Review Reports - Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (Anammox) Activities

Round 1

Reviewer 1 Report

This manuscript reported the short- and long-term effect of caffeine on anammox nitrogen removal. The topic is novel and the research helps to offer some instruction for coffee wastewater using anammox. However, the major revisions are still required.

1. Please give full names of the terms such as SCOD, bCOD, TN..., when they firstly appear.

2. Line 123-124 Why did you use the synthetic wastewater rather than coffee wastewater as the feed in this study？Table 1. Is there any nitrite contained in the coffee wastewater? Moreover, the caffeine concentration should be also given.

3. Please use the mathtype to edit the equation 2.

4. How to determine the SCOD and bCOD？

5. Fig. 1 Please give the reason for the higher TN removal at 50 mg/L caffeine dosage than the control with no caffeine.

6. The error bars should be indicated in all batch experiments (Figs1-3).

7. Line 217-219. The method of inhibition kinetics should be specified. Moreover, your obtained model results should be compared with other similar studies. doi.org/10.1016/j.scitotenv.2018.09.285

doi.org/10.1007/s11783-021-1433-3

8. In short-term experiment, the caffeine concentration reached 500 mg/L. However, only 2.5 mg/L caffeine was added in your long-term experiment. Why? Was there any relevance between two experiments？

9. Did you consider the role of the denitrification in TN removal contribution?

10. In my opinion, Only 28 days were not sufficient for affecting the change of microbial community, particularly autotrophic anammox species with the caffeine dosage condition during the long-term effect experiment.

11. The data for effluent nitrogen species were lacked during the long-term operation.

Author Response

Please give full names of the terms such as SCOD, bCOD, TN..., when they firstly appear.

The full names for the abbreviations have been added as suggested.

Soluble chemical oxygen demand (SCOD), biodegradable chemical oxygen demand (bCOD), total nitrogen (TN)

Line 123-124 Why did you use the synthetic wastewater rather than coffee wastewater as the feed in this study？ Table 1. Is there any nitrite contained in the coffee wastewater? Moreover, the caffeine concentration should be also given.

We have addressed this comment below.

Synthetic wastewater was used in order to control other parameters, such as ammonium (NH₄⁺) to nitrite (NO₂^-) ratio (1:1.32), high alkalinity and other trace elements necessary for anammox culture growth. Caffeine concentration of 5 mg/L was added to synthetic wastewater. The analytical results for coffee wastewater, including nitrite concentration, has been added.

Please use the mathtype to edit the equation 2.

It has been edited as suggested.

How to determine the SCOD and bCOD？

In this work, for SCOD analysis, we used a nylon membrane (0.45 μm) to filter sample first. After the sample was filtered, COD was determined by following the method described in Standard Methods for the Examination of Water and Wastewater (2005). However, we did not directly analyze the value for bCOD. We calculated the bCOD value by assuming that the BOD:bCOD ratio was 1.6:1.

Fig. 1 Please give the reason for the higher TN removal at 50 mg/L caffeine dosage than the control with no caffeine.

We have addressed this comment below:

A caffeine dosage of 50 mg/L did not affect anammox activity. However, the TN removal at 50 mg/L caffeine dosage was higher than the control with no caffeine because our error from analysis on both NH₄⁺ and NO₂^- concentrations when we took sample at caffeine dosage of 50 mg/L. The nitrogen removal efficiency, we calculated by using [(NH_{4 (inf)}⁺ plus NO₂^-_(inf)) – (NH_{4 (eff)}⁺ plus NO₂^-_(eff))]/[(NH_{4 (inf)}⁺ and NO₂^-_(inf))].

6.The error bars should be indicated in all batch experiments (Figs1-3).

It has been added error bars in the Figs 1-3 as suggested.

Line 217-219. The method of inhibition kinetics should be specified. Moreover, your obtained model results should be compared with other similar studies. doi.org/10.1016/j.scitotenv.2018.09.285

doi.org/10.1007/s11783-021-1433-3

We have addressed this comment as follows.

Chen et al. (2019) studied effect of quinoline on anammox activity. Quinoline is one type of nitrogen heterocyclic compounds that are found in refractory coking wastewater. They found that only 13.1 mg/L of quinoline concentration would affect anammox activity. A non-competitive model was able to be used in this case. By comparison quinoline is much more toxic than caffeine on anammox activity. Li et al (2021) investigated the effect of pyridine (that is also found in refractory coking wastewater) on anammox activity. They used a non-competitive model to explain anammox activity and reported the K_i of pyridine at 135.19 mg/L.

