Effect of yeast addition on the biogas production performance of a food waste anaerobic digestion system

Food waste contains numerous easily degradable components, and anaerobic digestion is prone to acidification and instability. This work aimed to investigate the effect of adding yeast on biogas production performance, when substrate is added after biogas production is reduced. The results showed that the daily biogas production increased 520 and 550 ml by adding 2.0% (volatile solids; VS) of activated yeast on the 12th and 37th day of anaerobic digestion, respectively, and the gas production was relatively stable. In the control group without yeast, the increase of gas production was significantly reduced. After the second addition of substrate and yeast, biogas production only increased 60 ml compared with that before the addition. After fermentation, the biogas production of yeast group also increased by 33.2% compared with the control group. Results of the analysis of indicators, such as volatile organic acids, alkalinity and propionic acid, showed that the stability of the anaerobic digestion system of the yeast group was higher. Thus, the yeast group is highly likely to recover normal gas production when the biogas production is reduced, and substrate is added. The results provide a reference for experiments on the industrialization of continuous anaerobic digestion to take tolerable measures when the organic load of the feed fluctuates dramatically.

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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) In this paper, effects of yeast addition on the biogas production performance of a food waste (FW) anaerobic digestion system were comprehensively investigated. Generally, the topic and the data analysis are significant, the results obtained would make a sound contribution to the fields of sludge treatment and anaerobic digestion. However, in the reviewer's opinion, there are some major and minor shortcomings needed further attention via a major revision, with the detail as follows: 1. What is the role of yeast in the anaerobic digestion? I find there are a contradictory statement about it in the introduction. Firstly, the author stated " the AD process with FW often results in the accumulation of volatile organic acids (VFAs) in the reaction system and the acidification reduces methane yield and destroys the stability of the digestive system" in Page 3, Line 17-20. Then in Page 4, Line 10-12, the authors stated " The addition of yeasts during anaerobic digestion can increase the acid production rate of anaerobic digestion and shorten the time of anaerobic digestion". Both statements obviously are contradictory. What exactly is the mechanism for enhanced digestion of FW led by yeast? It should be confirmed. 2. In this study, the yeast was added in twice. What is the reason? and how to chose the added time? Meanwhile, the characteristic of the yeast Saccharomyces cerevisiae should be provided. 3. Except for the yeast, some other pretreatment technologies are benefit to the anaerobic digestion of FW and they should be summarized and compared in the introduction. Some recent references maybe helpful, such as Reviews in Environmental Science and Bio-technology, 2019, 18(4):771-793(https://doi.org/10.1007/s11157-019-09515-y); Environmental Science and Pollution Research, 2019, 26(14): 13984-13998 (https://doi.org/10.1007/s11356-019-04798-8) 4. In this study, the substrate also was added secondly. Why? There are only one equation, so using 3.1 to label is improper. 5. Numerous grammar errors distributed across the paper, resulting in poor readability. I think the article in its current state is unable to be accepted unless the article will be re-written by some of the co-authors who are skillful in English.

Decision letter (RSOS-200443.R1)
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. 2. Elaborate with more published articles on the mechanisms of yeast action on substrates and other operational factors during biogas production.

Materials and methods
1. Use more technical term not "domestication". 2. For full disclosure, the source of the yeast and glucose should be stated (whether purchased or manufactured). 3. Is the AD reaction batch or continuous? Kindly state and was the AD reaction replicated?
If yes, how many times? And then the digestion period is not stated. 4. Edit the statement in line 9,10 and 11, page 6.
5. Indicate how did the authors determine yeast and glucose dosages and addition intervals, any previous studies or experiments to support your decision. 6. What is the implication of the second yeast and glucose addition, is it economically viable if implemented on commercial scale?

Results and discussion
1. "… fermentation were determined ( Figure 2)" in line 20, page 6, how was it "determined" or do you mean "recorded"? 2. Provide citation for "Under standard conditions, 1 g of protein (calculated as VS) can produce 992 mL of biogas in anaerobic fermentation" in line 10 and 11, page 7.
3. In section 3.1, there should be proper discussion of facts with adequate citation. Results should be stated earlier before discussions with literature information to avoid missing out salient discoveries in the study. 4. This study is not a review article; therefore, emphasis must be on the results of this study, then supporting literatures and such statements to justify the results.

