Bio Ethanol Production from Rice Straw Saccharification via Avicelase Gene in E. coli Recombinant Strain

Referee  1

1.       The grammar and format and spelling need to be checked as

Line 24: (bioethanol)

Line 74: correct the format

Line 157: (most)

Line 231: (showed)

Line 295: should be deleted

Unify the beginning format of each paragraph

Unify the format of “Figure” capture in the text

Done

Done

Done

Done

Done

Done

Done

2.       For the all used chemicals, identify the manufacture and country of origin

Done

3.       Section “2.5 cloning of avicelase gene”: put the web site in the reference and referred their reference number in the text

Done

4.       In section “2.6.2. effect of inorganic nitrogen and pH on avicelase activity”: Explain why 3 days were chosen as the incubation period.

Done

5.       In line 123: What is your evidence of reaching the required level of washing?

until reaching neutral pH in the washed water (Added in text)

6.       What is the device used in the centrifugation process (model, manufacture and country of origin)?

(Cooling centrifuge, HERMLE, Z 326K, Germany) (Added in text)

7.       Explain what procedures are used to avoid contamination of the sample at the different stages

Added in the text

8.       Tabel4: Why weren't the following days studied until the highest value of activity was reached?

The treatments were up to one week but results were declined after the third day.

Added in text “. After the third day the avicelase activities were declined gradually (data not shown).”

9.       In section “3.5.2. Effect of inorganic nitrogen and pH on avicelase activity” and section “3.5.3. Effect of organic nitrogen and pH on avicelase activity” Explain the reason and cite previous studies

Strategies for plant wastes-degradation concentrate on using efficient cellulolytic microorganisms strains individually or in a combination. Utilizing different cellulolytic microorganisms requires identifying the degradation conditions that best fit to all used strains for best synergistic action.

The research team’s overall aim is to use different cellulolytic microorganisms and different clones for efficient rice straw saccharification. For this purpose the present study aimed to clone avicelase gene and to identify its expression in different growth conditions.  It also aimed to saccharification rice straw by avicelase and uses the produced sugars for ethanol production.

10.   In line 308 and 309: explain how the ethanol concentration was calculated.

Ethanol yield content of the fermented samples was measured by determination of consumed sugar after fermentation and converted it to ethanol following the equation: 1 g glucose produce 0.51 g ethanol which equal 0.51 X 100/81 = 0.62 ml ethanol as described in (23).

11.   The conclusion needs to be enhanced

Done

12.   Many of the references are old. Newer and useful references in the same field can be cited such as (https://doi.org/10.3390/polym13213762, https://doi.org/ 10.3390/en15176131, and https://www.mdpi.com/1422-0067/23/3/1777

Done

13.   It is recommended to add the effect of the pretreatment on the composition of the used rice straw

Done

Referee  2

ü  The introduction is essential. It needs to be expanded.

The literature references provided in the introduction are outdated and need to be revised. Indeed, the topic proposed by the authors is well covered in more recent literature.

Done

ü  Lines 36-37 “Most of these straws are buried, decayed in the fields or burned, which cause waste and pollution”.

Please, support this information with recent literature references.

Three recent references were added:

John  et al., 2022, Patel et al., 2021, Salleh et al., 2019

ü  37-38 “Biological hydrolysis of cellulosic substrates using microbial cellulolytic enzymes is widely recognized as an ecologically clean process”.

Compare chemical and biological methods and briefly explain their advantages and disadvantages to deepen the discussion. Please, support information with recent literature data.

Added: “Cellulosic substrates could be hydrolyzed by chemical and biological means. Chemical hydrolysis is rapid and efficient process, but it is energy-intensive, produced hazardous waste, and can inhibit downstream processes. Biological hydrolysis is a more environmentally friendly alternative, carried out under milder conditions and with lower energy requirements. However, it can be slower and dependent on microorganisms' activity, limiting throughput and scalability, with challenges in maintaining consistent enzyme production. Recent studies showed that biological hydrolysis is a promising approach for producing sugars from cellulosic substrates, including the use of mixed microbial cultures and genetically engineered bacteria, which could become an increasingly important method for producing sustainable and renewable fuels and chemicals (Choi et al., 2021) and (Zhang et al., 2020).”

ü  Lines 39-41 “Degradation of cellulose into glucose and then its utilization as an energy source for bioconversion into various useful materials have been studied intensively [1]”.

Please, support this information with a more recent literature reference.

Done:

(AbdElhafez et al., 2022)

ü  Lines 42-43 “A set of enzymes are needed for the complete degr-adation of cellulose [Bhat and Bhat, 1997] [2]”.

Please, delete [Bhat and Bhat, 1997]. Regardless, the literature reference is outdated. Replace it or add a more recent literature reference.