However, in this case, we could not distinguish between an un-competitive model (expectation that caffeine is able to bind site away from the substrate binding site) and a non-competitive model (expectation that caffeine is able to bind free enzyme and substrate from the binding site). For this reason, the mechanism of caffeine inhibition on anammox activities could not be described clearly. Further investigation on the mechanism of caffeine inhibition is needed.

In short-term experiment, the caffeine concentration reached 500 mg/L. However, only 2.5 mg/L caffeine was added in your long-term experiment. Why? Was there any relevance between two experiments？

We have addressed this comment below:

In the short-term or acute experiment (only one-time addition of caffeine), we used low to high caffeine concentrations in order to determine at which caffeine concentration one might observe an effect on anammox activity. On the other hand, in the long-term experiments we used a low caffeine concentration (as one would expect from a coffee wastewater), but added it daily, because we wanted to determine the effects of a low amount of caffeine over time.

Did you consider the role of the denitrification in TN removal contribution?

No, because our cultures consist primarily of anammox bacteria rather than denitrifying bacteria.

In my opinion, Only 28 days were not sufficient for affecting the change of microbial community, particularly autotrophic anammox species with the caffeine dosage condition during the long-term effect experiment.

The reviewer’s opinion might be correct. However, because the doubling time of anammox bacteria is thought to be around 11-14 days, we thought that our experimental time could be sufficient in order to observe inhibition by caffeine dosage.

The data for effluent nitrogen species were lacked during the long-term operation.

The reviewer’s understanding is correct. Nitrate concentration of each effluent during long-term experiment has not been shown. Both NH₄⁺and NO₂^-concentrations were reported and these nitrogen forms were deceased. However, we analyzed nitrate concentrations only a few times and those nitrate concentrations followed the anammox stoichiometric ratios (0.26 mol of NO₃^-produced) according to the equation (1). For this reason, we assumed that anammox activities could be defined by observing only the decrease of NH₄⁺and NO₂^-concentrations.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript submitted by Wongphoom et al. presents the anammox activity under caffeine and COD wastewater. The topic is very interesting because effect of caffeine on anammox activity was not understand so far. The authors should consider the following points before the publication.

Major comment

1. English in the manuscript must be checked by native speaker.

2. Define the bCOD, TCOD, SCOD in the text.

3. L74-77, the authors should show the dominant anammox species and their abundance (population) of the enrichment culture.

4. L121, it is not clear that detailed of wastewater. Is it from a factory or shop? Please describe the details because this is the key point in this manuscript.

5. Table 1, why the wastewater contains such high nitrite (NO2-)? Did you add it? It is unusual situation. Also add the concentration of caffeine here.

6. L196 and L198, there are 5 plots in figure 1 and 2, but only 4 values in the sentences.

7. Figs 4 and 5, how calculate the % inhibition in Fig. 5? Based on the values in Fig. 4 because SAA was dropped on 14 days.

8. Please add the mechanism and discussion of the inhibition of anammox activity by the presence of caffeine.

9. Please add the discussion on the color derived from coffee of wastewater.

10. It is not clear anammox population was not changed even though the anammox activity was decreased.

Minor comment

L46, the formation of NO3- is true based on the anammox stoichiometric ratios (0.26 mol of NO3- produced) according to the equation (1).

L82-83, EDTA is doubly described. Please check the concentrations carefully.

L105 and others, the unit of mMol is not correct. I think it should be mmol/L or mM.

L118, add period “.” in the end of sentence.

L145 and equation (4), NH4+.

L154, did you measure BOD? bCOD?

Fig. 2, in the second plot (caffeine concentration is 50 mg/L) why inhibition (%) was zero even though the SAA decreased from 0.62 to 0.53?

L229, mg/L.

L277, technique.

Author Response

Major comment

English in the manuscript must be checked by native speaker.

All text in the manuscript has been carefully rechecked by experts fluent in idiomatic English.

Define the bCOD, TCOD, SCOD in the text.

The full names for the abbreviations of bCOD, TCOD, SCOD have been added as suggested.

Soluble chemical oxygen demand (SCOD), biodegradable chemical oxygen demand (bCOD), total chemical oxygen demand (TCOD).

L74-77, the authors should show the dominant anammox species and their abundance (population) of the enrichment culture.

The dominant anammox species in this work would be Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida.

L121, it is not clear that detailed of wastewater. Is it from a factory or shop? Please describe the details because this is the key point in this manuscript.

It has been explained as suggested.

Coffee wastewater from this work was collected from the Amazon Café, the most popular coffee shop in Thailand.