Conclusion
1. Revise this conclusion section to reflect the essence of this study and the findings. 2. "Especially, activated yeast was supplemented twice when AD process was inhibited with few daily biogas generation (nearly zero)" this statement in line 20 and 21, page 11 could be misleading, while it may be true for your second addition, Fig. 2a denied the near zero yield statement for the first addition. Please revise.
3. Edit the language in line 21, page 11 and line 1, page 12.

Dear editors:
Thanks for your careful review with regard to our manuscript "Effect of yeast addition on the biogas production performance of a food waste anaerobic digestion system" (Manuscript ID: RSOS-200443).
These comments are very helpful for us to revise and improve our manuscript. We have made changes in the text based on the comments of two reviewers and also re-examined the manuscript. Revised parts are highlighted in Yellow in the paper. Point-to-point responses to the comments are listed as flowing (Responses to reviewers' questions in Blue words; Revisions in Red words).
We appreciate for your consideration of our work, and hope that the responses and the revision could be acceptable.

Response to Reviewer 1
In this paper, effects of yeast addition on the biogas production performance of a food waste (FW) anaerobic digestion system were comprehensively investigated. Generally, the topic and the data analysis are significant, the results obtained would make a sound contribution to the fields of sludge treatment and anaerobic digestion. However, in the reviewer's opinion, there are some major and minor shortcomings needed further attention via a major revision, with the detail as follows:

Q1:
What is the role of yeast in the anaerobic digestion? I find there are a contradictory statement about it in the introduction. Firstly, the author stated " the AD process with FW often results in the accumulation of volatile organic acids (VFAs) in the reaction system and the acidification reduces methane yield and destroys the stability of the digestive system" in Page 3, Line 17-20. Then in Page 4, Line 10-12, the authors stated " The addition of yeasts during anaerobic digestion can increase the acid production rate of anaerobic digestion and shorten the time of anaerobic digestion". Both statements obviously are contradictory. What exactly is the mechanism for enhanced digestion of FW led by yeast? It should be confirmed.

Replies:
Thank you for your careful review and valuable comments of this paper.

Appendix B
The role of yeast in anaerobic digestion is that yeast can accelerate reaction rates in the hydrolysis stage of organic matter and promote the hydrolysis of food waste. And the addition of yeast promotes the hydrolysis of substrates and converts the degradable organic matter of substrates into neutral ethanol instead of acid propionic acid or butyric acid, which reduces the load of VFA in the system and provide more potentially available energy to methanogens, thereby improving the system's stability. should be provided.

Replies:
The purpose of adding yeast for the first time is to investigate the promotion effect and mechanism of yeast on anaerobic digestion of food waste. With the increase of anaerobic fermentation time, the daily biogas production of the two groups decreased significantly. Because the accumulation of volatile fatty acids in the system inhibits the activity of methanogens and further weakens their ability to metabolize VFAs, thus reducing the methane yield. In order to investigate the effect of yeast on reducing the load of VFAs, improving the system's stability and methane production, yeast was added on the 12th day of fermentation when the daily biogas production was significantly reduced.
The purpose of the second addition was to investigate whether the decrease of gas production to 0 was due to the depletion of substrate or the deactivation of methanogens.
On the 37th day of fermentation, the daily biogas production of both experimental groups decreased to 0, so yeast and glucose were added for the second time on the 37th day of fermentation.
The yeast is Angel active yeast (produced by Angel Yeast Co., Ltd., Hubei, China), which is used as an additive to the anaerobic digestion system. Yeast contains 38-60% protein, 25% -35% carbohydrate, 4-7% fat and 6-15% nucleic acid. The reason for adding yeast twice, adding time and the characteristic of the yeast are supplemented in the revised manuscript.

