Lignocellulose Degrading Weizmannia coagulans Capable of Enantiomeric L-Lactic Acid Production via Consolidated Bioprocessing

Round 1

Reviewer 1 Report

The manuscript is well-written and nicely executed, results are well-explained. The manuscript discusses possibly synthesizing lactic acid in a consolidated mode using Weissmania coagulans. However, the following suggestions would be helpful to improve further and revise the manuscript.

  1. Line 93-94: Please mention the temperature range for growing Bacillus coagulans for lactic acid production.

2.      Please specify the purpose of using Locust bean gum in this study.

3.      Section 2.5.2. Please elaborate on how and which LCB substrates were used as unpretreated biomass. What procedures/protocols were used to sterilize the biomass before adding it into the medium?

4.      Line 262: Please edit the line to clearly include how many microbes were identified initially, how many produced yellow zones, etc.

5.      Can the authors justify the reasoning behind why all the microbes grown on Thermoaciduran agar and XYP agar were W. coagulans? Please add references to previous studies or provide a good discussion.

6.      It is interesting to see LA production under static conditions. Did the authors consider the effect of agitation on LA synthesis?

7.      Please consider including a table comparing lactic acid yields from MA42, P13, and S5 compared to previous literature numbers, considering including numbers from other thermophilic microbes (if used any for LA production), and comparing yield numbers in this study to the LA yields, etc. reported in earlier studies also using W. coagulans.

8.      Line 285: Please include protocols to grow the bacteria anaerobically in the material and methods.

9.      Please include graphs highlighting the growth profiles of MA42, P13, and S5, aerobically and anaerobically, in different media substrates listed in Table 2. This will help visualize the data and interpret growth comparisons through quantitative numbers rather than qualitative ones.  

10.  Line 289: Correct Cellulse to Cellulose.

11.  Line 292: Please include the materials and methods of the procedure used to perform uncontrolled and controlled pH conditions (pH 6-7) during the progress of the experiments.

12.  Line 293: Please add units to the LA numbers.

13.  Line 294: Please specify how other acids were identified.

14.  Table 3 and Figure 2: Please consider representing enzyme activities in U/ml (the standard for expressing enzyme activity) rather than mU/ml. If the enzyme units are expressed in U/ml, the observed enzyme activities of the three strains are very low (less than 1 U/ml). With such low enzyme activity, please justify the growth of the isolates on untreated LCB biomasses. Please include relevant discussion in the manuscript.

15.  Please consider writing reasoning (advantages or disadvantages) associated with synthesizing lactic acid using Thermophiles compared to mesophiles. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Manuscript entitled “Lignocellulose Degrading Weizmannia coagulans Capable of 2 Enantiomeric L-Lactic Acid Production via Consolidated Fermentation” by authors Punnita Pamueangmun, Aliyu Dantani Abdullah, Md. Humayun Kabir, Kridsada Unban, Apinun Kanpiengjai, Joachim Venus, Kalidas Shetty, Chalermpong Saenjum and Chartchai Khanongnuch is a very interesting and comprehensive work that includes the isolation of new strains of bacteria that can degrade lignocellulosic raw material, use almost all the carbohydrates produced by the hydrolysis, and produce homofementatively L-lactic acid with high enantiomeric purity in a, so-called, consolidated bioprocess.

This scientific article is well writen but contains issues that need clarification.

Comments:

Only in title you are using expression consolidated fermentation, in text is used consolidated bioprocessing (CPB)

L24 instead of substrate, pls use raw material…

L34-35 please rewrite, not clear enough (“properties of lignocellulolytic enzyme properties…”)

L101 interesting comment, but it is based on starch hydrolysis, example with lignocellulose degradation and fermentation of lignocellulosic hydrolysates would fit better.

L120 please state the purity of chemicals used

L126 please add compositions of nutrient agar used, or name the manufacturer

L131 add comma after xylose

Section 2.3. please try to rewrite entire section to be more clear to the reader, considering all media (liquid or agar) and conditions used for experiments…

L155 -156 in the sentence “In these experiments, cultivation of yeast was performed in liquid media based on XYP broth” please delete yeast and write bacteria.

Additionally, you are saying that cultivations were performed on media based on XYP broth. Please be more accurate (e.g. xylose from XYP broth was substituted with… and all other components of the media were as written in section 2.1?)

L165-L171 this section should come before section at L159-165

L169 u can’t state that pH was constantly adjusted every 6 h of fermentation… (pH can be constantly adjusted if automatic adjustment of pH is used, as in bioreactors) please rewrite…

L177-L178 please rewrite, not clear, “the supernatant was determined” --> was used for determination of…

L179 the substrates are mentioned in section 2.1., please describe method or give reference for enzyme activity determination

L189 5 g of ground agricultural residue can not be only carbon source in XYP medium if XYP is defined as xylose, yeast extract, peptone medium. It is not clear if the medium consists of ground agricultural residue + peptone + YE or ground agricultural residue + xylose + YE + peptone…  please check and rewrite

L198-199 please rewrite sentence, not clear enough

L210 unpretreated is not needed in the title, please remove

L213 how did u test pH before and after addition of NaOH solution, 6h in lactic acid production phase is a relatively long time without pH adjustment

L231 same comment for XYP broth as in line L189

L240 u are referring to a crude enzyme (supernatant from fermentations)? Please rewrite

L250 same comment as for L240

L268 add medium (YPX broth) used due to clarity.

L284 add ”in medium with D-glucose…”

L292 pH contion --> pH condition

L294 how did u determine concentration of acetic and formic acid and ethanol? The method used isn’t described in M&M section.

L405 “non-pretreatment” not needed in title

Fig 3 comment, did u consider that lactic acid is produced from carbohydrate available in the broth and not from lignocellulosic raw material given the very little amount produced

L423-424 not clear enough, revise ,”cellooctaose, the most complex cellulosic substrate to lactic acid”

L444 There is no table 4, u are probably referring to table 5, please check and revise

Comment on Table 4(5)

Did u determine the total carbohydrate content of alkali or acid pretreated lignocellulosic raw materials? Pls show data. (It is well known that during alkali and acid pretreatment certain amount of carbohydrate is hydrolyzed and is part of the liquid phase. Therefore, total carbohydrate content of pretreated lignocellulose isn’t the same as total carbohydrate content of non-pretreated lignocellulose.)

In section 2.6.2 u only state that fermentation is conducted the same as in 2.6.2.

Pls check your reference list because references in text and the one in reference list do not match.

e.g.

Gandini et al., (2017) isn’t given in Reference list

Tomas-Pejo et al., 2011 isn’t given in Reference list

Baadhe, R.R.; Potumarthi, R.; Mekala, N.K. Influence of dilute acid and alkali pretreatment on reducing sugar production from 620 corncobs by crude enzymatic method: A comparative study. Bioresour. Technol. 2014, 162, 213-217, 621 doi:10.1016/j.biortech.2014.03.117. isn’t given in text

and many others

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for taking into account the concerns and editing the manuscript accordingly. One thing that can further improve the manuscript includes adding some text relating this work to the previous work published in the literature. Please consider strengthening the discussions further to bring out the novelty of this work over previously explored and published texts.

Author Response

Thank you for the valuable suggestion. We have added the discussion in comparison to the previous reports which seem to be similar to our work as the yellow highlighted. The further experiment on the mutagenesis of W. coagulans to enhance L-lactic acid from the selected lignocelluloses has been also suggested (line 514-521 and 537-541).

Reviewer 2 Report

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Author Response

Your kind suggestions and acceptance of our revised manuscript are really appreciated.