Residual Gas for Ethanol Production by Clostridium carboxidivorans in a Dual Impeller Stirred Tank Bioreactor (STBR)

Round 1

Reviewer 1 Report

Line 41: Typo: Pre-pre treatment

Lines 72-74: What are the advantages of adding surfactants over the other mentioned strategies? Why the authors decided to investigate this strategy?

Line 89: How the syngas composition was determined?

Lines155-160: Syngas components utilization/variation was not monitored by any means during the experiments? Could the authors provide a justification for this choice?

Figure 3&4: Concentration of metabolites was measured for only three time points, after 20, 100 and 120 hours of fermentation. Could the author provide a rationale for choosing these time points? And the reason for not sampling between 20 and 100h?

 

I would also suggest to add a Discussion section, with the relevance of these findings, how they compare to previous studies and future perspectives.

 

Author Response

Line 41: Typo: Pre-pre treatment

This correction was performed.

 

Lines 72-74: What are the advantages of adding surfactants over the other mentioned strategies? Why the authors decided to investigate this strategy?

It is a simple and cheap approach. The price of tween80 at MilliporeSigma (https://www.sigmaaldrich.com/US/en) is around US$30/liter, which could be reduced when industrial amounts are commercialized. The hollow fiber membrane reactor (HFMR), which hinders high mass transfer coefficient has drawbacks related to scale-up into commercial size HFMBR and membrane fouling (Yasin et al., 2014). STR are simple to operate, but the increase in agitation speed and gas flow rate can cause cell damage and increase in energy costs. Therefore, the addition of a surfactant to STR can maintain simplicity and increase mass transfer. This justification was added to the introduction section (lines 81-83).

 

Line 89: How the syngas composition was determined?

The gas was ordered from White Martins Praxair Inc. with this specific composition based on the gas obtained by pyrolysis of urban waste. This technology has been developed by Main Engineering Srl and Innova Renewable Energies. This information was added to the document (lines 93-95).

 

Lines155-160: Syngas components utilization/variation was not monitored by any means during the experiments? Could the authors provide a justification for this choice?

Our goal was to evaluate the effect of tween 80 on ethanol production. Although syngas components profile would help to explain some results, the unavailability of the analytical equipment to determine these components when the experiments were performed, made us decide to focus on product formation only.

 

Figure 3&4: Concentration of metabolites was measured for only three time points, after 20, 100 and 120 hours of fermentation. Could the author provide a rationale for choosing these time points? And the reason for not sampling between 20 and 100h?

In our previous studies, we have identified that ethanol production by this strain in ATCC® 2713 medium increases gradually until 24 h, then stabilizes [5]. In other culture medium, it starts to increase again after 70 h [5]. So, we decided to sample serum bottle fermentations at strategic points (24 h – the first peak, 96 h – the second peak, and then 120 h, to verify final stabilization) to avoid volume reduction This information was added to the text (lines 227 – 231).

 

I would also suggest to add a Discussion section, with the relevance of these findings, how they compare to previous studies and future perspectives.

We agree with the reviewer and this section was added, with a comparison to previous studies and possible outcomes of these findings (lines 338-373).

Author Response File: Author Response.pdf

Reviewer 2 Report

I have carefully read the manuscript and found that this work is important for the development of the pure and applied research. In my opinion, manuscript entitled: “Residual gas for ethanol production by Clostridium carboxidivorans in a dual impeller stirred tank bioreactor (STBR)” might be of interest to a specific group of readers. The authors presented the possibility of producing ethanol and higher alcohols from syngas using the bacteria Clostridium carboxidivorans and supplementation with a surfactant. The positive effect of the addition of compounds lowering the surface tension on the efficiency of ethanol production by increasing the solubility of gases (CO, CO2) in the substrate was demonstrated. The usefulness of the bioreactor culture in the production of alcohols from syngas was also indicated. However, the manuscript needs some improvements in the form of:

#1: Line 41: Please correct the word "pretreatment".

#2: Please indicate the novelty of the presented research in relation to the research of other authors. Please also explain why only Tween 80 was used as a surfactant and not another industrial surfactant.

#3: Please indicate in section 2.1. how was syngas stored (pressurized cylinder?).

#4: Please add in section 2.5.2. HPLC analysis details. Please add the range of compounds analyzed, method of quantification (ESTD or ISTD), injection, and method parameters.

#5: Section 3.1.1: The biomass concentration was highest at the 10th hour of cultivation. How is it possible that the biomass concentration has decreased? It should be understood that the viability of the cells has decreased or the biomass has been diluted. The concentration of biomass cannot decrease.

#6: In bioreactor studies, there is a clear lack of control in the form of culture without the addition of Tween 80. Please comment on this.

#7: It is worth adding a preliminary mass balance and presenting an economic account of the production of ethanol and higher alcohols from syngas.

#8: In the discussion, please indicate the differences between the presented studies and the studies of other authors. Please indicate the novelty aspect.

