Biocrude production through Maillard reaction between leucine and

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Biofuels and Biomass

Biocrude production through Maillard reaction between leucine and glucose during the hydrothermal liquefaction Yi Qiu, Aersi Aierzhati, Jun Cheng, Hao Guo, Weijuan Yang, and Yuanhui Zhang Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.9b01875 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 31, 2019

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Highlight 1) Leucine and glucose were used as model molecules to investigate the Maillard reactions between microalgal proteins and carbohydrates. 2) Pyrazine derivatives repolymerized from feedstock degradation products consisted of 84.2% biocrude. 3) Maillard reactions gave over fivefold increase on biocrude production (47.6 wt.%).

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Biocrude production through Maillard reaction between leucine and glucose during the hydrothermal liquefaction Yi Qiua, Aersi Aierzhatib, Jun Chenga*, Hao Guoa, Weijuan Yanga, Yuanhui Zhangb aState

Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China

bDepartment

of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign,

Urbana, IL 61801, USA

Abstract In this work, leucine and glucose were used as model molecules to investigate the Maillard reaction between proteins and carbohydrates during hydrothermal liquefaction (HTL) of microalgae. The main pathway of Maillard reaction between leucine and glucose via HTL was related to the reaction of deaminated leucine with cyclic oxygenated compounds from degraded glucose to produce pyrazine derivatives. GC-MS results revealed that N&O-heterocyclic compounds, organic acids, and carbonyls/imine/amine were the main components of both biocrude and aqueous fraction. The optimal reaction temperature, reaction time, leucine to glucose weight ratio and solid concentration for biocrude production are 320 °C, 60 min, 2:5, and 20 wt.%, respectively. The obtained biocrude (higher heating value of 38.07 MJ/kg), presented large amount of pyrazine derivatives (84.2% peak area), yielded 47.6 wt.% of leucine and glucose feedstock.


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Key words: Maillard reaction; Hydrothermal liquefaction; Biocrude; Leucine; Glucose 

Corresponding author: Prof. Dr. Jun Cheng, State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou



310027, China. Tel.: +86 571 87952889; Fax: +86 571 87951616. E-mail: [email protected].

1. Introduction With increasing demand for energy overlapping the reduction of fossil resources and an increased amount of organic wastes, the proposed energy-related solutions continuously increase. For instance, the hydrothermal processes are often applied to convert wet biomass into valuable solid, liquid, and gaseous fuels. Hydrothermal liquefaction (HTL) refers to the thermochemical conversion of carbonaceous resources into oily substances in hot pressurized sub/supercritical water. During HTL, the biomass is decomposed into four valuable products, i.e., a crude-like bio-oil with higher heating values up to 35 – 40 MJ/kg, a combustible solid residue called ‘char’, an aqueous phase containing light polar platform chemicals, and a CO2-rich gaseous phase also containing certain amounts of hydrogen and light hydrocarbons 1. Microalgae are promising feedstocks for renewable energy and production of valuable chemicals. HTL of microalgae to produce biofuels is beneficial in terms of high conversion efficiency due to the properties of the reaction medium, sub/supercritical water. Hence, drying pretreatment is avoided and the energy consumption is reduced

2, 3.

During the HTL process of microalgae, the intermediate

products of carbohydrates and proteins degradation react with each other, process


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known as Maillard reaction. The yields of bio-crude are 5 – 25 wt.% higher than the lipid content of the microalgae depending on the biochemical composition 4, 5. Kruse et al. found that the Maillard reaction involves the generation of free radical scavengers, which decreases the rate of free radical chain reactions that are highly relevant for gas formation 6, 7. Therefore, the gas yield is lower in the presence of proteins or amino acids compared with the systems free of these compounds. In other words, the amino acids derived from the hydrolysis of the proteins promoted the conversion of the carbohydrates into the liquid fuel. This phenomenon was reported by many other papers for the pyrolysis process. Wang et al. performed single pyrolysis and co-pyrolysis of microalgal components (castor oil, soybean protein, and glucose were used as model compounds) to reveal the interaction between various feedstocks by thermogravimetric analysis and biocrude evaluation 8. However, these studies focused on the Maillard reaction during pyrolysis conversion. As for hydrothermal liquefaction process, Biller and Ross used biochemical components as model molecules (e.g., albumin and a soya protein, starch and glucose, the triglyceride from sunflower oil and two amino acids), microalgae and cyanobacteria with different biochemical contents as feedstock 4. All these feedstocks were liquefied at 350 °C, 200 bar, 1 M Na2CO3, and 1 M formic acid to predict the behavior of microalgae with different biochemical composition. Gai et al. studied the conversion of two low-lipid content microalgae by hydrothermal liquefaction in subcritical water between 200 °C and 320 °C and proposed the general reaction network involved in the conversion of those lipids during HTL process 9. However,


