Research on catalytic pyrolysis of algae based on Py-GC/MS

In order to improve the quality of catalysis products of algae, composite molecular sieve catalyst was prepared by digestion and crystallization of HZSM-5 to reduce the oxygen content of the catalytic products. According to the analysis of the pyrolysis products, the best preparation conditions were chosen of tetra propylammonium hydroxide (TPAOH) solution 2.0 mol l−1, cetyltrimethylammonium bromide (CTAB) solution 10 wt%, crystallization temperature 110°C, digestion–crystallization time: 24–24 h. The results indicate that the main function of catalysts is to promote the conversion of alcohols into hydrocarbons by reducing energy barriers. Catalysed by the composite molecular sieve, the content of alcohols in the pyrolysis products decreased from more than 30% to less than 10%, the content of hydrocarbons increased from 20% to nearly 60%, while all the adverse components remained at a low level, which indicates that the catalytic pyrolysis products are of high quality. The great deoxidation effect of composite molecular sieves is not only due to the expansion of the range of organic matter during re-pyrolysis, but also the increasing of the residence time of pyrolysis products inside the structure for the external mesoporous structure.


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The discussion seems to be superficial, therefore more in-deep discussion is required to improve the scientific aspect . Conclusions. Some critical guidance in the evaluation of the present state and in the further study is given in this section. The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: (There are no comments.) RSC Scientific Editor: Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) This manuscript describes that effect of hierarchical micro-mesoporous composite molecular sieve HZSM-5 / MCM-41 zeolite on the quality of bio-oil from Py-GC/MS of algae. After reading the manuscript, I think it is a very interesting work that deserves publication in this journal. It is should be accepted after reasonably reply and revise the following suggestions: 1. In the analysis section, additional references are needed to further explain the changes in catalytic performance; 2. Please repeat the experiment at least three times and add an error bar to the diagram; 3. In conclusion section, please add the comparison with other references to illustrate the superiority of catalysts in this study; 4. Conduct elemental and industrial analysis on algae; 5. Why not use quantitative analysis and only use peak area to explain the results; 6. The database can not only use NIST MS library database, but also need to be compared with other references； 7. Please amend the language in the articile.

Reviewer: 2
Comments to the Author(s) 1-What were the highlight of your work? 2-What is your research novelty? 3-The introduction part need to rewrite using updated references and the most important articles on your research area 4-Summary need to be rewritten in more scientific way. 5-What is the heating rate is it 20000 or 20C/s 6-Are the reported results (not including the characterization results) in your paper reproducible? Are those come from a single experiment since the number of runs are not clearly indicated. 7-The discussion seems to be superficial, therefore more in-deep discussion is required to improve the scientific aspect . Conclusions. Some critical guidance in the evaluation of the present state and in the further study is given in this section. It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email. The quantification method in the present paper assumes the compounds identified by GC-MS to have a similar response factors, resulting in the use of total areas as proxies for yields. This assumption is justified by the similar nature of the products obtained in each case, since the products of non-catalytic pyrolysis are typically oxygenated organic molecules, and the products of catalytic pyrolysis are typically hydrocarbons. Therefore, we expect that differences in calibration response factors for compounds in a single experiment are not large enough to cause significant deviations in the results, in such a way that the trends and conclusions from the experiments reported in the present work are representative of the process. The conclusion part of the original manuscript lacks scientific nature, and the analysis part of the original manuscript has been modified to add the part of mechanism analysis, so we have rewritten the conclusion part according to the current analysis and discussion part. In addition, we have corrected other unqualified parts of languages.
Reviewer: 2 Comments to the Author(s) 1-What were the highlight of your work?
The most important part of this study is the application of composite molecular sieve catalyst in algae pyrolysis. Algae is considered as a new generation of biomass fuel with potential, and the content of oxygen compounds in its direct pyrolysis products is high, which is not conducive to the direct utilization of bio-oil. In order to reduce the oxygen content of its pyrolysis products, we applied the composite molecular sieve to the catalytic pyrolysis of algae, and evaluated the performance of the catalyst according to the analysis results of the pyrolysis products of algae, so as to determine the best conditions of preparation of the catalyst.
At the same time, through the analysis of the products, it is found that catalytic pyrolysis is mainly to deoxidize and hydrogenate the alcohols in the products. Through the catalytic pyrolysis of the composite molecular sieve catalyst, the content of hydrocarbons in the catalytic products increased from 20% to nearly 60%, while the content of the adverse components remained at a very low level, indicating that the bio-oil had a high quality.

