Biotransformation of Citrus Waste-I: Production of Biofuel and Valuable Compounds by Fermentation

Round 1

Reviewer 1 Report

The review describes the “Citrus waste as a renewable feedstock for production of biofuel and valuable compounds via biotransformation”. The objectives of this review is highly impactful and timely, since the waste is a problem with the environment and can be utilized as a raw materials for the production of different types of chemical such as bioethanol, biogas, biofuels, organic acids, enzymes etc. This manuscript is well written and I recommend for a minor revision. My comments are appended below.

1.) The authors must mention the estimated cost for the production of ethanol or fuel production by waste and should be somehow compared with the traditional/conventional method. It is very essential for the industrial application.

2.) In the manuscript, some scientific facts are written without experimental evidence and cross-referencing. Therefore it is highly recommend to add some more references. For instance in page 3 line 118-120, page 5 line 158-159, page 15 line 365-368 and so on.

3.) Please improve the sentence, page 15 page 347-348.

Author Response

Response to Reviewer comments

Reviewer 1.

Dear Reviewer

First, we greatly appreciate you for reviewing our manuscript.

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The review describes the “Citrus waste as a renewable feedstock for production of biofuel and valuable compounds via biotransformation”. The objectives of this review is highly impactful and timely, since the waste is a problem with the envirnment and can be utilized as a raw materials for the production of different types of chemical such as bioethanol, biogas, biofuels, organic acids, enzymes etc. This manuscript is well written and I recommend for a minor revision. My comments are appended below.

1.) The authors must mention the estimated cost for the production of ethanol or fuel production by waste and should be somehow compared with the traditional/conventional method. It is very essential for the industrial application.

Response: The production of ethanol and biogas from citrus processing waste in particular is still in the stage of laboratory scale production and a lot to be done in this regard in order to bring it to the scale of industrial production. We have added an additional section (appended below) on economic aspects of the citrus peel waste-to-ethanol production along with recovery of limonene as a by-product. The concepts for a successful and profitable operation of a functional biorefinery that could generate reasonable revenue are under progress and focus of this review article is to bring all the recent progresses documented in this direction so far at one place. The recent market size of different chemicals (that can be derived from the processing of citrus waste) are recorded in Table 3. The market size shown here is of total production from all kinds of resources and not particularly from the citrus waste feedstock. This is to emphasize that the production from citrus waste resources can add up to the current size and to the overall contribution from natural resources.

 Section 5. Economic aspects

Production of bioethanol from the citrus processing wastes is accompanied by the simultaneous production of d-limonene, methane (biogas) and pectin at commercial scale which ad up to economic benefits. Apart of these, the capital generated from these by-products is utilized in procurement of enzymes, acids and microbial strains required for hydrolysis, and to some extent paying for the utility bills involved in energy, electricity and apparatus/equipment maintenance. Improved technologies and continuous innovations have helped immensely to scale up the production of bioethanol and by-products efficiently. Although, the production is still in its demonstration phase. According to assumptions based on calculations, a plant with production capacity of ~152,000 m³ of ethanol per year where the ethanol price is USD 475.51 per m³ provides a net gain of revenues of USD 90.00 per m³ without d-limonene. On the other hand the gain is USD 169.00 per m³ when d-limonene was included as a co-product [1,2]. Furthermore, it is estimated that a citrus processing waste based biorefinery would require ~3.3 million tonns of fresh waste every year to make the process economically profitable.

