Tolerance of Kluyveromyces marxianus Under Acetic Acid-, Isoamyl Alcohol-, Hydrogen Peroxide-, and Ethanol-Induced Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work aimed to evaluate the response of K. marxianus under acetic acid-, isoamyl alcohol-, hydrogen peroxide-, and ethanol-induced stress. The high biotechnological potential of these microorganisms determines the relevance of this work. The differences obtained when comparing two tests for determining the toxic effect of stress factors on yeast are also important.

The results obtained are new, which determines the novelty of the work. The topic of the work corresponds to the topic of the journal.

The presented manuscript is a logical, consistent presentation of the material, written in an understandable language. The experimental methodology is described in sufficient detail. The presented figures / graphs are understandable. Statistical data processing was carried out by the authors. The formulated conclusions correspond to the presented results. The cited sources are relevant.

 

Author Response

We appreciate your positive comments on our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper analyses K. lactis for stress responses when treated with isoamyl alcohol, acetic acid, hydrogen peroxide, and ethanol stress.  Cytotoxic and fermentation assays were performed, including dose-response curves to develop IC50 and IC30 curves.  It also demonstrates incongruences between the assay types and describes these.  The experiments are straightforward and conclusions seems sound.  However, the paper seems terse and without true analysis and conclusions that move the science forward.

I would suggest in general in the discussion describing that although you're not sure of the exact reasons for the toxicity, digging a bit more into the potential biochemical pathways and suggesting some more possible reasons, perhaps looking at the Pichia literature to make some inferences.

And perhaps adding some very short experiments at the IC50 looking at enzyme analysis or qPCR of AAT for the isoamyl treatment, ADH for ethanol and so forth, or perhaps a single enzyme assay for all yeast treatments such as ADH to try to get at mechanism of inhibition would strengthen the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper of Acosta-Cuevas et al " Tolerance of Kluyveromyces marxianus..." is interesting and presented some relevant information  on the resistance of K marxianus to different stress agents. However this paper contains some errors and  should be revised in  many points. First of all the References  should be completely rewritten since in many of them the journal name or the pages are lacking and in some there  are wrong indications.
Other poins:
Introduction:

line 34  pen-toses  should be pentoses
line 35  acids shoul be changed in "organic acids"
line 53-55 resveratrol is not a metabolite produced by S. cerevisiae, The yeast produces glycogen, threalose and glycerol in response to osmotic stress!!
line 67 which are the cited "protective molecules" ?

Mat and Methods:

line 95  give the composition of YPD medium

lines 141-144,  really m is the number of duplication for hour, that is 1/Td, where Td is the doubling time!
the growth rate constant is given by:

K= 2,303 ( log M2 –log M1) /  t2-t1 ; being K the constant of exponential growth

N=N₀ e ^Kt

and is equal to  ln2/Td

in addition is not correct to measure the growth rate (or m ) starting from the cell density at time =zero, but is should be measured during the phase of exponential growth. If you look the the data of  Fig.2 (A) it is clear that the growth rate of the cultures in presence of ethanol and of hydrogen peroxyde is the same of the control one,  (i.e the slope between  5 and 8 hours is the same of the control strain  between time 0 and 6 hours) with and initial delay.

Results

line 190  acetic acid causes , not ethanol

lines 220 225,  why rezasurin? the indirect method used the MTT assay as described in the Mat and Meth section!

Table 2 , please specify that m is  1/Td (duplication for hour) and not the rate constant of growth...
Indeed the  Maximum rate of growth for K.marxianus was  0.56 h-1 ( FEMS Yeast Research, Volume 7, Issue 3, May 2007, Pages 422–435, https://doi.org/10.1111/j.1567-1364.2006.00192.x  a 30°C)
and also from your data of Fig2 a value of 0.54 h-1 can be calculated. (this is K = ln2/Td).
In addition the values reported for growth in presence of ethanol and of hydrogen peroxyde are wrong if they are calculated during the exponential growth phase that starts at 4 hr (just after a delay..) and are quite similar to the value of control culture.

line 339   such as growth of mammalian cells (not medical research..)

References.

Most of the references reported are incomplete or wrong, for example  which is the Journal of Ref1 ? and of Ref 4 etc.. but inb many others is lacking the Journal and or the page number..

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing my concerns.  This paper can be published now.

Author Response

Dear reviewer, we greatly appreciate your insightful comments. They helped us greatly improve our manuscript. Thank you very much.

Reviewer 3 Report

Comments and Suggestions for Authors

The  paper of  Acosta-Cuevas et al. "Tolerance of Kluyveramyces marxianus..." has been revised carefully and the authors responded in a positive way to all the questions raised by the presen referee.
There is only a minor question/modification: In the legend of Table 2 (line 360) the definition of  is not correct, since according to  definition of  in the Eq 2 ,  should be is equal to ln2/Td and not 1/Td.

Author Response

REVIEWER’S COMMENT 1. There is only a minor question/modification: In the legend of Table 2 (line 360) the definition of µ is not correct, since according to  definition of µ in the Eq 2 , µ should be is equal to ln2/Td and not 1/Td.

OUR COMMENT. Thak you for your comment. We agree.

MODIFICATION. 

1. The text: "the inverse of the duplication time" was replaced by "equal to Ln2 times the inverse of the duplication time" [Lines 360-361].