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they did not investigate the interaction between glucose and amino acids (compounds of carbohydrates and proteins degradation) during HTL process. Peterson et al. studied the kinetics and mechanism of a Maillard-type reaction using glucose-glycine mixtures as feedstock in hydrothermal process and found that the presence of glucose always resulted in higher glycine destruction 10. On the other hand, the glycine caused the increase or decrease of glucose conversion depending on the initial concentration of glycine. Although progresses in understanding the reactions occurring during the biomass conversion by HTL process are already made, more studies are still needed to understand the anomalous behavior observed under hydrothermal conditions. Fan et al. used lactose, maltose, and lysine as model molecules and tested them individually and in binary mixtures to evaluate the Maillard reaction network 11. They found that the presence of carbohydrates in the mixtures (e.g., lysine - carbohydrates) led to bio-oil yields exceeding those obtained from the conversion of single substances (10 – 39 wt.%). The reaction scheme involving the key chemical compounds was proposed based on the results provided by FT-IR and NMR spectroscopies. The analysis of the functional groups of those compounds revealed that the carbohydrates are hydrolyzed into glucose before the reaction with lysine to produce biocrude. However, the principal amino acids in the algal feedstock are glutamic acid (15.0 - 17.4%), aspartic acid (10.9 - 13.4%), leucine (10.6 - 12.0%), valine (8.2 - 10.7%) and alanine (8.1 10.0%) 12 and lysine was not an ideal model compound for the protein in microalgae. In this study, based on preliminary experimental results, leucine and D-glucose reaction has been chosen for detailed investigation, because its ability to produce high


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biocrude amount with glucose. The effects of the reaction temperature, feedstock ratio, reaction time, and solid concentration of feedstock on biocrude oil yield were studied

through

the

hydrothermal

liquefaction.

Gas

chromatography-mass

spectrometry (GC-MS) was used to identify the components of both biocrude and aqueous fraction obtained from HTL. After detailed analysis of the experimental results, a predicted reaction pathway of leucine and D-glucose via HTL was proposed.

2. Materials and methods 2.1 Materials The model compounds used in this study, glucose and leucine, were of analytical grade and purchased from Sigma-Aldrich. GCMS-grade dichloromethane (DCM, CH2Cl2) purchased from Sigma-Aldrich was used to recover the bio-oil from the reaction vessel.

2.2 Hydrothermal liquefaction experiment The HTL reactions were performed using 30 mL stainless steel cylinder batch reactors in a tube furnace (Thermo Scientific Lindberg Blue M). Various feedstock ratio (leucine: glucose of 7:0, 4:3, 3:4, 2:5, 1:6, and 0:7 ), feed solutions concentrations (5, 7.5, 10, 15, and 20%), reaction temperatures (280, 300, 320, and 340°C) and reaction time (10, 20, 40, 60, 90 min) were tested. In a typical HTL experiment, the bath reactors loading with feedstock was sealed with high-pressure valves and then purged with nitrogen gas to replace the air in the chamber (the initial


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pressure of 0.50 MPa). The reactor was then transferred into the tube furnace which had been heated up to the desired reaction temperature. It took about 3–4 min for the reactor surface to reach the preset HTL temperature. The reaction temperature was maintained for a certain retention time corresponding to experimental design13-15. And the final pressure in reactors was about 0.90 MPa. After the reaction was completed, the reactor was quenched with room temperature cooling water for 3 min to stop the reaction immediately. The gas product was collected from the out let of the high-pressure valve. To recover the mixtures of desired oil product and aqueous phase left in the reactor after the HTL process, DCM was dispensed into the reactors for extraction of biocrude. The recovered mixture containing the DCM, aqueous phase, and solid residue were vacuum filtered using a Whatman 55 mm glass-fiber filter (Whatman, Cat, No. 1822-055) through a pre-weighted filter paper (Whatman No.4). The solid residues were quantified by weight differences of filter papers before and after filtration. The aqueous and DCM phases were then separated in a separatory funnel. Biocrude was collected after evaporation of the DCM solvent and quantified by weight. The experiments were performed in duplicate for all conditions to determine the average values and standard deviations. When the standard deviation from the two HTL tests was larger than 3%, a third independent HTL test would be added16. Biocrude yields =