Research aspects to be focused on to scale up the production can be summed up in the following points; (a) Pretreatment of citrus processing waste and hydrolysis of the same; enzyme loading, enzymatic reaction rates, yields of fermentable sugars, ethanol and overall co-production of limonene, methane, pectin and biologically active compounds; (b) kinetics and thermodynamics of pre-treatment processes on citrus waste: degradation of waste as a function of temperature and time; (c) upgradation and modernization of pre-treatment process in terms of yielding appropriate particle size of the raw material, surface area and morphology for enhanced action of hydrolysis and efficient results; (d) development of suitable processes to recover and obtain the by-products and important chemicals, such as polyphenols, carotenoids, sugars pectin , d-limonene, methanol and galacturonic acid along with bio-ethanol; and (e) statistical records on critical process data for small-to-medium-to-large sized bio-refinery units linked to citrus/food processing plants with waste generation capacity of 36,000-360,000 tons per year [3]. The cost of raw materials or feed-stock is a crucial aspect in the production of biofuel from waste-biomass. Besides this the cost involved in the maintenance of the different apparatuses and instruments processing unit(s) in the bio-refinery or the reactor, chemicals, enzymes, microbial strains and culture facilities, utilities (steam, water, electricity, etc.), consumables, equipment installation, labour, overhead charges and taxes of the state. During production of ethanol from biomass, lignin is generally obtained as a residue is consumed to generate steam and electricity, thereby reducing the utility costs considerably. However, during the production of ethanol from citrus waste biomass lignin is not recovered as a residue, but recovery of limonene as a valuable solvent and a co-product is economically significant (Figure 13).

Figure 13. Citrus based ethanol economies; A comparative analysis of Gross revenue (a) production cost for the citrus peel waste-to-ethanol process with and without the recovery of limonene (capacity: 25 million gal/year) (b). Adapted from [2].

A bioethanol processing unit installed adjacent to huge citrus food processing industries across the world is established to produce 10 million gallons of ethanol per annum. This suggests that production of ethanol from citrus waste biomass is beneficial. However, installing a small unit would be more expensive and disadvantageous [2,4]. In the current time, it is yet to establish an installation of industrial scale ethanol production based on citrus waste biomass and limited to laboratory demonstration only. Patsalou et al. reported an effiecient biorefinery strategy for the production of bioethanol and methane employing three different strains of yeast, viz., Pichia kudriavzevii KVMP10 and Kluyveromyces marxianus and Saccharomyces cerevisiae. They proposed a zero-waste strategy by combining multiple extraction steps along with feremntation processes to produce ethanol and methane. This includes removal of limonene and essential oils in the first step using distillation of the hydroxylates post pretreatment. The remaining residue was dried and subjected to dilute acid hydrolysis and extraction of pectin. Simultaneously, ethanol was removed by distillation. The remaining residue was then subjected to fermentation by different yeast strains in separate tanks (for comparison) to produce ethanol. Post ethanol production, the residue was again treated with dilute hydrolysis and subjected to anaerobic digestion for the production of methane. P. kudriavzevii KVMP10 was observed to produce the highest yield of 30.7 g/l from the fermentation process conducted at 42 ºC followed by 18.6 g/l by K. marxianus and 8.6 g/l by S. cerevisiae. The methane production was 342 mLgvs-1 (volatile solid)[5].

Limonene is a green chemical which has minimal or no adverse effects to health or environment. It is generally recognized as safe in applications by Food and Drug Administration (FDA). It has aromatic properties and used as a stabilizing agent in non-alcoholic beverages, fruit juices, ice-creams, cakes and bakery items. Besides, it has therapeutic properties and used in traditional medicine and skin care products due to the presence of nutritive compounds in its composition. In the recent years, limonene is also finding applications in end user industries, such as automobile, aerospace, wood and marine industries and the global demand is on continuous rise. Pectin finds applications as gelling agent, and a variety of food products, such as jam, jellies, yogurts and deserts. It reduces cooking time, improves texture of the processed food and enhances its shelf life. It is also used in wound healing preparations, medical adhesives and other pharceutical products. Pectin have been observed to rise in demands in the European and Asia-Pacific countries, particularly in the food and beverage industries based on processed and packaged food. Galacturonic acid is obtained from oxidation of galactose, a sugar, and a main componentof pectin. In pectin it exists in polymeric form, i.e., polygalacturonic acid. It is in high demands for its application in chemical industries, as laboratory reagent, and personal care products. The global organic acid market is expanding due to the rising usage of organic acids in food, beverages, cosmetics, chemical and pharmaceutical industries, animal feed as a substitute of antibiotic growth promoters (AGP) due to its antioxidant properties, preservation, acid regulation and flavor enhancement. Furthermore, extraction of a number of bioactive compounds, such as flavonoids, pectin, galacturonic acid, carotenoids, etc., which possesses commercial value in terms of their applications in food and pharmaceutical industries, renders the production of ethanol from citrus waste biomass more beneficial in terms of economic advantages [6-13].

