Lactic Acid Production Using Sugarcane Juice as an Alternative Substrate and Purification through Ion-Exchange Resins
, Luciana Porto de Souza Vandenberghe * and Carlos Ricardo SoccolRound 1
Reviewer 1 Report
This work is very good with interesting results, and open a new gates for other researchers in the field.
I have few comments to be addressed:
L63 correct (thr)
L196: replace secreted by produced.
L217-218: the authors should calculate the actual concentration of staring LA and recalculate the real recovery percentage (63%) not based on the 96h production titer.
Author Response
Reviewer #1:
Comments and Suggestions for Authors
- This work is very good with interesting results, and open a new gate for other researchers in the field. I have few comments to be addressed:
- L63 correct (thr)
Response: Thank you. The typo was corrected.
- L196: replace secreted by produced.
Response: Thank you for this suggestion, the word was replaced.
- L217-218: the authors should calculate the actual concentration of starting LA and recalculate the real recovery percentage (63%) not based on the 96 h production titer.
Response: Thank you for this observation. It was assumed that what is being suggested is to consider the initial LA concentration and the “converted” LA concentration. Usually, the LA concentration in the fermented broth is very low, where the predominant form is the precursor calcium lactate (it is mentioned in L177-178, “from 120 hours onwards, a solid calcium lactate was observed, compelling the recovery and analysis steps more difficult”). If this assumption corresponds to the suggestion, the difference between “initial-free LA” and “converted LA” values, considering the theorical yield, are available in Table 2. Also, a description was added in L175-176. In L227-228, the best condition of LA production, LA productivity during fermentation kinetics, and the recovery percentage were highlighted. This fragment was rewritten.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The article " Lactic acid production using sugarcane juice as alternative substrate and purification through ion exchange resins" has been well described from the authors. Although the topic does not represent a novelty in this biotechnology field, the down streaming process is still an open discussion area in terms of efficiency and costs. Therefore I will recommend the publication of the above mentioned article after a small point/clarification
In the introduction the authors described the actual methods for LA purification: what is missing in the publication a comparison in terms of cost/energy and time between the different approaches ( it also can be referred to previous publication etc).
Moreover, it will interesting to verify the efficiency of the resins after each use in order ( if will be always equal to the first round or not)
Author Response
Reviewer #2:
Comments and Suggestions for Authors
- The article " Lactic acid production using sugarcane juice as alternative substrate and purification through ion exchange resins" has been well described from the authors. Although the topic does not represent a novelty in this biotechnology field, the down streaming process is still an open discussion area in terms of efficiency and costs. Therefore, I will recommend the publication of the above-mentioned article after a small point/clarification:
- In the introduction the authors described the actual methods for LA purification: what is missing in the publication a comparison in terms of cost/energy and time between the different approaches (it also can be referred to previous publication etc).
Response: Thank you for the suggestion, a brief economic analysis was added in Table 6. A more detailed energetic and economic analysis in the present conditions is in progress.
- Moreover, it will be interesting to verify the efficiency of the resins after each use in order (if will be always equal to the first round or not).
Response: Thank you for the observation, it is very insightful. Further assays with these resins in the present conditions are being studied.
Author Response File: Author Response.docx
Reviewer 3 Report
The present work describes a method for producing LA by fermentation. It also compares the use of resins to the conventional precipitation method used to recover LA from the fermentation broth.
The abstract is clearly written.
The introduction argues the topic well and allows even non-experts in the field to understand.
Material and methods
Not all sections are clear, as discussed below. Some improvements could make reading easier.
Section 2.3.1
Was the medium just 20g/l sugarcane juice and 25g/l yeast extract? Was 20g/L sugar cane juice or reducing sugars contained in it?
On line 94 the authors mentioned "inoculation rate": what did they mean?
Figure 1: wanting to be picky, “Fermented broth” is not a step but the substrate of the process. The author could modify in “Fermentation” or delete the surrounding square
Section 2.4: could the section be renamed “conventional LA recovery”?
Line 111: did the author intended “clarification” by “precipitation”?
Section 2.5.3. Could the authors provide a better description of the reactor (volume, impeller,…)?
Table 1 should be moved to the introduction or, better, to the discussion section because it is a description of the resins and not a material or method
Statistic: did the authors used any software?
Results and discussion
Lines 171- 172: was cell growth evaluated?
Table 2: Can the authors explain why total sugar decreases during the first 48 hours while increasing afterward?
Line 181: after how many times has Karp reached the presented result?
Conclusion
Lines 322-323 In the present work, different pH conditions were not studied, but only one. Therefore, it is not possible to state, with this study alone, that "the optimal production conditions are linked to a pH close to neutrality"
With respect to the literature mentioned, what is the novelty of the present work?
Do the authors think that the recovery method could be applied at industrial scale?
It is possible a direct comparison between all the recovery methods described, considering also an economical analysis?
Author Response
Reviewer #3:
Comments and Suggestions for Authors
- The present work describes a method for producing LA by fermentation. It also compares the use of resins to the conventional precipitation method used to recover LA from the fermentation broth.
- The abstract is clearly written.
- The introduction argues the topic well and allows even non-experts in the field to understand.
Material and methods
- Not all sections are clear, as discussed below. Some improvements could make reading easier.
Response: Thank you. Some points were clearly described as follows.
