Acetaldehyde Stimulation of the Growth of Zymomonas mobilis Subjected to Ethanol and Other Environmental Stresses: Effect of Other Metabolic Electron Acceptors and Evidence for a Mechanism
Round 1
Reviewer 1 Report
This manuscript reported that acetaldehyde stimulates shorting the lag phase of growth in Zymomonas mobilis exposed high ethanol concentration. Possible mechanism of the phenomena shown in this manuscript has still not been understood. However, effects of acetaldehyde on growth of ethanol-exposed Z. mobilis would be interesting.
For publication, revision of this manuscript is required.
Comments
- Metabolic pathways including acetaldehyde metabolism and other carbon metabolisms with redox equivalents should be shown as a figure. In addition, further discussion should be described based on the figure.
- It is better to discuss why acetaldehyde was effective for shorting the lag phase of growth rather than the increasing the growth rate.
- How or why do stresses disturb intracellular redox balance in Z. mobilis? This should be discussed more. If possible, data of restoration of redox balance disturbed by ethanol with acetaldehyde and other compounds should be shown based on the results shown in this manuscript.
- It is not clear why the effect of acetaldehyde on growth of Z. mobilis exposed to ethanol stress can contribute to fermentation of lignocellulosic biomass. Authors should show the effect of acetaldehyde on growth of Z. mobilis exposed to stresses due to compounds in lignocellulosic biomass hydrolysate and discuss more.
Author Response
Please see attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript presents an interesting study related to the former articles from this group related to methabolic Acetaldehyde stimulation of Zymomonas mobilis growth. I think it is good contribution that can have aplication not in sintetic media but in lignocellulosic hydrolysates.
My coments are:
Mat and Methods
2.1 Organism, media and inoculum preparation.
“Cells were grown under either aerobic or anaerobic conditions. Anaerobically grown cells were cultivated in 450 ml medium in modified 500 ml Erlenmeyer flasks as described before [8], while aerobically grown cells where cultivated in 250 ml medium in 1 L Erlenmeyer flasks stoppered with cotton wool and baffled with stainless steel coils (3.5 cm diameter, 30 turns per coil)...
- What about agitation, incubator, … steril conditions?
“Unless other wise specified the data shown are the means of triplicate fermentations.”
-This information is not related to inoculum preparation – reorganize the Mat and Methods sections and provide respective detailed information.
change g/l by G/L
2.2.”
Authors never explain how & when they introduced the additives, made the sampling…
Please complete with detailed information: explain which fermentation assays were made!
2.3 The length of the lag phase (lag time) was determined by an extrapolation method mentioned previously [16].
- Provide respective minimum information in order to readers comprehension
Results
“To determine the effect of acetaldehyde on glycolysis in the absence of growth, acetaldehyde was added to washed high cell density cultures of Z. mobilis in ethanol-containing buffer that did not permit growth (Figure 4)”
Authors must explain how thei wash cells in Mat and Methods.
Fig 3 and 4 must be presented in the same format as fig 5 since the former ones are not easy to read and understand!
Discussion
To better understand the discussion it would help to have a metabolism draw with the enzymes which activity was discussed.
Author Response
Please see attachment
Reviewer 3 Report
The authors described the effect of acetaldehyde addition to enhance Zymomonas mobilis tolerance to ethanol stress. The acetaldehyde reduce the lag phase of ethanol-stressed Z. mobilis culture. The acetaldehyde acts as a metabolic electron receptor and stimulates glycolysis in non-growing bacteria. Acetaldehyde addition enhances slightly stressed-bacteria growth.
- Line 42: the sentence “For this…….weir” misses a parenthesis
- Line 83: in the precedent paragraph, the anaerobic culture was performed in 500 ml Erlenmeyer with 450 ml of medium, why the authors didn’t use the same proportion to analyze glucose consumption in non-growing bacteria.
- Line 87: there is title layout problem
Author Response
Please see attachment
Round 2
Reviewer 1 Report
The manuscript has been properly revised according to the comments from reviewers.