The summary of different processes carried out in a biorefinery dealing with citrus wastes to produce biofuel, biogas and important chemicals of potential commercial values are presented in Figure 14. The recent progresses in this field are summarized in Table 3.

Table 3.  Important chemicals of potential commercial value and their application

2.) In the manuscript, some scientific facts are written without experimental evidence and cross-referencing. Therefore it is highly recommend to add some more references. For instance in page 3 line 118-120, page 5 line 158-159, page 15 line 365-368 and so on.

Response: The above-mentioned recommendations by the reviewer have been included in the revised manuscript. The pretreatment section has been hugely modified and references have been carefully incorporated.

3.) Please improve the sentence, page 15 page 347-348.

Response: The suggested correction has been carefully incorporated.

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We are grateful to you for the valuable comments again.

Sincerely yours,

Prof. Sunghun Cho

School of Chemical Engineering

Yeungnam University

Gyeongsan, Korea 38541

Tel) +82-53-810-2535

E-mail) [email protected]

1. Pourbafrani, M.; Forgács, G.; Horváth, I.S.; Niklasson, C.; Taherzadeh, M.J. Production of biofuels, limonene and pectin from citrus wastes. Bioresource Technology 2010, 101, 4246–4250.
2. Zhou, W.; Widmer, W.; Grohmann, K. Economic analysis of ethanol production from citrus peel waste. Proceedings of the Florida State Horticultural Society 2007, 120, 310-315.
3. Rivas-Cantu, R.C.; Jones, K.D.; Mills, P.L. A citrus waste-based biorefinery as a source of renewable energy: technical advances and analysis of engineering challenges. Waste Management research 2013, 31, 413-420.
4. Mazumdar, A.; Srivastava, R. Citrus Peel Gasification using Molten Sodium Heat Pipes; Hemispheric Center for Environmental Technology, Florida International University, Miami, Florida 33174 Miami, Florida, 2004.
5. Patsalou, M.; Samanides, C.G.; Protopapa, E.; Stavrinou, S.; Vyrides, I.; Koutinas, M. A Citrus Peel Waste Biorefinery for Ethanol and Methane Production. Molecules 2019, 24, 2451-2466.
6. Research, H. Limonene Market Size and Forecast, By Application (Food & Beverage, Pharmaceutical, Personal Care, Industrial) and Trend Analysis, 2015 - 2025; 2019.
7. Reports, V. Lactic Acid Market Size USD 1156.5 Million by 2026 at a CAGR 2.5%; Bangalore, India, 2020.
8. Reports, M. Global 5- hydroxymethylfurfural (5-HMF) Market Report 2018; 2018; p 123.
9. Report, M.R. Pectin Market by Type (HM Pectin, LM Pectin), Raw Material (Citrus fruits, Apples, Sugar beet), Function, Application (Food & beverages, Pharmaceutical & Personal Care Products, Industrial Applications), and Region - Global Forecast to 2025. Report code FB 7357 2019, 181.
10. Report, M.A. Succinic Acid Market Analysis By Application (1,4 BDO, Resins, Coatings, Dyes & Inks, Pharmaceuticals, Polyurethanes, Food, Plasticizers, Cosmetics, Solvents & Lubricants, De-icing Solutions) And Segment Forecasts to 2022; 2016; p 93.
11. Report, M. Prebiotic Ingredients Market - Forecast(2021 - 2026); 2019.
12. Mahato, N.; Sinha, M.; Sharma, K.; Koteswararao, R.; Cho, M.H. Modern Extraction and Purification Techniques for Obtaining High Purity Food-Grade Bioactive Compounds and Value-Added Co-Products from Citrus Wastes. Foods 2019, 8, 523(521-579).
13. Mahato, N.; Sharma, K.; Sinha, M.; Baral, E.R.; Koteswararao, R.; Dhyani, A.; Cho, M.H.; Cho, S. Bio-sorbents, industrially important chemicals and novel materials from citrus processing waste as a sustainable and renewable Bioresource: A review. Journal of Advanced Research 2020, 23, 61-82.
14. Watch, M. Limonene Market Analysis with Top Countries Data, Market Size, Key Players, Applications, Trends and Forecasts to 2026. 2021.
15. Report, E.M. Enzymes Market Size Worth $17.2 Billion By 2027 | CAGR: 7.1%; 2020.
16. Report, M. Enzymes Market Type (Protease, Carbohydrase, Lipase, Polymerase and Nuclease, and Other Types), Source (Microorganisms, Plants, and Animals), Reaction Type (Hydrolase, Oxidoreductase, Transferase, Lyase, and Other Reaction Types), and Application (Food and Beverages, Household Care, Bioenergy, Pharmaceutical and Biotechnology, Feed, and Other Applications) - Global Opportunity Analysis and Industry Forecast, 2017-2024. 2018, 344.
17. Market, C.A. Citric Acid Market by Form (Anhydrous and Liquid), Application (Food, Pharmaceuticals, and Cosmetics), Function (Acidulant, Antioxidant, Preservative, and Sequestrant), and by Region (North America, Europe, Asia-Pacific, and RoW) - Global Forecast to 2020; 2015.
18. Report. Protein Extracts from Single Cell Protein Sources Market by Source (Algae, Yeast, Bacteria, Fungi), by Application (Animal Feed, Biotechnology, Agriculture & Fertilizers), by Geography (U.S., Canada, China, India, Japan, Germany, U.K., France, Italy, Spain, Brazil, Mexico, South Africa) – Global Market Size, Share, Development, Growth and Demand Forecast, 2013–2023; 2018; p 127.
19. Report. Protein Extracts from Single Cell Protein Sources Market by Source - Global Market Size, Share, Development, Growth and Demand Forecast, 2013-2023; 2018; p 127.