2-What is your research novelty?
At present, there are many studies on the catalytic pyrolysis of composite molecular sieve catalysts in lignin biomass, but its application in algae pyrolysis is not enough. However, lignin biomass is quite different from algal biomass in composition, and the results of catalytic pyrolysis of many lignin biomasses may not be applicable to algal biomass. In preparation of catalyst, different from NaOH used in previous studies, this study used mild alkaline TPAOH to modify HZSM-5. Previous studies involving catalysts modified by an alkali treatment have focused on alkali-treated HZSM-5 at low concentration (<1.0 mol/L) of inorganic base. The high concentration (2.0 mol/L) of TPAOH solution is more effective for HZSM-5 treatment than the low concentration base solution (< 1.0 mol/L). Moreover, the modified catalyst does not require NH4NO3 ion exchange. The 2.0 mol/L TPAOH modified HZSM-5 led to produce the highest aromatic selectivity and catalytic activity from CFP of algae. In addition, the higher concentration such as 2.0mol/L was used to improve the reaction rate, so as to ensure the digestion effect and improve the preparation efficiency.
3-The introduction part need to rewrite using updated references and the most important articles on your research area

Appendix B
Thank you very much for your kind comment. We refer to the researches on the catalytic pyrolysis of algae and the catalysis of composite molecular sieve in the past three years, especially the influential reports on such researches in some authoritative journals and learn the current research achievements in this field. At the same time, the statement of the premature research results in the introduction is deleted and the research achievements in recent years are sorted out and supplemented in the introduction section to improve the timeliness and credibility of this article. Thank you very much for your kind comment. We have rewritten the summary part to make it scientific. And the content of the summary part was supplemented according to the modification of the results and discussion part. In order to demonstrate the repeatability of the experimental results, the experiments were carried out for three times in the past, and the experimental results were basically the same. In the original draft, we selected the data with the most obvious trend to illustrate the results. In the revised draft we integrated the results of three experiments, took the average value and added error bars to each group of data to explain the experimental results, so as to increase the credibility of the experimental results.

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The discussion seems to be superficial, therefore more in-deep discussion is required to improve the scientific aspect. Conclusions. Some critical guidance in the evaluation of the present state and in the further study is given in this section.

Thank you very much for your kind comment.
In the discussion part of experimental results, we analyzed the specific components of the pyrolysis products and found that the catalysis was mainly the conversion of alcohols into hydrocarbons. At the same time, through the comparison of specific product content, the difference of catalytic performance between the catalyst prepared in this experiment and the ordinary molecular sieve catalyst was found. In addition, by referring to other studies, it is shown that the catalytic mechanism is the reduction of energy barrier. Meanwhile, through the comparison of products and the results in other literatures, it is speculated that alcohols are mainly deoxidized, hydrogenated and dehydrated in the process of catalysis, and the reaction pathways of some substances are described. Finally, high nitrogen content is the problem of algae pyrolytic oil. The composite molecular sieve has no effect on nitrogen content although its deoxidation effect is obvious. How to reduce the nitrogen content in the pyrolysis products of algae and its combined with composite molecular sieves to improve the quality of algae pyrolysis products can be one of the later research directions.

1.Summary
In order to improve the quality of catalysis products of algae, composite molecular sieve catalyst was prepared by digestion and crystallization of HZSM-5 to reduce the oxygen content of the catalytic products. According to the analysis of the pyrolysis products, the best preparation conditions were chosen of TPAOH solution 2.0mol/L, CTAB solution 10wt%, crystallization temperature 110℃, digestion-crystallization time: 24-24h. The results indicate that the main function of catalysts is to promote the conversion of alcohols into hydrocarbons by reducing energy barriers. Catalyzed by the composite molecular sieve, the content of alcohols in the pyrolysis products decreased from more than 30% to less than 10%, the content of hydrocarbons increased from 20% to nearly 60%, while all the adverse components remained at a low level, which indicates that the catalytic pyrolysis products are of high quality. The great deoxidation effect of composite molecular sieves is not only due to the expansion of the range of organic matter during re-pyrolysis, but also the increasing of the residence time of pyrolysis products inside the structure for the external mesoporous structure.