It is replaced by [Kavitha et al., 2021, Kavitha et al., 2022]

ü  Lines 43-46 “The cellulase family consists of three types, endo-β-1,4-glucanase (EC 3.2.1.4), exo-β-1,4-glucanase (cellobiohydrolase or avicelase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) [3]. These enzymes act synergistically and hydrolyze cellulose chain into single sugars.”

Please, support both sentences with recent literature data. Please, provide a brief overview of cellulase enzymes as well as commercial products (also as cellulase cocktails) currently on the market and their costs.

References were added: [Kavitha et al., 2021, Kavitha et al., 2022]. And [John et al., 2022].

A paragraph was added: Cellulases have many roles in different fields including environment and industry. They play a vital role in drifting the carbon cycle from various cellulose biomass (Meng and Ragauskas, 2014). They also have an important applications in the textile industry (Singh et al., 2007), food, paper and pulp, laundry, waste management and biofuel production (Kuhad et al., 2010; Anna-malai et al., 2016).

* Cellulose market report was added Cellulase Market Outlook (2022 – 2032)

ü  Lines 47-49 “The first cellulase gene was cloned in 1982 [4], most of cellulase genes have been expressed in E. coli, S. cerevisiae and Pichia pastoris [5-9], but the expressed cellulase activity was low”.

Please, provide data, available from the recent literature, on the low activity of expressed cellulases.

This sentences was replaced with “

The first cellulase gene was cloned in 1982 [4], most of cellulase genes have been expressed in E. coli, S. cerevisiae and Pichia pastoris [5-9]. The cellulase was expressed in these hosts with different activities.  More molecular and biotechnology studies are still needed for their commercial application such as using expression vectors (Cabrita et al., 2006) and hosts to produce high levels of cellulase.

Please refer to “The recombinant P. pastoris had avicelase activity in the supernatant of 0.1276 U/mL. [33]” in 3.5 section.

ü  Lines 49-50 “It is needed to select different vector and host to produce high levels of cellulase for commercial use”.

Please, support this information with recent literature references.

ü  Lines 51-52 “Cellulosic biofuels are not produced at a competitive level yet, due to the high cost of processing with the currently available technologies [10]”.

One of the reasons why biofuels from the cellulosic matrix are not competitive is the high cost of hydrolytic enzymes used in the saccharification step. Please provide a more detailed explanation of how the production of enzymes using recombinant DNA technology could be advantageous for reducing the cost of the biofuel production process.

Add: “Using strong promoters in expression vectors is one of the promising approaches for high production levels of cellulases genes products and hence reducing their production cost. In another approach, by using the appropriate vector cellulases genes could expressed directly in yeast strains and acquire them the cellulolytic activity to utilize cellulose and convert it to bioethanol.”

ü  Lines 52-55 “To utilize cellulolytic enzymes in the degradation of cellulosic materials industrially, it is important to study the enzymatic characteristics of these enzymes and to clarify the mechanisms of the synergistic effects among the enzymes”.

This concept needs to be clarified.

A new sentence was added:” Cellulose hydrolysis requires the synergistic action of several classes of enzymes, and achieving the optimum secretion of these simultaneously remains a challenge (Mattam et al., 2022).”

ü  Line 67 “LB medium [14] was used for bacterial growth”.

Since the acronym LB is used for the first time in the article, it must be explicitly stated.

Done

ü  Lines 71-73 “An equal E. coli inoculum was used to inoculate each medium by inoculated the corresponding amount to reach OD 620 0.01 / 20 ml from an overnight culture (Huss [12]. This inoculum contains about 1.3 X 105 CFU/ml”.

Please, delete "(Huss", before of "[12]".

Done

ü  Line 74 “Screening for avicelase production”

Is this the title of a subparagraph? Please, Follow the guidelines for authors on how to indicate subparagraphs.

Done

ü  Line 77 “They were inoculated on BHM medium supplemented with…”

Since the acronym BHM is used for the first time in the article, it must be explicitly stated.

Done

ü  Line 85 “The resulting samples were then cooled to in ice water..”

Please, explain the need to perform this step.

As mentioned by the cited reference this step was done to stop the reaction.

ü  Lines 104-106 “E. coli 104 DH5α (pAvi-168) was grown on different media including complex medium (LB medium) and minimal medium (BHM medium) supplemented with different nitrogen sources using different shaking conditions”

What are the reasons for using different shaking conditions?

Shaking condition increase oxygen level and homogenate the culture contents.

Added at section 3.5

ü  Lines 152 “Ethanol yield content of the fermented samples was measured as described in [21]”.

It is not enough to indicate the reference. Please, describe the method/process used to measure ethanol levels in fermented media.