Author Response File: Author Response.docx

Reviewer 2 Report

The present review reports a comprehensive study of the recent literature on citrus waste recovery and its biological transformation for the production of valuable chemicals and biofuels. Indeed Citrus waste have been extensively investigated for the extraction of bioactive molecules and limonene and can be further utilized to produce ethanol, biogas and fuels. The authors should better outline the importance of the present review. The high number of studies recently reported in literature testify the attention of the scientific community and of the post processing industry to recover valuable materials and chemicals.

The manuscript is in line with the scope of the journal, and the number of the literature reported are reasonable, but the authors should also mention, at least in the introduction section of other possibility of biofuel production from citrus waste. Pyrolysis, gasification and hydrothermal carbonization have been investigated as thermochemical upgrade of citrus waste and represent  valid alternatives for the production of solid biofuels and recovery of valuable chemicals.  Consider to include relevant literature as:

Pyrolysis: Volpe, M., et al.: Upgrade of citrus waste as a biofuel via slow pyrolysis,(2015) Journal of Analytical and Applied Pyrolysis, 115, pp. 66-76. DOI: 10.1016/j.jaap.2015.06.015.

Gasification: Volpe, R., et al.  Catalytic effect of char for tar cracking in pyrolysis of citrus wastes, design of a novel experimental set up and first results,(2016) Chemical Engineering Transactions, 50, pp. 181-186. ;

Hydrothermal carbonization (HTC): Saha, N.; et al: Cationic Dye Adsorption on Hydrochars of Winery and Citrus Juice Industries Residues: Performance, Mechanism, and Thermodynamics. Energies 2020, 13, 4686.

English language should be checked and revised carefully before considering for publication.

Author Response

Response to Reviewer comments

Reviewer 2.

Dear Reviewer

First, we greatly appreciate you for reviewing our manuscript.

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The present review reports a comprehensive study of the recent literature on citrus waste recovery and its biological transformation for the production of valuable chemicals and biofuels. Indeed Citrus waste have been extensively investigated for the extraction of bioactive molecules and limonene and can be further utilized to produce ethanol, biogas and fuels. The authors should better outline the importance of the present review. The high number of studies recently reported in literature testify the attention of the scientific community and of the post processing industry to recover valuable materials and chemicals.