Revisions in revised manuscript (Page 6, Lines 2-5):
In the yeast group, 2.0% (calculated as VS) of activated Saccharomyces cerevisiae was added when fermentation gas production had drastically reduced (day 12 of fermentation) to investigate the effect of yeast on reducing the load of VFAs, improving the system' s stability and methane production.
(Page 6, Lines 8-10): In order to investigate whether the reduction of gas production to zero in the later stage of anaerobic fermentation is due to the depletion of substrate or the deactivation of methanogens, yeast and glucose were added for the second time. Replies: Thank you for your valuable advice, which is very helpful to our writing.
We added the summary and comparison of pretreatment technology in the introduction of the revised manuscript (Page 3, Lines 21-23; Page 4, Lines 1-6).

Revisions in revised manuscript (Page 3, Lines 21-23; Page 4, Lines 1-6):
Previous studies have identified several methods to enhance methane fermentation efficiency using FW substrate pretreatments, including mechanical, thermal, chemical, and biological pretreatments [12,13]. Mechanical pretreatment enhances anaerobic process by increasing specific surface area. Li et al. studied the effect of heat pretreatment on the degradation of organics in FW, found that heat pretreatment increased the stagnation period of protein degradation (35-65%), and the cumulative gas production also increased linearly [14]. However, these methods require costly Replies: In this study, the substrate was added for the second time, because the daily biogas production in the later stage of fermentation has decreased to 0, which may be caused by the depletion of substrate in the anaerobic digestion system. And this paper wants to study whether adding yeast can restore biogas production when adding substrate after biogas production is reduced. The equation is corrected on Page 8, Line 7、Line 12.
Original text (Page 8, Line 7、Line 12): The calculation formula for protein biogas production is shown in equation (3.1).

Revisions in revised manuscript:
The calculation formula for protein biogas production is shown in equation (1).

Revisions in revised manuscript:
In this work, activated yeast was added to the fermentation broth of anaerobic fermentation of FW.

Q3:
Elaborate with more published articles on the mechanisms of yeast action on substrates and other operational factors during biogas production.
Replies: Thank you for your valuable advice, which is very helpful to our writing.
In the introduction of the revised manuscript, we quoted more published articles, and elaborated the mechanism of yeast action on substrates and other operational factors during biogas production. (Page 3, Lines 21-23; Page 4, Lines 1-6). Q5: For full disclosure, the source of the yeast and glucose should be stated (whether purchased or manufactured).

Replies:
Thanks for your comment. The source of the yeast and glucose and the characteristic of the yeast are supplemented in the revised manuscript.

Q6:
Is the AD reaction batch or continuous? Kindly state and was the AD reaction replicated? If yes, how many times? And then the digestion period is not stated. Q8: Indicate how did the authors determine yeast and glucose dosages and addition intervals, any previous studies or experiments to support your decision.
Replies: Thanks for your comment. We have researched the eff ect of ethanol prefermentation on organic load rate and stability of anaerobic digestion systems and published these results in other papers [1][2][3]. In these studies, we determined the dosage of yeast and glucose and the activation method of yeast. The yeast was first inoculated into 100 mL sterilized growth medium (2% glucose) with a mass ratio of 2% (W/V medium) in a 250 mL flask for activity recovery. This pre-cultivation was performed anaerobically at 35 ℃ for 2 h with mixing at 150 rpm using a rotary shaker.
The addition time of yeast and glucose is selected according to the situation of anaerobic fermentation system. In order to investigate the effect of yeast on reducing the load of VFAs, improving the system' s stability and methane production, yeast was added on the 12th day of fermentation when the daily biogas production was significantly reduced. On the 37th day of fermentation, the daily biogas production of both experimental groups decreased to 0, so yeast and glucose were added for the second time on the 37th day of fermentation. We have supplemented this part in the revised manuscript (Page 6, Lines 3-7; Lines 9-11).
1. Wu C, Wang Q, Yu M, Zhang X. 2015 Effect of ethanol pre-fermentation and inoculum-to-substrate ratio on methane yield from food waste and distillers'grains. Saccharomyces cerevisiae was added when fermentation gas production had drastically reduced (day 12 of fermentation) to investigate the effect of yeast on reducing the load of VFAs, improving the system's stability and methane production. The method for the yeast addition and activation has been mentioned in previous research [15].
(Page 6, Lines 9-11): In order to investigate whether the reduction of gas production to zero in the later stage of anaerobic fermentation is due to the depletion of substrate or the deactivation of methanogens, yeast and glucose were added for the second time(No biogas generation, 37 d).