Section 2.3.1
- Was the medium just 20g/l sugarcane juice and 25g/l yeast extract? Was 20g/L sugar cane juice or reducing sugars contained in it?
Response: Thank you for the question, this medium was optimized in our previous research, and it was composed of diluted sugarcane juice in a concentration of 20 g/L of total reducing sugars and addition of 25 g/L of yeast extract, which was favorable for inoculum preparation. The word “diluted” was added in L94, making it more understandable.
- On line 94 the authors mentioned "inoculation rate": what did they mean?
Response: Thank you for this question. It was meant to the proportion of inoculum fixed at 10% v/v (from our previous research - Oliveira et al. [19]). This detail was added in L96.
- Figure 1: wanting to be picky, “Fermented broth” is not a step but the substrate of the process. The author could modify in “Fermentation” or delete the surrounding square
Response: Thank you for this observation. Indeed, it is an inlet, and Figure 1 was modified.
- Section 2.4: could the section be renamed “conventional LA recovery”?
Response: Definitely, the section was renamed. Thank you for the suggestion, it was very helpful.
- Line 111: did the author intended “clarification” by “precipitation”?
Response: Another helpful observation, thank you. The intention was to describe the clarification and precipitation processes. Both were previously optimized by our research group. This was added in L113-114.
- Section 2.5.3. Could the authors provide a better description of the reactor (volume, impeller,…)?
Response: Yes, thank you for the suggestion. The stirred tank (Tecnal TE-054MAG, with Tecnal TE-139 mechanical agitator) has a digital controlling system for temperature and mechanical agitation, 2L of total volume capacity, and propeller-type impeller for axial flow. These missing details were added to Section 2.5.3, L145-147.
- Table 1 should be moved to the introduction or, better, to the discussion section because it is a description of the resins and not a material or method
Response: Thank you for the suggestion, Table 1 was moved to Introduction, where it was suitable for the description mentioned before in the text (L66).
- Statistic: did the authors used any software?
Response: Thank you for the question. Yes, results were analyzed using the Software Statistica 5.0 (StatSoft, Tulsa, USA). This information was added to Section 2.7, L169-170.
Results and discussion
- Lines 171- 172: was cell growth evaluated?
Response: Thank you for the question. Cell growth was monitored but not quantified due to CaCO3 interference that makes conventional cell growth evaluation difficult. This information was added in L174.
- Table 2: Can the authors explain why total sugar decreases during the first 48 hours while increasing afterward?
Response: Thank you for the question. It is possible due to metabolic processes involved in the fermentation pathway. Usually, the initial consumption of the available sugars is linked to the energy necessary for the cell maintenance, this leads to a decrease in the total sugar concentration in the medium during the first 48 hours (sugar metabolism and pyruvate production). LAB convert pyruvate into LA generating energy for the cells, while LA accumulates in the medium, contributing to the decrease in pH. It is known that some LABs, especially Lactobacillus, are sensitive to low pH and may slow down their growth, the LA production and the sugar consumption. After 48 hours, LAB may adapt to the medium conditions via enzymatic regulation mechanisms, which allows them to retake the metabolism leading with a rapid acidification. A clear formation of a solid block of calcium lactate before 120 hours (data not shown), when considerable acidification of the medium occurred, causing analytical difficulties. Therefore, kinetics ended at this time. This explanation was added to discussion in L206-211 and L216-2019. That is why some unexpected variations of sugar concentration occurred.
- Line 181: after how many times has Karp reached the presented result?
Response: Karp et al. have studied lactic acid production in 2011. Yes, it is an old reference. However, this paper was the starting point of our research group in this subject. In this case, authors employed soybean molasses as substrate for lactic acid production by L. agilis.
Conclusion
- Lines 322-323 In the present work, different pH conditions were not studied, but only one. Therefore, it is not possible to state, with this study alone, that "the optimal production conditions are linked to a pH close to neutrality"
Response: Thank you. The “Conclusions” section was rewritten.
- With respect to the literature mentioned, what is the novelty of the present work?
Response: Thank you for this question. Positive perspectives are related to using a substrate that did not undergo pretreatment; it was possible to use it directly as substrate under submerged condition with simple adjustments. Anionic and cationic exchange resins were used simultaneously in contact with the sample in a stirred tank, for LA purification during one single step. This method is simple and stable regarding flow rates, time and temperature, and it does not need successive washings with chemical solvents reducing effluents’ generation. Another important point is that it allows the use of higher sample volumes in less time, and the resins’ recovery is facilitated via density difference using water as solvent. Scale-up process can be considered. This statement was added to conclusions (L339-356).
- Do the authors think that the recovery method could be applied at industrial scale?
Response: Thank you for this question. This study provides insights regarding process scalability. In fact, great perspectives were shown at bench scale. The usage of combined ionic exchange resins in contact with larger volumes of samples in a stirred tank could be an option for industrial processes, due to its feasibility considering operational volumes and controlling conditions. This method can be used for transitioning scales, to evaluate the resins’ performance. Future studies concerning the adsorption capacity of these resins, simultaneously, are envisaged. This statement was added to conclusions (L339-356).
- It is possible a direct comparison between all the recovery methods described, considering also an economical analysis?
Response: Thank you for the question, a brief economic analysis was added in Table 6. Detailed energetic and economic analysis in the present conditions is in progress.
Author Response File: Author Response.docx