Weight of biocrude × 100% Weight of leucine and glucose


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2.3 Analytical chemistry A CE440 elemental analyzer (Exeter Analytical, North Chelmsford, MA) was used to record the elemental analysis results. After the H, N and C contents were collected directly in the analyzer, the O content was able to be calculated directly based on weight differences. Prior to GC-MS analysis, the sample was diluted to desired concentration with DCM. An Agilent 6890 gas chromatograph equipped with an Agilent 7683B autosampler and an Agilent 5973 mass selective detector was used for the mixture analysis. The compounds were separated on a 60 m ZB-5MS column with 0.32 mm nominal diameter and 0.25 µm film thickness. The injection temperature was controlled at 250 °C. The oven temperature was initially set at 70 °C and increased to 300 °C with a rate of 5 °C·min-1 and held for 5 min. The detector temperature was set to 230 °C. Pentadecanoic acid was used as internal standard. The compounds were identified based on the NIST (NIST08) database.

3. Results and discussion 3.1 Maillard reactions between leucine and glucose The main components of bio-crude oil and aqueous fraction obtained by HTL of glucose with leucine and identified by GC-MS are listed in Tables 1 and 2. They are divided into several groups based on the functional groups. Thus, N&O-heterocyclic compounds, organic acids, carbonyls/imine/amine are the three principal groups. It is


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worth mentioning that they were described in detail previously

17.

The biocrude and

aqueous fraction analyzed in the current work are less complicated than those reported in the literature for which microalgae were used as feedstock

9, 18.

The purity of the

feedstock used in this study made it possible to investigate the Maillard reaction between leucine and glucose. Fig.1 shows the components of biocrude and aqueous fraction categorized by functional groups. The pyrazine derivatives were the major chemicals in the bio-crude oil sample. However, it was observed that the fraction of these derivatives changed with the reaction time. Relative peak area of 75.8%, 56.8%, 69.9%, 71.8% were characterized by GC-MS on the biocrude oil yielded during 20, 40, 60, and 90 min reaction time, respectively. The highest pyrazine derivatives fraction of 84.2% was obtained under optimal reaction conditions, i.e., 320 ºC, 60 min, leucine to glucose weight ratio of 2:5, solid concentration of 20%. This result is in contradiction with those reported by Chao et al. because of the lack of lipids in the feedstock

9, 19.

The analysis of the aqueous fraction showed that small organic acids

were the main components. Fewer N&O-heterocyclic compounds were also identified in the aqueous fraction, which is in well agreement with the literature 20. According to the experimental results and previous study on reaction pathways of hydrothermal liquefaction16, 19, 21, 22, the main reaction routes of the HTL reactions between leucine and glucose were proposed (Fig. 2). A: D-glucose degraded to produce cyclic oxygenates under hydrothermal environment, which can further polymerize by aldol splitting and cyclization to form phenol derivatives when hydrothermal temperature increased over 300 °C. However, the HTL of pure glucose


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yielded only 8.5wt.% (of feedstock) biocrude(Fig.3). B. The cyclodehydration of leucine molecules produced various cyclic dipeptides and cyclic imides. C. After the hydrolysis of protein into amino acids, two types of reactions can occur. The first one is the decarboxylation to generate carbonic acid and amide, whereas the second one is the amino acids deamination to ammonia and organic acid. These small molecules are then converted into pyrrole, pyrazine by ring-closing polymerization (Maillard reactions). D. Maillard reaction occurred between reducing sugars and amino acids obtained from carbohydrates hydrolysis and proteolysis, respectively. In this case, leucine reacted directly with glucose to obtain pyridine derivatives. E. Two kinds of reactions including decomposition and repolymerization happened to generate pyrazine 23, 24. Firstly, leucines were decarboxylated to produce amino compounds and carbon dioxide or were deaminated to form carboxylic acid and ammonia. Cyclic oxides were produced by degradation of glucose (as shown in Table 1 and 2, amino compounds, carboxylic acid, cyclic oxides, were detected in crude oil and aqueous fraction on GC-MS). At low temperature (i.e., 260 °C), these decomposition compositions repolymerized to generates the melanin polymer, which prevent the formation of biocrude oil. However, as the temperature increases over 280°C, melanin polymers will decompose into monocyclic compounds, such as pyrazines 10, 25. According to the following material proportion experiment, the oil produced from a mixture of leucine and glucose (weight ratio of leucine to glucose of 2:5) (36.7%) in the hydrothermal liquefaction reaction was far higher than those of pure leucine (17%) or glucose (8.5%) obtained under the same reaction conditions. And


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under optimal reaction conditions, the highest pyrazine derivatives fraction in biocrude reached 84.2%. It was reasonable to conclude that the Maillard reaction pathway D and E between leucine and glucose to generate abundant pyrazine derivatives were the main reaction pathways.