The manuscript is in line with the scope of the journal, and the number of the literature reported are reasonable, but the authors should also mention, at least in the introduction section of other possibility of biofuel production from citrus waste. Pyrolysis, gasification and hydrothermal carbonization have been investigated as thermochemical upgrade of citrus waste and represent valid alternatives for the production of solid biofuels and recovery of valuable chemicals.  Consider to include relevant literature as:

Pyrolysis: Volpe, M., et al.: Upgrade of citrus waste as a biofuel via slow pyrolysis,(2015) Journal of Analytical and Applied Pyrolysis, 115, pp. 66-76. DOI: 10.1016/j.jaap.2015.06.015.

Gasification: Volpe, R., et al.  Catalytic effect of char for tar cracking in pyrolysis of citrus wastes, design of a novel experimental set up and first results,(2016) Chemical Engineering Transactions, 50, pp. 181-186. ;

Hydrothermal carbonization (HTC): Saha, N.; et al: Cationic Dye Adsorption on Hydrochars of Winery and Citrus Juice Industries Residues: Performance, Mechanism, and Thermodynamics. Energies 2020, 13, 4686.

English language should be checked and revised carefully before considering for publication.

Response. We are grateful to the reviewer for the suggestion. We have included a paragraph as per suggestion to the introduction section of the revised manuscript and the suggested articles have been cited. After a detailed discussion with us authors on this, we came to agree on preparing a separate manuscript on this topic. As the number of reports published on the preparation of adsorbent materials for adsorption of dyes and heavy metals is huge. Hence, we have decided to divide the present manuscript in two parts, viz., 

1. “Biotransformation of Citrus Waste-I: Production of Biofuel and Valuable Compounds by Fermentation”,

 and

2. “Biotransformation of Citrus Waste-II: Biosorbent Materials for the Removal of Dyes and Heavy Metals from Polluted Water”

The added portion in the current revised manuscript is appended below in highlights.

Hydrogen is a clean fuel gas and presents a wide range of power options for domestic as well as industrial applications. Currently, hydrogen is produced from non-renewable resources, i.e., fossil fuels by means of steam reforming. In this process, the natural gas reacts with steam under high pressure at high temperatures in the presence of nickel-based catalyst. Alternatively, hydrogen can also be produced from renewable resources, such as biomass by means of gasification. In the process of gasification, hydrogen is produced via pyrolysis of citrus waste biomass in dried pelleted form in an electrically heated furnace. The resulted gas produced from the pyrolysis process is cooled and analyzed for gaseous products and its fuel properties [23,24]. Volpe et al. carried out torrefaction and slow pyrolysis experiments at 200-650 ºC to obtain bio-oil and biochar from lemon and orange peel wastes by thermal degradation in a horizontal fixed-bed pyrolysis reactor (FBR). Torrefaction of the peel residues in the temperature range 200-325 ºC produced fuels with energy densities of 1.56 and 1.58 from lemon peels and orange peels, respectively and found to be stable at a high temperature of 325 ºC. Pyrolysis of the peel residues at 400-650 ºC produced high energetic bio-chars and tars [25]. Pyrolysis is a thermo-chemical treatment to the residual biomass to convert it into solid biofuels which is a mix of solid(chars), vapors (tar) and gases. The tar yields of pyrolysis of ~2g of oven-dried lemon pulp were found to be ~ 0.4 g of chars [26]. In addition the citrus waste residues have also been investigated for its promising role in the adsorption of dyes and heavy metals from polluted waters [27].

