Advanced Monitoring and Control of Redox Potential in Wine Fermentation across Scales

Round 1

Reviewer 1 Report

While this work could bring interesting results on the application of air pulses and the monitoring thanks to the POR, it is based on insufficient scientific data and methodological procedures. The reviewer recommends that this article be completely revised so that it meets the standards of a scientific publication. Only then can an in-depth analysis of the results of this work be carried out.

L37-41 and L214-216: The reviewer disagrees with the assertion that the POR is independent of DO. Oxygen is a component of the ORP measurement.

L44-48: What pH change is expected ?

Table 1: Can the authors explain why their value of vvm (vol air per vol medium per minute), a widely used parameter of bioreactor aeration, changes: 0.18 for 100L, 0.024 for 1500L, 0.045 for 10000L ?

Chap 2.1: How POR electrodes are prepared before use (procedure ?) ?

Table 3.: How parameters are calculated, show details. For Viability constant, replace « l/g/h » by «l /g.h ». 

L208-209: What is the relationship between 54 µM and 0.86 mg/L oxygen.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In the reviewed original research article “Advanced Monitoring and Control of Redox Potential in Wine Fermentation Across Scales”, Nelson et al. apply redox alteration during wine fermentation at different scales (100 L, 1500 L and 10000 L). The redox potential of the fermentation broth (ORP) is kept above -40 mV by pulsing with pressurized air by a self-developed control algorithm. Similar approaches were pursued in previous publications. The novelty in this study concerns the application to industrial (10 m³) scale and the set-point of the ORP of -40 mV, which is lower than in previous works. Most data were gained from the 100 L scale, at which five fermentations (3 x uncontrolled ORP and 2 x controlled ORP) were conducted. Only single fermentations were performed at the 1500 L and 10000 L scales.

The article begins with a broad introduction to the topic, highlighting the role and importance of ORP in bioprocesses. Here, the focus is laid on wine fermentation. Furthermore, the necessity for large-scale experimental studies is pointed out – arguing that the dynamics of ORP response to artificial control systems of such can hardly be predicted from theory. After the introduction, the article continues with a brief Materials and Method section, focusing on the experimental set-up, particularly the ORP sensing and control system. Subsequently, the results are presented, separate from the discussion. In the results part, first, the ORP control applied to the different scales is presented. Afterwards, the effect of the altered ORP on the fermentation pattern across the different scales is outlined. The results section's final part quantifies the ORP control's effects at the 100 L scale by a fermentation parameter estimation. The wine fermentation model and the procedure for parameter estimation were also developed in previous work of the group. After the results section, the experimental data are discussed. Particular emphasis is placed on the ORP values and the underlying chemistry. Then, the prospective advantages of the artificial ORP increase are elaborated, highlighting the importance of the experimental results obtained from the large-scale fermentations. The article closes with a short conclusion, stating that the results indicate a positive effect of the ORP control on the overall fermentation performance. Moreover, the last sentence states that this is the first time ORP control has ever been applied to commercial-scale wine production.

As a general remark, the article is professionally written, and the overall appearance is quite satisfactory. Compared to other studies, the main novelty in this work concerns cultivation at the commercial scale, which is also clearly highlighted by the authors. Furthermore, the manuscript offers a clear structure making it easy to follow the topic and understand the work. Nonetheless, some questions and unclarities arose while reviewing the manuscript, which should be addressed and/or answered by the authors before the article might be accepted (major revise). I hope my comments below are helpful and will contribute to improving the quality of the manuscript.

General comments:

1. It is great to see that, already at such an early development stage, the process control is applied to a commercial scale. This might help address issues related to scale up early during process/technology development, which is often not possible in academic research. Nonetheless, readers of the manuscript might wonder why significant parts of the results and the discussion are focus on the 100 L scale. Were there no data available from control fermentations (conventional commercial scale wine fermentation with uncontrolled ORP) for comparison? In this regards, it is indeed difficult to evaluate the results of the fermentations at the 1500 L and 10000 L scale and the advantage of ORP control at these scales. Please comment if these data were unavailable and why they were not included in the manuscript. 

2. Significant parts of the introduction deal with the prospective influence of ORP on the formation of H2S. In this work, H2S was not quantified during fermentation and the issue was also not further discussed in the results or discussion part. Please explain why it was not quantified but addressed with an extra paragraph in the introduction section (lines 79-87).

3. The Material and Methods section lacks a description of the applied model and parameter estimation procedure for the wine fermentations (results presented in table 4). If it was subject to previous work, please summarize briefly and name the reference. 

4. In general, difficulties with the applied ORP control, especially regarding the limited redox buffer capacity of the wine fermentation broth, are thoroughly elucidated in the manuscript. Unfortunately, the work does not address the issue of increasing heterogeneity with increasing reactor size and its effect on the measured ORP signal and also the control itself. Please add a short paragraph to the discussion addressing this issue. 