3.2 The effect of the feedstock weight ratio on biocrude production The feedstock weight ratio of leucine and glucose was optimized under the following conditions, i.e., 300 ºC reaction temperature, 40 min reaction time, 4.2% solid concentration. Pure glucose produced little biocrude during HTL (weight ratio of produced biocrude to dry feedstock of 8.5%). However, the weight ratio of obtained biocrude to dry feedstock increased from 8.5% to 36.7% when feedstock ratios of leucine to glucose increased from 0:7 to 2:5 (Fig. 3). As the amino acids produced by the hydrolysis of proteins promoted the conversion of carbohydrates into biocrude oil by inhibiting gasification reaction6, 7, the presence of leucine promoted the consumption of cyclic oxygenates and therefore promoted the formation of pyrazine derivatives. According to the GC-MS analysis of obtained biocrude (Table 1), the main pyrazine derivative was 5-dimethyl-3-(3-methylbutyl)-pyrazine for which two leucine and two glucose molecules were necessary. These results were consisted with the elemental analysis in Table 3. The higher heating value of obtained biocude increase from 29.94 MJ/kg to 33.26 MJ/kg and 36.82 MJ/kg when the weight ratio of leucine to glucose decreased from 4:3 to 3:4 and 2:5 because of the production higher HHV bio-oil compositions. However, the weight ratio of converted biocrude to dry


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feedstock decreased from 36.7% to 17% as the feedstock ratio further increase to 7:0 due to the decrease of cyclic oxygenates. Based on these results, the best ratio of leucine to glucose for biocrude production is 2:5 (wt.%).

3.3 The effect of the hydrothermal temperature on biocrude production The biocrude yield slowly increased from 32.0 to 34.0% when the reaction temperature increased from 280 to 320 °C and then decreased to 31.0% when the temperature increased to 340 °C (Fig. 3). Hence, the HTL of leucine and glucose to produce biocrude is not very sensitive to the reaction temperature in the range of 280°C and 320 °C. However, the HHV of obtained biocrude increased from 32.85 MJ/kg to 36.57 MJ/kg during this temperature range. At lower temperature (260 °C), the Maillard reaction generated melanin polymer instead of biocrude oil. However, as the temperature increases to 280 °C, melanin polymers will decompose into monocyclic compounds, such as pyrazines 10, 25, which improved the HHV of biocude. The decrease of biocrude production at 340 °C is likely due to the decomposition of pyrazine resulting in the decrease of HHV to 36.31 MJ/kg, which is consistent with earlier studies

26-28.

Those studies showed that heavy biocrude (not soluble in

n-hexane) compounds substantially transform into smaller compounds when HTL temperature is above 320 °C. Thus, the optimal reaction temperature of 320 °C was selected for the HTL investigated herein.


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3.4 The effect of the solid concentration on biocrude production The effect of the solid concentration on the weight ratio of biocrude to dry feedstock at 320 °C and 60 min is shown in Fig. 4. The weight ratio of biocrude to feedstock increased from 33.0 to 41.0% when the solid concentration increased from 4.2 to 10% and slightly improved to 41.5% when solid concentration increased to 20%. As the HHV of obtained biocrude also improved from 36.96 MJ/kg to 37.16 MJ/kg, these results were in line with those already published in the literature

26, 29

showing that the increase in solid concentration of feedstock would promote the yield of the heavy biocrude. As Peterson et al. suggested for an energetically and economically efficient hydrothermal process, the target biomass loading should be in the range from 15 to 20 wt.% 30. Hence, an optimal solids concentration of 20% was determined for this study.

3.5 The effect of the hydrothermal liquefaction time on biocrude production The reaction time was optimized at leucine to glucose weight ratio of 3:4, 320 ºC reaction temperature, 4.2% solid concentration. Under these conditions, the weight ratio of biocrude to feedstock dramatically increased from 2.0 to 33.0% when the reaction time increased from 10 to 60 min and then slightly decreased to 32.0% when the reaction time further increased to 90 min (Fig. 4). During the first 60 min, the biocrude yield rapidly increased to 33% because of the Maillard reaction between leucine and glucose as well as the other side reactions during the hydrothermal


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liquefaction process. Considering the GC-MS results in Tables 1 and 2, the Maillard reaction was the main reaction during this process and pyrazine derivatives were the main compounds of biocrude (> 55.0 % relative peak area). These suggestions can be confirmed by the increase of biocrude HHV from 26.02 MJ/kg to 36.72 MJ/kg as hydrothermal time prelonged from 10 min to 60 min. When the reaction time was further raised to 90 min, the chemical degradation of biocrude over time decreased biocrude yield (32.0%) as well as its fuel properties (HHV=29.55 MJ/kg)

26, 31.