This article attempts to review the recent processes in the field of biomass energy, biofuels and biosorption of dyes and heavy metals by processing of the citrus waste biomass via biotransformation. The article also includes a detailed description of various useful compounds and materials that can be extracted commercially from citrus wastes. These chemicals find applications in the synthesis and production of various industrially important chemicals of commercial significance. The motivation behind this review is to emphasize the need of the hour to explore the possibilities of harnessing the hidden potential of producing valuable utilizable products out of citrus biowaste which go unnoticed and dumped usually. The review has been presented in two parts under titles “Biotransformation of Citrus Waste-I: Production of Biofuel and Valuable Compounds by Fermentation”, and “Biotransformation of Citrus Waste-II: Biosorbent Materials for the Removal of Dyes and Heavy Metals from Polluted Water”. The first part deals with achieving various product from fermentation of the citrus wastes, e.g., biofuels (ethanol, methane and biodiesel) and valuable compounds, viz., organic acids (citric, succinic, pyruvic, lactic, acetic), Vit-C, enzymes, single cell proteins and prebiotics. The second part deals with obtaining adsorbent materials for the adsorption of dyes and heavy metals from waste/ polluted-water employing thermochemical biotransformation methods.

The English language in the manuscript has been thoroughly revised.

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We are grateful to you for the valuable comments again.

Sincerely yours,

Prof. Sunghun Cho

School of Chemical Engineering

Yeungnam University

Gyeongsan, Korea 38541

Tel) +82-53-810-2535

E-mail) [email protected]

Author Response File: Author Response.docx

Reviewer 3 Report

The article deals with an important and topical topic.

Citrus waste as a renewable feedstock for production of biofuel. It was described very well. It is legible and understandable. The article collects and systematizes many (current) scientific works.

Abstract: I understand the nature of the work.

Keywords: are selected correctly.

Introduction: it is relevant and valuable in terms of content, you can only have comments that can be discussed with the authors:

1. Figure 1, whether the authors have the rights to publish the drawing, in my opinion, looks little scientific.
2. "The biofuels can reduce carbon dioxide emission by 80% compared to gasoline or petroleum fuel [12-14]." The sentence is true, but are the authors aware that very often the SI combustion engine powered by gasoline, after switching to gas (methane-based) fuel, reduces power and torque?

Chapters 2 to 5: are very well described and the attached tables and figures reflect the overview made very well.

The summary is also correct and substantively valuable.

The literature attached to the article is up-to-date based on scientific journals.

Congratulations to the authors for a job well done.

Author Response

Response to Reviewer comments

Reviewer 3.

Dear Reviewer

First, we greatly appreciate you for reviewing our manuscript.

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Reviewer 3.

The article deals with an important and topical topic.

Citrus waste as a renewable feedstock for production of biofuel. It was described very well. It is legible and understandable. The article collects and systematizes many (current) scientific works.

Abstract: I understand the nature of the work.

Keywords: are selected correctly.

Introduction: it is relevant and valuable in terms of content, you can only have comments that can be discussed with the authors:

1. Figure 1, whether the authors have the rights to publish the drawing, in my opinion, looks little scientific.

Response: The drawings is presenting the summarized viewpoint on environmental aspects of the citrus waste when left untreated. It can be processed and transformed adequately for obtaining valuable products and the environment can be saved from the consequences of pollution. The meaning is scientific. The graphic has been taken from a previously publish article by the same authors and can be republished with permission.

1. "The biofuels can reduce carbon dioxide emission by 80% compared to gasoline or petroleum fuel [12-14]." The sentence is true, but are the authors aware that very often the SI combustion engine powered by gasoline, after switching to gas (methane-based) fuel, reduces power and torque?

Response: The authors are aware of this issue. The biogas can be efficiently used for producing electricity and warming the houses during winter seasons, other than fueling vehicles. In addition, ethanol has another problem of corrosion of engine parts as it is hygroscopic and absorbs moisture. Therefore, in tropical countries this is a serious issue where the moisture level is usually very high.  The technology to achieve the best and desired results in terms of is in primitive stages and needs further investigations and newer models to address this problem

Chapters 2 to 5: are very well described and the attached tables and figures reflect the overview made very well.

The summary is also correct and substantively valuable.

The literature attached to the article is up-to-date based on scientific journals.

Congratulations to the authors for a job well done.

Response: The authors are very thankful to the reviewer for the efforts and time.

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We are grateful to you for the valuable comments again.

Sincerely yours,

Prof. Sunghun Cho

School of Chemical Engineering

Yeungnam University

Gyeongsan, Korea 38541

Tel) +82-53-810-2535

E-mail) [email protected]

Author Response File: Author Response.docx