2.Introduction
The global warming caused by fossil energy [1,2] has attracted worldwide attention, and as non-renewable energy, it is difficult for fossil energy to support the sustainable development of society in the future. The development of renewable energy has become a worldwide trend, such as solar, wind, tidal and biomass energy. According to relevant researches [3], biomass energy, a kind of clean energy, plays an important role in alleviating environment pollution problems. The utilization of biomass could be realized by means of biomass energy conversion technology which mainly includes liquefaction, gasification and pyrolysis. Pyrolysis [4] is considered as a promising technology, which refers to the technology that organic materials decomposed into solid, liquid and gas products (bio-char, bio-oil, non-condensable gas) at high temperature (300-1000℃) in an inert atmosphere. Nowadays researches on biomass pyrolysis mainly focus on wood biomass, while few on aquatic biomass. Take algae as example, algae belong to low-grade, oxygen-releasing autotrophic plants, with varieties of species and wide distribution. Moreover, the output of many kinds of algae in China ranks first in the world. Most algae belong to single-celled organisms [5], which means it will be easy to be improved, and can be cultivated by changing the cultivation conditions to produce species more suitable for pyrolysis [6,7]. Compared with the first generation of biofuels [8] (edible biomass, sugar and starch plants) and the second generation of biomass [9] (lignocellulosic biomass), algae has several prominent advantages [10]: (1) High photosynthetic efficiency, which is conducive to alleviate the greenhouse effect problem; (2) Nutrients (N,P) can be extracted from waste-water and returned to the soil by waste product of fertilization; (3) Algae has a short breeding cycle [13], and its process of breeding is easy to realize automation. (4) Algae does not need to occupy arable lands, and is less affected by seasons and regions. According to the analysis composition of algae, algae has high lipid accumulation, which is suitable for the decomposition of bio-oil by heat conversion technology. Considering above advantages, algae is a potential biomass material.
There are always problems with biological oils prepared by direct pyrolysis of biological substances, such as low calorific value, high acid content and low hydrocarbon content.
Therefore, measures should be taken to improve the quality of biological oils. One of common methods is to use catalysts, such as molecular sieve, metals and metal oxides. Among them, metal oxide has large pore diameter, strong water stability, and certain deoxidation performance, which is helpful to improve the stability of biological oil; Alkali metals mainly include sodium salt, potassium salt, calcium salt and their oxides; Microporous molecular sieve refers to molecular sieve with pore diameter less than 2nm, which has good deoxidation and aromatization properties. HZSM-5 [17][18][19], one common molecular sieve, has a microporous structure that allows pyrolysis steam to enter the interior for further pyrolysis. Under the catalysis of HZSM-5, the release of oxygen-containing gas (CO and CO2) shows a significant decline, and the conversion of furans to aromatic hydrocarbons may be promoted over strong acid site [17,18]. However, due to its small pore size [20], the yield of water and gas increased while the yield of organic matter decreased obviously.
Besides, catalysis of HZSM-5 may be deactivated due to the polymerization of a mass of oxycompound [21]. The mesoporous zeolite MCM-41 with a larger pore size provides lager surface area and more accessible reaction sites. It has been pointed that the mild acidity of MCM-41 catalyst provides an ideal environment for controlling the conversion of high molecular weight lignocellulosic molecules [18,20]. However, products will escape before complete re-pyrolysis because the pore size is too large [18]. Therefore, the utilization of fracture properties of macroporous catalysts and the re-framing properties of microporous molecular sieve catalysts as catalysts for biomass catalytic pyrolysis has received extensive attention [18]. In order to improve the quality of bio-oil from algae, existing studies have shown that oxygen content and acid compound of biological oil can be reduced when catalyzed by Ni supported zeolites [11], while nitrogen content of bio-oil can be reduced when catalyzed by Mg-Al layered double oxide/ZSM-5 composites with a Mg/Al molar ratio of four(MgAl4 -LDO/ZSM-5) [12]. Hydrothermally carbonized [13] can increase the maximum weight loss rate of algae during pyrolysis and can be combined with catalytic pyrolysis to realize the reduction of nitrogen content in biological oil; ZSM-5 catalytic copyrolysis [14] can be a favorable process to enhance the yield of upgraded bio-oil. At present, composite molecular sieve catalyst is widely used in lignin biomass [15,16] and shows relatively superior catalytic performance. Previous studies have used the addition of macroporous and mesoporous molecular sieves to LOSA-1 to improve the selectivity of low carbon olefins and aromatic hydrocarbons [18]. Meanwhile, the mixture of HZSM-5 and MCM-41 has been studied to improve the catalytic pyrolysis effect of fresh straw [22].
However, few researches have conducted on the composite of mesoporous and microporous molecular sieve and its application on proteinaceous biomass.
In this study, a hierarchical micro-mesoporous composite molecular sieve HZSM-5/MCM-41 with external mesoporous and internal microporous was developed through digestion and reassembly of molecular sieve HZSM-5, so as to meet re-pyrolysis requirements of pyrolysis steam in a wider range and improve the quality of pyrolysis products. With the support of Py-GC/MS [23] (pyrolysis-gas chromatography/mass spectrometry), this study intends to explore the catalytic pyrolysis products of algae and find suitable catalysts for catalytic pyrolysis to improve the quality of pyrolysis oil.
Pyrolysis products were determined by Py-GC/MS [23]. The main characteristics of spirulina can be seen in Table 1. The content of oxygen was calculated from the difference of 100% and the mass ratio of C, H, N, S. TPAOH (Tetra propylammonium hydroxide) was dissolved into 50ml ultrapure water with a certain mass fraction to prepare TPAOH solution, and then 10g HZSM-5 was added, stirred and heated in water bath at 40℃ for 1h. Next CTAB (Cetyltrimethylammonium Bromide) solution of a certain concentration was mixed with HZSM-5 solution and heated in the water bath at 26℃ for 1h. Then, it was poured into the reactor for 24h at a certain temperature for resolution. After adjusting the pH of the solution to 8.50 [2] by dilute sulfuric acid solution (5%), it was poured into the reactor for 24h for crystallization. After the solution was filtered and dried, dried samples were heated at 550℃ for 6h, after which the preparation of catalyst composite molecular sieve HZSM-5/MCM-41 was completed.