The

results revealed that 60 min is the optimum reaction time for biocrude production by HTL between leucine and glucose. Considering all the results mentioned above, the optimal condition for biocrude production via HTL between leucine and glucose (2:5 leucine to glucose weight ratio, 320 ºC reaction temperature, 20% feedstock solid concentration, and 60 min reaction time) produced the optimal biocrude oil yield of 47.6 wt.%. The GC-MS results of optimal condition were as shown in Fig.1, Table 1 and Table 2. At the optimal condition, pyrazine derivatives consisted of 84.2 % of relative peak area in obtained biocrude, giving the highest HHV of 38.07 MJ/kg.

4. Conclusion In summary, this work showed that the Maillard reaction played a significant role in the interaction between proteins and carbohydrates during hydrothermal liquefaction (HTL). Insight into Maillard reactions in microalgae during the HTL process was obtained by selecting leucine and glucose as model molecules. The main


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chemical compounds of biocrude oil and aqueous fraction after HTL reactions were N&O-heterocyclic compounds, organic acids, and carbonyls/imine/amine, due to the simple compositions of the feedstock. The main reaction pathway of Maillard reaction based on key chemical compounds identified by GC-MS was also proposed. The optimal reaction conditions (leucine to glucose weight ratio of 2:5, reaction temperature of 320 ºC, reaction time of 60 min, and feedstock solid concentration of 20%) yielded 47.6 wt.% biocrude oil which was over five times of the biocrude yield obtained through HTL of pure glucose (8.5%). High content (84.2%) of pyrazine derivatives in the obtained biocrude gave the highest HHV of 38.07 MJ/kg. However, further investigation, i.e. different feedstocks or other research methods (quantum chemistry calculation, etc.), on the reaction mechanism of HTL is necessary for a better scientific understanding on Maillard reactions.

Acknowledgement This work was supported by the National key research and development program-China (2017YFE0122800), National Natural Science Foundation China (51476141).


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and model enhancement of food waste hydrothermal liquefaction with combined effects of biochemical composition and reaction conditions. Bioresource Technology 2019, 284, 139-147. 17. Chao, G.; Zhang, Y.; Chen, W. T.; Peng, Z.; Dong, Y., Energy and nutrient recovery efficiencies in biocrude oil produced via hydrothermal liquefaction of Chlorella pyrenoidosa. Rsc Advances 2014, 4, (33), 16958-16967. 18. Cheng, J.; Huang, R.; Yu, T.; Li, T.; Zhou, J.; Cen, K., Biodiesel production from lipids in wet microalgae with microwave irradiation and bio-crude production from algal residue through hydrothermal liquefaction. Bioresource Technology 2014, 151, 415-418. 19. Gai, C.; Zhang, Y.; Chen, W.-T.; Zhang, P.; Dong, Y., An investigation of reaction pathways of hydrothermal liquefaction using Chlorella pyrenoidosa and Spirulina platensis. Energy Conversion and Management 2015, 96, 330-339. 20. Sudasinghe, N.; Dungan, B.; Lammers, P.; Albrecht, K.; Elliott, D.; Hallen, R.; Schaub, T., High resolution FT-ICR mass spectral analysis of bio-oil and residual water soluble organics produced by hydrothermal liquefaction of the marine microalga Nannochloropsis salina. Fuel 2014, 119, 47-56. 21. Saber, M.; Nakhshiniev, B.; Yoshikawa, K., A review of production and upgrading of algal bio-oil. Renewable and Sustainable Energy Reviews 2016, 58, 918-930. 22. Leng,

L.;

Li,

H.;

Yuan,

X.;

Zhou,

W.;