Added :” Ethanol yield content of the fermented samples was measured by determination of consumed sugar after fermentation and converted it to ethanol following the equation: 1 g glucose produce 0.51 g ethanol which equal 0.51 X 100/81 = 0.62 ml ethanol as described in (23).”

ü  Lines 136-152.

It is not necessary to divide the paragraph "2.10. Fermentation of bioethanol" into five short subsections/subparagraphs. Please, assemble these subsections/subparagraphs into one paragraph (into 2.10).

Done

I suggest improving this paragraph.

ü  Lines 157-165.

Several concepts in this paragraph are already discussed in the introduction, so they are redundant. Regardless, this information should be emphasized in the introduction rather than in paragraph 3.

Done

ü  Lines 199-200. Figure 1

Please improve the figure so that caption is easier to understand.

Figure 1 was improved

ü  Lines 211 “Results confirmed the existence of plasmids in all tested E. coli transformants”.

Please explain the results obtained in more detail.

Two sentences were added:

The existence of different DNA bands in each tested transformant represents the different plasmid DNA forms of one plasmid such as supercoiled, open circular or linear plasmid.

   Results obtained from Fig. 4 confirmed the existence of only one plasmid in each tested transformant, in spite of its different forms showed in Fig. 3. Figure 4 also presented the avicelase gene (lower DNA bands) in all tested transformants where the EcoRI cuts the recombinant plasmid twice at the border of the insert (Fig. 1).

ü  Lines 247-270 §3.5. “Optimized condition for avicelase expression”

Please, provide a table summarizing the results obtained under different experimental conditions.

The suggested Table is not easy to make since different parameters were studied. Instead a paragraph could be more useful.

Added:” The highest avicelase expression was found at the 2nd day of incubation with avicel which produced 50 IU/L, followed by using rice straw for two days (40 IU/L). Using LB medium with shaking for three days produced 39 IU/L. The best inorganic nitrogen source was found to be (NH4)2NO3 at pH 6 (about 1.4 IU/ mg protein, followed by the same compound at pH 7 and with (NH4)SO4  at pH 8 (more than 1.2 IU/ mg protein). The best organic nitrogen was malt extract at pH 7 which produced about 0.45 IU/ mg protein.”

ü  Lines 302-310 §3.6. Bioethanol fermentation

Please, describe the calculation method for estimating fermentation efficiency.

Ethanol yield content of the fermented samples was measured by determination of consumed sugar after fermentation and converted it to ethanol following the equation: 1 g glucose produce 0.51 g ethanol which equal 0.51 X 100/81 = 0.62 ml ethanol as described in (23).

ü  The "conclusions" paragraph is very weak. Please, highlight the main outcomes, the benefits that this research study can provide in the production process of bioethanol from the cellulosic matrix, and possible future perspectives.

Done

ü  Please, check for any language or grammatical errors.

Done

ü  Please, follow the “instructions for authors” provided by the journal to improve the manuscript template.

Done

Referee  3

the article is suitable for publication in Cleantech. There are some flaws that need to be clarified:

Thank you

- The novelty of the work is not clearly stated in the introduction. Review .

Done

- in the results, insert more literature in order to compare the results obtained.

Done

- conclusion is too short and does not portray the work;

Done

Moderate English changes required

The article has been proofread by the, English citizen Mr. Islam Diaa Eltayyar, 1 Rathbone Square, London. W1T 1FB , [email protected]

Lines 38-45: Support this information with recent literature

Terasawat, A.; Phoolphundh, S. Simultaneous Biological Pretreatment and Saccharification of Rice Straw by Ligninolytic Enzymes from Panus neostrigosus I9 and Commercial Cellulase. J. Fungi 2021, 7, 853. https://doi.org/10.3390/jof7100853

Takano, M., Hoshino, K. Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus. Bioresour. Bioprocess. 5, 16 (2018). https://doi.org/10.1186/s40643-018-0203-y

Line 109: The word "medium" is repeated twice

Done

- Line 175 Paragraph 2.10. Please enter information relating to the matrix (rice straw) used (also using literature data) for cellulose, hemicellulose, lignin, ash, carbohydrate (glucose, and others such as xylose, arabinose, mannose and galactose) content.

Rice straw is a lignocellulosic material essentially constituted of cellulose (24.0%), hemicelluloses (27.8%), and lignin (13.5%), with important amounts of ash (17%) that correspond mostly to silica (Rosado et al., 2021

-Lines 202-203: Please consider using an equation editor to write the math formula.

Done

- Line 378: Please discuss paragraph 3.6. Bioethanol fermentation better

Done

- Please consider reporting standard errors in Table 4, Table 5, Figures 7 and 8

Done

I would like to sign my review report

Thank you