Pyrolysis experiment
According to relevant studies, the temperature of pyrolysis by Pyroprobe 5200 pyrolyzer [35] (CDS Analytical) in this study was 600℃ [25,26] and the pyrolysis time was 20s. Pour 0.5mg catalyst, 0.5mg biomass raw material and 0.5mg catalyst into the quartz capillary tube successively, and use quartz wool to separate each layer and seal both ends.
The heating rate was set at 20,000℃/s [36], and the tube was kept at the reaction temperature for 20s. High-purity helium (99.999%, Nanjing Maikesi Nanfen Special Gas Co.,

Relative content
In the present work, the relative content ( content R ) of organic pyrolytic products are calculated using a semi-quantitative method based on the area percentages of the chromatograph peaks and defined as follows [19,27]:  However, when the concentration of CTAB keeps growing, a decline can be seen in the content of hydrocarbons in the pyrolysis products. It is possible that the hexagonal structure starts to become disordered, and the damage to the microporous structure of HZSM-5 is excessive [28]. When the concentration of CTAB is 10wt%, the damage to microporous structures of HZSM-5 is relatively weaker, and the MCM-41 structure can be assembled on the surface. Therefore, its catalytic effect is the best, with the highest content of hydrocarbon of 59.17% and the lowest content of acid substances of 2.79%. At the same time the content of aldehydes and ketones is low, which ensures the stability of pyrolysis products effectively.

Influence of different temperatures of crystallization on catalytic effect
The temperature of resolution during the preparation of the molecular sieve was 110 ℃, and the temperature of crystallization were 90 ℃, 100 ℃, 110 ℃ and 120 ℃.

Fig. 3 Distribution of catalyst products at different crystallization temperatures
As the Fig. 3 shows, when the crystallization temperature is lower than 110 ℃, the increase of temperature promoted the increase of the content of hydrocarbons in the pyrolysis products. It may due to that the increase of temperature promotes the reaction between dissolved crystal nucleus and template agent, which is conducive to the assembly of MCM-41 structures on the external surface of HZSM-5 and the formation of composite molecular sieve structures. However, with the temperature keeps increasing, decline can be seen in the content of hydrocarbon and increase can be found in the content of acids and alcohols. The explanation for this phenomenon may be that when the temperature further increases, the reaction between template ion and silicate root is unduly violent, which promotes the formation of amorphous material and destroys the assembly of mesoporous structures [27].
When the crystallization temperature is 110℃, the content of hydrocarbon reaches the highest (59.17%), while the contents of acids (2.79%), aldehydes (1.08%) and ketones  In this study, the digestion-crystallization time was respectively adjusted to 12-36h, 24-24h, 36-12h. As can be seen from Fig.4, when the digestion time was 12h and the crystallization time was 36h, due to the short digestion time, the dissolved sialic acid radical is few, which hindered the assembly of MCM-41 in the next step [27]. However, when the digestion time is 36h and the crystallization time is 12h, although a large amount of sialic acid radical is dissolved, the crystallization time is too short to fully assemble the mesoporous structure, and the mesoporous structure is poor in order. When the digestion time and crystallization time are both 24h, the mesoporous structure is formed and the microporous structure is less destroyed, and the ratio between the two competitive processes of digestion and crystallization is relatively appropriate. As can be seen from Fig. 4, when the digestion-crystallization time is 24-24h, the hydrocarbon content reaches the highest level, and acids, aldehydes and ketones all reach the lowest level, indicating that the composite molecular sieve HZSM-5/MCM-41 catalyst has a great catalytic pyrolysis effect.