Q9:
What is the implication of the second yeast and glucose addition, is it economically viable if implemented on commercial scale?

Replies:
Thanks for your comment. The purpose of the second addition was to investigate whether the decrease of gas production to 0 was due to the depletion of substrate or the deactivation of methanogens. On the 37th day of fermentation, the daily biogas production of both experimental groups decreased to 0, so yeast and glucose were added for the second time on the 37th day of fermentation. We have supplemented this part in the revised manuscript (Page 6, Lines 9-11). According to our research results, after the second addition of yeast and glucose, the daily biogas production is still significantly increased, which indicates that the addition of yeast is conducive to the rapid recovery of biogas production in the anaerobic system. Therefore, if it is implemented on a commercial scale, it is economically feasible when the substrate in the anaerobic digestion system is not exhausted and the methane bacteria are not inactivated.
Revisions in revised manuscript (Page 6, Lines 9-11): In order to investigate whether the reduction of gas production to zero in the later stage of anaerobic fermentation is due to the depletion of substrate or the deactivation of methanogens, yeast and glucose were added for the second time.

Comments for Results and discussion
Q10: "… fermentation were determined ( Figure 2)" in line 20, page 6, how was it "determined" or do you mean "recorded"?
Replies: Thanks for your comment. We have made changes on the Page 7, Line19 of the revised manuscript. Results should be stated earlier before discussions with literature information to avoid missing out salient discoveries in the study.

Replies:
Thank you for your valuable advice, which is very helpful to our writing.
In Section 3.1 of the revised manuscript, we had a supplementary discussion of facts with adequate citation (Page 8, Lines 8-21).

Revisions in revised manuscript (Page 8, Lines 8-21):
The addition of activated yeast can drastically promote the anaerobic digestion of methanogenesis in FW. In other study, yeast was added to the substrate for ethanol prefermentation in the sequencing batch methane fermentation of food waste. The results showed that methane production in the EP group (254 mL/g VS) was higher than in the control group (35 mL/g VS) [25].
It was consistent with the results of this study.
This effect may be attributed to the reduction in the production of VFAs by the addition of activated yeast. Because of the glycolytic metabolic pathway in S. cerevisiae, the end products are mainly ethanol and acetic acid after yeast addition [27]. Moreover, the acidification in AD system is considered as the problematic inhibition of biogas production. Thus, FW substrate was mainly metallised into ethanol by additional yeast, instead of VFAs. The ethanol could be gradually converted into acetic acid, which can be easily used by methanogens, thereby increasing methane production and enabling the stable operation of anaerobic fermentation [4].
And the addition of yeast can improve the relative abundance of methane-producing bacteria in Replies: Thank you for your valuable advice, which is very helpful to our writing.
We have revised sections 3.2 and 3.3 of the revised manuscript. After the revision, we first emphasized the results of this study, and then provided literatures and statements to prove the rationality of the results.
Revisions in revised manuscript (Page 9, Lines 5-16): As shown in Figure 3-a, the pH values of the two experimental groups gradually decreased but were never less than 6.5. Therefore, acidification did not occur during anaerobic fermentation. This phenomenon also explains why the daily output of biogas in the early stage of fermentation in Figure 2-a did not decrease to zero, even if it decreased. After 8 days of anaerobic fermentation, the pH of the two groups of experiments rapidly increased probably because of the buffering effect of the system itself. After the first addition of yeast or glucose, the pH value of the yeast group did not significantly differ from that of the control group, which was above 7.5. According to current research reports, due to the differences in the nature of the additives, the pH of a methane fermentation system that can maintain stable operation is in the range of 6.5-8. the addition of activated yeast on day 12 of fermentation, the alkalinity of the system increased rapidly (Figure 3-b). The TVFA/TA ratio continued to decrease, and the yeast system began to produce a large amount of gas. The TVFA/TA value of the yeast group was lower than that of the control group, indicating that the addition of yeast to the anaerobic digestion system can adjust alkalinity and reduce TVFA concentration, thereby improving the stability of anaerobic digestion. The TVFA/TA values of the two groups of experiments never exceeded 0.4. This result also proved that the system was not acidified, and the reduction in gas production before the addition of yeast or glucose may be caused by substrate exhaustion. Similar findings have been found in previous studies. The ratio of TVFA/TA can be used as an early warning of digestive system imbalance. TVFA/TA shows the ratio between compounds that causes a decrease in pH and compounds that maintain alkalinity in the system. This index sensitively reflects the ability of the anaerobic digestion system to withstand acidification. When TVFA/TA exceeds 0.4, the acidification of the anaerobic system is about to be destabilised. When TVFA/TA exceeds 0.6, the acidification of the anaerobic system will be completely imbalanced [23]. Comparing the two groups' parameters, such as pH, alkalinity, TVFA concentration and TVFA/TA ratio, revealed that these parameters followed the same trend. However, the alkalinity of the yeast group was slightly higher than that of the control group, the TVFA and TVFA/TA of the yeast group were slightly lower, thereby indicating that the addition of activated yeast improved the stability of anaerobic digestion.