Huang,

H.,

Bio-oil

upgrading

by

emulsification/microemulsification: A review. Energy 2018, 161, 214-232. 23. Demirbaş, A., Mechanisms of liquefaction and pyrolysis reactions of biomass. Energy Conversion and Management 2000, 41, (6), 633-646. 24. Trevelyan, W. E.; Harrison, J. S., STUDIES ON YEAST METABOLISM .1. FRACTIONATION AND MICRODETERMINATION OF CELL CARBOHYDRATES. Biochemical Journal 1952, 50, (3), 298-303. 25. Déniel, M.; Haarlemmer, G.; Roubaud, A.; Weiss-Hortala, E.; Fages, J., Energy valorisation of food processing residues and model compounds by hydrothermal liquefaction. Renewable and Sustainable Energy Reviews 2016, 54, 1632-1652. 26. Valdez, P. J.; Nelson, M. C.; Wang, H. Y.; Lin, X. N.; Savage, P. E., Hydrothermal liquefaction of Nannochloropsis sp.: Systematic study of process variables and analysis of the product fractions. Biomass and Bioenergy 2012, 46, 317-331. 27. Valdez, P. J.; Savage, P. E., A reaction network for the hydrothermal liquefaction of Nannochloropsis sp. Algal Research 2013, 2, (4), 416-425. 28. Tian, C.; Liu, Z.; Zhang, Y.; Li, B.; Cao, W.; Lu, H.; Duan, N.; Zhang, L.; Zhang, T., Hydrothermal liquefaction of harvested high-ash low-lipid algal biomass from Dianchi Lake: Effects of operational parameters and relations of products. Bioresource Technology 2015, 184, 336-343. 29. Jena, U.; Das, K. C.; Kastner, J. R., Effect of operating conditions of thermochemical liquefaction on biocrude production from Spirulina platensis. Bioresource Technology 2011, 102, (10), 6221-6229. 30. Peterson, A. A.; Vogel, F.; Lachance, R. P.; Fröling, M.; Jr, M. J. A.; Tester, J. W., Thermochemical biofuel production in hydrothermal media: A review of sub- and supercritical water technologies. Energy & Environmental Science 2008, 1, (1), 32-65. 31. Huang, Y.; Chen, Y.; Xie, J.; Liu, H.; Yin, X.; Wu, C., Bio-oil production from hydrothermal liquefaction of high-protein high-ash microalgae including wild Cyanobacteria sp. and cultivated Bacillariophyta sp. Fuel 2016, 183, 9-19.


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figures and Tables Fig.1 Compositions of (A) biocrude and (B) aqueous fraction through Maillard reactions between leucine and glucose in hydrothermal liquefaction Fig.2 Proposed main reaction pathway of Maillard reaction between leucine and glucose in hydrothermal liquefaction Fig.3 Effects of hydrothermal liquefaction temperature and feedstock ratio on biocrude production Fig.4 Effects of hydrothermal liquefaction time and solid concentration of feedstock on biocrude production Table 1 GC-MS analysis on major chemical compositions of biocrude product via HTL of glucose and leucine Table 2 GC-MS analysis on major chemical compositions of aqueous fraction via HTL of glucose and leucine Table 3 Elemental analysis of biocrude obtained through various hydrothermal reactions.


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Page 19 of 28

A 80

20 min 40 min 60 min 90 min optimal condition

% of Total Relative Peak Area

70 60 50 40 30 20 10

Ca rb on yls

/Im ine /

Am ine

ac id s rg an ic O

O

th er N& O

Py r

-h et er oc y

cli c

az ine

co m po un ds

de riv at ive s

0

B 20 min 60 min 90 min optimal condition

60

% of Total Relative Peak Area

50

40

30

20

10

co c cli cy &O -h et er o

O 2 C

N

Am id es /im in e/ ca rb on yls

c

ac i

m po un ds

d

0

or ga ni

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Fig.1 Compositions of (A) biocrude and (B) aqueous fraction through Maillard reactions between leucine and glucose in hydrothermal liquefaction. Note: optimal condition referred to weight ratio of leucine to glucose 2:5, hydrothermal temperature 320 ºC, solid concentration 20%, hydrothermal time 60min. The other conditions referred to weight ratio of leucine to glucose 3:4, hydrothermal temperature 320 ºC, solid concentration 4.2%.


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Fig.2 Proposed reaction pathways of hydrothermal liquefaction between leucine and glucose


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Page 21 of 28

Hydrothermal temperature( C) 45

Weight ratio of biocrude to feedstock through hydrothermal liquefaction (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

280

300

320

340

40 35 30 25 20 15

Hydrothermal temperature

10

Feedstock weight ratio

5 7:0

4:3

3:4

2:5

1:6

Feedstock weight ratio (leucine:glucose)

0:7

Fig.3 Effects of hydrothermal liquefaction temperature and feedstock ratio on biocrude production. (conditions: reaction time 40 min, solid concentration 4.2%, weight ratio of feedstock leucine to glucose 3:4 for various temperatures, reaction temperature 300ºC for various feedstock ratios.)


Energy & Fuels

Hydrothermal liquefaction time (min) 0

Weight ratio of biocrude to feedstock through hydrothermal liquefaction (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

20

40

60

80

100

45 40 35 30 25 20 15

Solid concentration of feedstock

10

Hydrothermal liquefaction time

5 0 5

10

15

20

Solid concentration of feedstock (%)

Fig.4 Effects of hydrothermal liquefaction time and solid concentration of feedstock on biocrude production. (conditions: weight ratio of leucine to glucose 3:4, hydrothermal temperature 320 ºC, solid concentration 4.2% for various hydrothermal time, hydrothermal time 60 min for various solid concentrations.)