Comparison of pyrolysis and catalytic pyrolysis products
In this study, direct pyrolysis of algae and catalytic pyrolysis by HZSM-5 and composite molecular sieve HZSM-5/MCM-41 were conducted. The distribution of pyrolysis products is shown in Fig.5.
In the term of direct pyrolysis, due to the high content of nitrogen in algae, the content of nitrogen-containing organic compounds is significantly higher than that of herbaceous biomass, which is consistent with the previous research [25]. Due to the low lignin content of algae, the phenolic substances in their pyrolysis products are also few, accounting for only 3%. Hydrocarbon content is relatively high, up to 25%, and so as to alcohols, up to 30%, which may be due to the high axunge content of algae themselves. At the same time, contents of acids and aldehydes were also lower, at 5% and 5% respectively. It can be found that the bio-oil generated by direct pyrolysis of algae has high quality, indicating that algae is a promising biomass raw material. re-pyrolysis, the hydrocarbon content in the products is significantly increased, alcohols are significantly reduced, and aldehydes are also reduced to some extent. Catalyzed by HZSM-5, acid substances increase slightly, from 4% to 5%; The hydrocarbon content increase significantly from 15% to 28%; Alcohols decrease significantly from 35% to 20%; The content of aldehydes also drop, from 5% to 2%; Esters also fall, from 5% to 3%. It can be considered that through the catalytic pyrolysis of HZSM-5, due to the appropriate pore size of HZSM-5, parts of oxygen-containing organic compounds further undergoes deoxidation during re-pyrolysis, and main components are some alcohols of macromolecules [26,29].
The content of nitrogen-containing compounds is hardly affected, indicating that HZSM-5 could not promote the removal of nitrogen from nitrogen-containing organic compounds.
It can be considered that due to the special structure of the external mesoporous and internal micropores of the composite molecular sieve HZSM-5/MCM-41, more oxygencontaining organic substances undergo deoxidation reaction during further pyrolysis inside the catalyst, while alcohols are also the main organic constituents for re-pyrolysis. Similarly, the content of nitrogenous organic compounds is not affected, indicating that the removal of nitrogen could not be achieved by just adjusting the aperture.  The contents of alcohols and hydrocarbons in direct pyrolysis and catalytic pyrolysis products are shown in Table 2. Compared with direct pyrolysis products, micromolecular alcohol content in pyrolysis products catalyzed by HZSM-5 shows a significant decline, for example, the content of cyclopropane ethanol, 3-Pentyn-1-ol and 4-Cyclopentene-1,3diol, trans-decreases respectively from 6.8%, 1.04% and 1.63% to 0, 0.4% and 0, which indicates that sufficient hydrogenation, deoxidization, dehydration reaction happens to these compounds. The addition of HZSM-5 helps to reduce the energy barrier due to hydrogen bond and van der Waals force, thus promoting the occurrence of repyrolysis. And the possible pathways [30,31] of their hydrodeoxidation reactions are show in Fig.6. As to macromolecular alcohols such as Ethanol, 2- Compared with bio-char, Ni/SBA-15 [12] and activated carbon [13]  by reducing the content of alcohols. However, the adverse components do not change significantly due to their low content, and the content of N-containing compounds is hardly affected. After catalytic pyrolysis by HZSM-5/MCM-41, the alcohols decrease significantly again, and the content of adverse components also decrease, but the content of nitrogencontaining organic matter is not affected. After analysing the specific substances, it is found that HZSM-5 had great hydrodeoxidation performance on micromolecular alcohols, but the catalytic effect was limited by its pore size. The external mesoporous structure of HZSM-5 / MCM-41 not only widen the reaction range of hydrodeoxidation reaction, but also increase the residence time of pyrolysis products in the internal structure, making deoxidation hydrogenation reaction more complete. Through the analysis of the products, it is found that the alcohols are mainly converted into hydrocarbons by hydrogenation, deoxidation and dehydration under the catalysis. Finally, high nitrogen content is the problem of algae pyrolytic oil. The composite molecular sieve has no effect on nitrogen content although its deoxidation effect is obvious. How to reduce the nitrogen content in the pyrolysis products of algae and its combined with composite molecular sieves to improve the quality of algae pyrolysis products can be one of the later research directions.

Funding Statement
This work was sponsored by the National Natural Science Foundation of China (No.51776042).

Competing Interests
We have no competing interests.