Author Response

Reviewer 2

I have carefully read the manuscript and found that this work is important for the development of the pure and applied research. In my opinion, manuscript entitled: “Residual gas for ethanol production by Clostridium carboxidivorans in a dual impeller stirred tank bioreactor (STBR)” might be of interest to a specific group of readers. The authors presented the possibility of producing ethanol and higher alcohols from syngas using the bacteria Clostridium carboxidivorans and supplementation with a surfactant. The positive effect of the addition of compounds lowering the surface tension on the efficiency of ethanol production by increasing the solubility of gases (CO, CO2) in the substrate was demonstrated. The usefulness of the bioreactor culture in the production of alcohols from syngas was also indicated. However, the manuscript needs some improvements in the form of:

#1: Line 41: Please correct the word "pretreatment".

This correction was performed.

#2: Please indicate the novelty of the presented research in relation to the research of other authors. Please also explain why only Tween 80 was used as a surfactant and not another industrial surfactant.

The novelty was indicated in the new section: “Discussion” (lines 338-373). Tween 80 is frequently used for microbial cultures since it is known to be non-toxic to cells. Carbon monoxide fermentations with several surfactants was tested before and Tween 80 was the best for cell growth (Bredwell et al 1997). Besides, previous studies showed “… significant increase (120%) in carbon monoxide mass transfer coefficient when Tween^® 80 and/or PFC (perfluocarbon) were added to water.” (lines 74 to 76).

Bredwell MD, Telgenhoff IMD, Barnard IS, Worden RMI (1997) Effect of Surfactants on Carbon Monoxide Fermentations by Butyribacterium methylotroph icum. 63:

#3: Please indicate in section 2.1. how was syngas stored (pressurized cylinder?).

Yes. The gas was stored in pressurized cylinder and this information was added to this section (lines 93-95).

 

#4: Please add in section 2.5.2. HPLC analysis details. Please add the range of compounds analyzed, method of quantification (ESTD or ISTD), injection, and method parameters.

This information was added to section 2.5.2 as suggested by the reviewer.

 

#5: Section 3.1.1: The biomass concentration was highest at the 10th hour of cultivation. How is it possible that the biomass concentration has decreased? It should be understood that the viability of the cells has decreased, or the biomass has been diluted. The concentration of biomass cannot decrease.

This growth profile has been shown for syngas batch fermentations with C. carboxidivorans in other works (Fernández-Naveira et al 2016a; Fernández-Naveira et al 2016b; Fernández-Naveira et al., 2019). Since syngas is not continuously fed in serum bottles, cell growth reaches stationary phase, when there is a balance between growth and death of cells. Fernández-Naveira et al 2016a showed that ethanol causes inhibition of cell growth. So, without substrate supply and with a toxic compound being produced, it is possible that cell death overlaps cell growth and autolysis may occur, reducing turbidity (Ogata et al., 1980). This information was added to the text (lines 183-190).

Fernández-Naveira, Á., Abubackar, H. N., Veiga, M. C., & Kennes, C. (2016a). Efficient butanol-ethanol (BE) production from carbon monoxide fermentation by Clostridium carboxidivorans. Applied microbiology and biotechnology, 100(7), 3361-3370.

Fernández-Naveira, Á., Abubackar, H. N., Veiga, M. C., & Kennes, C. (2016b). Carbon monoxide bioconversion to butanol-ethanol by Clostridium carboxidivorans: kinetics and toxicity of alcohols. Applied microbiology and biotechnology, 100(9), 4231-4240.

Fernández-naveira Á, Veiga MC, Kennes C (2019) Selective anaerobic fermentation of syngas into either C 2 -C 6 organic acids or ethanol and higher alcohols. Bioresour Technol. https://doi.org/10.1016/j.biortech.2019.02.018

Ogata, S., Choi, K. H., & Hongo, M. (1980). Morphological changes during conversion of Clostridium saccharoperbutylacetonicum to protoplasts by sucrose-induced autolysis. Microbiology and immunology, 24(5), 393-400.

#6: In bioreactor studies, there is a clear lack of control in the form of culture without the addition of Tween 80. Please comment on this.

The goal of bioreactor fermentation was to verify whether product formation was still possible with Tween 80, mechanical agitation and gas supply, which did not occur in bottle flasks. Tween 80 can form too much foam depending on its concentration, culture medium and gas flow rate. We have shown that these problems did not occur, and product formation was possible, which was our goal. This information was added to the text (lines 366-369).

 

#7: It is worth adding a preliminary mass balance and presenting an economic account of the production of ethanol and higher alcohols from syngas.

An economic study at this point would be too incipient (not enough data) and it was not the scope of the present study.

 

#8: In the discussion, please indicate the differences between the presented studies and the studies of other authors. Please indicate the novelty aspect.

A discussion section was added to the manuscript with a comparison to previous studies indicating the novelty aspect (lines 338-373).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Manuscript corrected as recommended by the reviewer and ready for publication.