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Table 1 GC-MS analysis on major chemical compositions of biocrude product via HTL of glucose and leucine

Compound name

Molar weight (g/mol)

CAS #

Formula

Structure

Retention Time (min)

Peak area (%) 20min

40min

60min

90min

Optimal*

N&O-heterocyclic compounds Methyl pyrazine

94

109-08-0

C5H6N2

6.53

/

/

/

/

2.68

2,5-Dimethyl-pyrazine

108

123-32-0

C6H8N2

8.10

3.38

/

/

/

12.67

Trimethyl-pyrazine

122

14667-55-1

C7H10N2

10.16

3.58

2.55

/

2.45

/

2-Ethyl-6-methylpyrazine

122

13925-03-6

C7H10N2

10.16

/

/

/

15.22

Isoamyl pyrazine

150

40790-22-5

C9H14N2

14.89

7.35

/

4.78

5.73

/

2-isoamyl-6-methylpyrazine

164

91010-41-2

C10H16N2

16.83

11.13

21.58

12.24

17.70

14.78

2,5-Dimethyl-3-(3-methylbutyl )-pyrazine,

178

18433-98-2

C11H18N2

18.79

56.21

25.67

45.33

44.15

37.56

2,3-Dimethyl-5-isopentyl pyrazine

178

18450-01-6

C11H18N2

19.08

1.10

1.35

/

3.16

/

2,3,5-Trimethyl-6-isopentyl pyrazine

192

10132-43-1

C12H20N2

20.62

/

8.17

/

1.05

/


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 24 of 28

2-Ethyl-3-methylpyrazine

122

15707-23-0

C7H10N2

25.75

/

/

7.54

/

1.31

2-(2-Methylpropyl)-3,5-di(1-m ethylethyl)pyridine

219

7033-68-3

C15H25N

22.40

0.95

10.10

/

2.94

/

3-Hydroxy-6-methylpyridine

109

1121-78-4

C6H7NO

21.40

/

/

1.26

/

/

2,6-dimethylpyridine-3-ol

123

1122-43-6

C7H9NO

22.17

/

/

3.68

/

/

3,6-bis(2-methylpropyl)pipera zine-2,5-dione

226

1436-27-7

C12H22N2O2

32.08

/

/

1.04

/

/

Isovaleric acid

102

503-74-2

C5H10O2

6.56

1.95

/

1.06

1.01

1.99

4-methyl-Pentanoic acid

116

646-07-1

C6H12O2

8.73

0.92

/

/

/

/

3-methyl-N-(3-methylbutyl) butan-1-imine

155

35448-31-8

C10H21N

11.49

/

1.06

1.82

1.37

/

N-pentylpentan-1-amine

157

2050-92-2

C10H23N

12.29

/

/

7.25

7.01

/

2-Isopropyl-5-methyl hex-2-enal

154

35158-25-9

C10H18O

12.88

3.02

1.80

/

1.16

/

N-(3-Methylbutyl)acetamide

129

13434-12-3

C7H15NO

13.55

/

/

/

3.43

/

Organic acids

Carbonyls/Imine/Amine


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Energy & Fuels

Triisopentylamine

227

645-41-0

C15H33N

19.32

/

1.35

/

/

/

4-Amino-2,3-xylenol

137

3096-69-3

C8H11NO

19.98

/

8.3

/

/

/

4-(diethylamino)benzoic acid

193

5429-28-7

C11H15NO2

19.99

/

3.45

/

/

/

Opt*: weight ratio of leucine to glucose 2:5, hydrothermal temperature 320 ºC, solid concentration 20%, hydrothermal time 60min. The other conditions referred to weight ratio of leucine to glucose 3:4, hydrothermal temperature 320 ºC, solid concentration 4.2%. “/” was referred to not detected.


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 26 of 28

Table 2 GC-MS analysis on major chemical compositions of aqueous fraction via HTL of glucose and leucine Retention Time (min)

Peak area (%) 20min

60min

90min

Opt*

CO2

1.86

27.5

11.39

33.4

3.60

91010-41-2

C10H16N2

11.67

2.54

/

/

/

178

18433-98-2

C11H18N2

12.08

1.32

/

/

/

Tetrahydrofurfuryl Alcohol

102

97-99-4

C5H10O2

17.45

/

/

/

/

α-HYDROXYγ-BUTYROLACTONE

102

19444-84-9

C4H6O3

17.54

2.77

1.71

/

/

28564-83-2

C6H8O4

18.35

/

/

/

/

5625-44-5

C10H18N2O2

18.74

2.59

/

/

/

>19.00

10.78

15.91

18.79

16.95

20.25

/

10.86

4.24

12.73

Compound name

Molar Weight (g/mol)