Specific comments:

1. Line 39: “…and it is independent of the concentration of oxygen in fermentation media“. This is not 100% correct. Oxygen is also an important participator in several redox reactions and exerts a strong influence on the overall ORP value (some papers have elucidated this in detail, check, for example, doi: 10.1016/0740-0020(84)90062-5). Most aerobic bioprocesses, usually conducted at DO levels above 20%, have measured ORP values in the range between +200-400 mV. Here, oxygen is (indirectly) the main contributor to the overall ORP value (because of its high reduction standard potential), but the exact value depends on other chemical species present in the fermentation broth. Therefore, please correct the statement "...and it is independent of the concentration of oxygen in the fermentation medium". If this does exclusively apply to wine fermentations, please distinguish in this case and explain in more detail.

2. Line 41 (regarding reference [13]): In the cited work from Dahod, the main argumentation is that ORP values for bioprocess control might be more applicable than DO-based process control since the signal of DO probes is often susceptible to failure or erratic signals at industrial scale. The paper is from 1982, and the especially the use of modern optical pO2-sensors makes this less of a problem. Please comment and rephrase.

3. Line 44: „….in redox potential at 25 °C“ Replace with "at standard conditions" (which do cover not only temperature but also pressure).

4. Lines 96-120: this paragraph describes the two main reaction steps for ORP control/alteration by adding air bubbles. Here, it is outlined that the rate-limiting step differs between scales and that the responsiveness of ORP control by sparging cannot be predicted from theory alone. This might be valid, but is it possible to say something about the approximate scale at which the reaction regime changes from gas/liquid transfer to mixing time? Are there any characterizations of conventional wine fermenters available from the literature or previous works? This might help to give more recommendations on using the developed ORP control for different scales. Please comment. 

5. Table 1: change “KPa” to “kPa”

6. Figures 1, 2 and 3: The three figures provide a sound overview of the experimental set-up, but please elaborate in more detail on the role of the additional installations in the 100 L scale (Cap temperature probe, Inner screen, Pumpover line, Brix Meter, Outer Screen, Probe Screen).

7. Lines 130-133 (also in relation to general comment no. 4): „Oxidation-reduction potential (ORP) probes (243050, Hamilton Company; Reno, NV, 130 USA) were placed in the fermentors to monitor the redox potential in real-time and export 131 the values into a database.” Was only one probe installed even at the 1.5 and 10 m³ scales? If so, where were they positioned and did the distance to the sparger position differ between the different scales? Together with the response time of the used ORP probes, might this have influenced the used control algorithm? Please comment.

8. Lines 137-138: “(previous unpublished work determined the solenoid opening time to achieve a desired redox response).” Please elaborate more on the controller. What kind of controller was used (PID? Neural network)? How was the controller implemented (via Labview, self-programmed, others)? Why was the opening time set as the controller input and not the air supply pressure?

9. Lines 165-166: „A redox potential setpoint of -40 mV is also expected to limit the chemical reduction of elemental sulfur to H2S at wine pH.” Why was this expected? Please comment.

10. Figure 5 c: As indicated by the redox signal, the lag phase at the 1.5 m³ appears to be approximately one day longer than in the others cultivations. What was the reason for this? Please comment.

11. Lines 196-198: “In the replicated 100 L fermentations, the specific maintenance rate was slightly higher in the controlled redox potential case, Table 4. The viability constant was also slightly lower.” This is no scientific language, please rephrase.

12. Lines 198-199: “This maintenance rate effect is similar to that found in an earlier study, where the fermentations were controlled at -20 mV [21]” In the cited study, the ORP was controlled >200 mV and not at -20 mV. Please comment and correct if necessary.

13. Table 4: Was the parameter estimation conducted with one dataset including the replicates or separate for all of them? If it was conducted separately for each fermentation run, please give the standard deviation (at least for the 3 x 100 L uncontrolled case).

14. Lines 214-216: “The redox potential is not affected by the dissolved oxygen concentration since oxygen is unreactive by itself and only electron transfer reactions that have favorable kinetics are involved in the establishment and changes of the redox potential of the solution.” What about redox-active species, which are produced (and might be excreted) by the yeasts during fermentation? Does their effect/contribution on/to the overall ORP increases over time or is there any quantitative evidence (maybe also from earlier studies) that those contributions to the overall ORP can be neglected? Please comment.

15. Line 217: „The redox buffer capacity of all other fermentation media reported…” Remove "all" - these are only a few examples from the literature and there might be other cases in which the buffer capacity is comparable to those in wine fermentation.

16. Lines 274-276: “One option in the future might be to control the redox potential during only part of the fermentation, for example only until yeast growth is completed which is generally indicated by the time of the peak fermentation rate.” Why would this be beneficial? What is the proposed influence of the increased ORP on yeast metabolism and process performance? Please comment.

17. Lines 286-288 (also in relation to specific comment no. 14): “This is interpreted as being due to the formation and release of reduced glutathione during yeast growth and a slowing effect of the peak decline due to the concentration of ethanol.” Only Glutathiones? What about other redox-active species excreted by the yeasts? Can they be neglected? Please comment.

18. Lines 280-300: What about more advanced control strategies, such as neural networks? Might they be of future use in this case? Please comment.

19. Lines 309-310: “Previous work has shown the response to the addition of air to differ between grape juices.” Please add the reference to the previous work.

20. Line 323: Remove the “.”. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The paper has been improved. Accept in present form.

Reviewer 2 Report

The authors have addressed all points raised and revised (improved) the manuscript accordingly. Furthermore, the point-by-point answers of the authors are much appreciated. The manuscript might now be accepted in its present form.