(Page 11, Lines 3-23):
The main VFAs components detected in the two groups were acetic, propionic and butyric acids (Figures 4-a and 4-b). Valeric acid, isovaleric acid and iso-butyric acid were also detected at low concentrations. The acetic acid concentration of the yeast group reached a maximum of 7.97 g·L-1 on day 8, and then began to decrease with the consumption of butyric acid. From days 11 to 15, the concentration of butyric acid decreased rapidly. Daily biogas production also increased ( Figure 2-a). Propionic acid gradually accumulated with the AD progression of fermentation, thereby propionic acid-type fermentation was considered to be established. In anaerobic fermentation, VFAs containing more than two carbon chains cannot be directly used as a substrate by methanogens, and thus, which are easily accumulated during fermentation [3]. Propionic acid is a common short-chain fatty acid.
From the perspective of metabolism, propionic acid is usually converted into acetic acid and hydrogen. However, acetic acid generation by propionic acid and butyric acid is an endothermic reaction that is difficult to perform thermodynamically, and the conversion of propionic acid to acetic acid is difficult. It was suggested that propionic acid is a disadvantageous substrate for microorganisms [19]. Therefore, when propionic acidtype fermentation is occurred, the utilisation of organic acids in the methanogenic phase is inhibited and acids accumulation is promoted, which adversely affects methanogenesis. It was found that high temperature and alkaline pH are favourable for propionic acid generation [34]. On the other hand, the yield of VFAs, especially propionic acid, was also suggested to be enhanced by adjusting the pH level. Further, the key enzyme activity associated with propionic acid formation was the highest, when the system pH was set at 8.0 [3]. After 12 days of fermentation, the propionic acid concentration of the yeast group was lower than that of the control group, indicating that the addition of yeast can play a certain preventive role when propionic acid fermentation has be occurred in the anaerobic fermentation system.

Comments for Conclusion
Q14: Revise this conclusion section to reflect the essence of this study and the findings.
Replies: Thank you for your valuable advice, which is very helpful to our writing.
We have revised conclusion section of the revised manuscript.

Revisions in revised manuscript (Page 12, Lines 15-21):
The effect of yeast addition on AD of FW was investigated in this study. The results showed that the addition of yeast can restore and promote the biogas production by anaerobic digestion.
Moreover, AD with yeast addition exhibited a high VFAs consumption rate and low propionic acid concentration, which prevented the excessive acidification phenomenon.
By adding yeast, FW was converted into ethanol as a slow-release substrate, instead of VFAs. The ethanol could be gradually converted into acetic acid, which can be easily used by methanogens, thereby increasing methane production and enabling the stable operation. Therefore, yeast addition was suggested as a feasible approach to maintain a stable AD system.

Q15
: "Especially, activated yeast was supplemented twice when AD process was inhibited with few daily biogas generation (nearly zero)" this statement in line 20 and 21, page 11 could be misleading, while it may be true for your second addition, Fig. 2a denied the near zero yield statement for the first addition. Please revise.