CAS #

Formula

44

124-38-9

164

Structure

Carbon dioxide Carbon dioxide N&O-heterocyclic compounds 2-Methyl-6-(3-methyl-butyl)-py razine 2,5-Dimethyl-3-(3-methylbutyl) -pyrazine

4H-pyran-4-one, 2,3-dihydro-3,5144 dihydroxy-6-methyl 3,6-di(propan-2-yl)piperazine-2,5198 dione Pyridinol derivatives 5-Hydroxymethyldihydrofuran188 2-one Amides/imine/carbonyls

140156-47-4

C8H12O5


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Energy & Fuels

5-Methyl-2-hexanone

114

110-12-3

C7H14O

2.68

4.15

1.33

/

/

3-Methylbutanal

86

590-86-3

C5H10O

2.68

/

/

/

/

3-Methyl-N-(3-methylbutyl) butan-1-imine

155

35448-31-8

C10H21N

5.23

3.69

/

/

/

hydroxyacetone

74

116-09-6

C3H6O2

7.38

1.50

/

/

/

Formic acid

46

0-00-1

CH2O2

2.68

/

/

7.03

/

Acetic acid

60

64-19-7

C2H4O2

9.39

15.6

30.70

10.05

50.08

Propionic acid

74

79-09-4

C3H6O2

10.54

/

1.67

/

1.79

Isovaleric acid

102

503-74-2

C5H10O2

12.15

7.88

7.58

5.97

3.83

Isocaproic acid

116

646-07-1

C6H12O2

13.64

5.27

11.52

11.87

2.28

Pyruvic acid

88

127-17-3

C3H4O3

17.46

3.64

/

/

/

4-oxopentanoic acid

116

123-76-2

C5H8O3

18.78

/

/

/

2.18

Organic Acid

Opt*: leucine to glucose weight ratio of 2:5, hydrothermal temperature 320 ºC, solid concentration 20%, hydrothermal time 60min.The other conditions referred to weight ratio of leucine to glucose 3:4, hydrothermal temperature 320 ºC, solid concentration 4.2%. “/” was referred to not detected.


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

Table 3 Elemental analysis of biocrude obtained through various hydrothermal reactions. C

H

N

Oa

HHVb

(%)

(%)

(%)

(%)

(MJ/kg)

Temperature(°C), leucine:glucose=3:4, solid concentration =4.2%,40 min 280

70.52±1.37

8.06±0.12

7.51±0.11

13.92

32.85

300

71.64±0.37

8.66±0.08

7.62±0.23

12.10

34.41

310

73.17±1.10

9.38±0.01

7.91±0.06

9.55

36.41

320

73.33±1.21

9.43±0.19

7.84±0.22

9.41

36.57

340

73.06±1.03

9.37±0.23

7.75±0.13

9.83

36.31

Weight ratio of leucine to glucose (%), solid concentration =4.2%, 300°C, 40min 4:3

68.04±0.89

7.265±0.02 5.49±0.19

19.21

29.94

3:4

71.93±0.99

7.83±0.18

7.85±0.06

12.41

33.26

2:5

72.28±0.86

9.91±0.05

7.94±0.08

9.87

36.82

Hydrothermal time(min), leucine:glucose=3:4, solid concentration =4.2%, 300°C 10

59.77±0.56

7.49±0.12

5.42±0.03

27.32

26.02

20

69.535±0.37 8.045±0.11 8.27±0.18

14.16

32.46

60

73.36±0.17

9.53±0.08

7.74±0.29

9.39

36.72

90

62.80±0.23

8.58±0.15

6.62±0.03

22.01

29.55

Solid concentration(%), leucine:glucose=3:4, 300°C, 40min, 10

73.28±0.21

9.70±0.12

7.77±0.21

9.26

36.96

20

72.78±0.13

10.00±0.07 7.67±0.12

9.56

37.16

Solid concentration=20 %,leucine:glucose=3:4, 320°C, 60min Optimal condition

74.65±0.45

9.99±0.09

7.43±0.08

a

7.94

38.07

Oxygen content was calculated by subtraction of carbon, hydrogen and nitrogen contents from 100 wt.%. bThe higher heating value (HHV) was estimated with the Dulong formula(Rizzo et al., 2013): HHV (MJ/kg) = 0.338C + 1.428(H-O/8) + 0.095S, where C, H, O, and S were weight percentages of elemental compositions in materials.


Biocrude production through Maillard reaction between leucine and

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