Table 1, why the wastewater contains such high nitrite (NO2-)? Did you add it? It is unusual situation. Also add the concentration of caffeine here.

We have addressed this comment below.

Yes, we added NO₂^-and caffeine concentrations into synthetic wastewater. This is because we were able to control other parameters as optimum substrates for anammox cultures growth such as nitrite concentration, alkalinity other trace elements. All of these reagents are very important for anammox cultures growth.

L196 and L198, there are 5 plots in figure 1 and 2, but only 4 values in the sentences.

It has been edited as suggested.

For caffeine concentrations of 0, 50, 250, 350 and 500 mg/L, the specific anammox activities (SAA) were 0.663, 0.527, 0.436, 0.311 and 0.300 mg-N/mg-VSS/d, respectively, as indicated in Figure 2. The SAA inhibition under the caffeine concentrations of 0, 50, 250, 350 and 500 mg/L were 0%, 0%, 10%, 32% and 33%, respectively, see Figure 2

Figs 4 and 5, how calculate the % inhibition in Fig. 5? Based on the values in Fig. 4 because SAA was dropped on 14 days.

We have addressed this comment below.

The percentage of inhibition was calculated by using the relationship between [(TN_inf – TN_eff)/TN_inf]x100. Occasionally there were errors when taking sample for analysis. Using SAA would be more accurate and practical rather than using only percentage of inhibition. This is because SAA depends on the amount of anammox biomass, TN_inf and TN_eff , see Equation (2). However, in our experiment, we tried to control the same amount of anammox cultures but at times it was very difficult.

Please add the mechanism and discussion of the inhibition of anammox activity by the presence of caffeine.

We have addressed this comment below.

In this case, the authors could not distinguish between an un-competitive model (expectation is that caffeine is able to bind site away from the substrate binding site) and a non-competitive model (expectation is that caffeine is able to bind free enzyme and substrate from the binding site). For this reason, the mechanism of caffeine inhibition on anammox activities could not be described clearly. Further investigation on the mechanism of caffeine inhibition is needed.

Please add the discussion on the color derived from coffee of wastewater.

We have addressed this comment below.

After using the biological treatment process to remove both organic matter and nitrogen from coffee wastewater, However, the treated effluent of coffee might not be directly discharged to the environment because of color remaining in coffee wastewater. A chemical treatment (adsorption process) using activated carbon might be indicated to complete the process .

It is not clear anammox population was not changed even though the anammox activity was decreased.

We have addressed this comment below.

Figure 7, it is clearly shown that addition coffee wastewater did not significantly change abundances of anammox bacteria (ranging from 6.82 x 10⁹ to 1.04 x 10¹⁰ copies/g-sludge) at day of 21, 28, and 36.

L46, the formation of NO3- is true based on the anammox stoichiometric ratios (0.26 mol of NO3- produced) according to the equation (1).

It has been corrected as suggested.

L82-83, EDTA is doubly described. Please check the concentrations carefully.

It has been rechecked and edited as suggested.

This synthetic wastewater was prepared using (NH₄)₂SO₄ (210 mg N/L) and NaNO₂ (273 mg N/L). Other minor ingredients in the synthetic wastewater per liter were 1250 mg KHCO₃, 7.5 mg FeSO₄.7H₂O, 26.25 mg Na₂EDTA.2H₂O, 225 mg KH₂PO₄, 150 mg MgSO₄.7H₂O, 186 mg CaCl₂.2H₂O, 0.06 mg Na₂O₃Se.5H₂O, 0.165 mg MoNa₂O₄.2H₂O, 0.187 g CuSO₄.5H₂O, 0.322 mg ZnSO4.7H₂O, 0.742 mg MnCl₂.4H₂O, 0.18 mg CoCl₂.6H₂O and 0.142 mg NiCl₂.6H₂O.

L105 and others, the unit of mMol is not correct. I think it should be mmol/L or mM.

It has been corrected as suggested.

L118, add period “.” in the end of sentence.

It has been corrected as suggested.

L145 and equation (4), NH4+.

It has been corrected as suggested.

L154, did you measure BOD? bCOD?

We analyzed directly for the BOD value, but we did not analyze for bCOD. We assumed that bCOD = 1.6 BOD.

Fig. 2, in the second plot (caffeine concentration is 50 mg/L) why inhibition (%) was zero even though the SAA decreased from 0.62 to 0.53?

We have addressed this comment below:

A caffeine concentration of 50 mg/L did not affect anammox activity but the SAA at caffeine concentration of 50 mg/L is decreased as compared with the control (without caffeine concentration). SAA depends on the amount of anammox biomass in equation (2). In the experiment, we tried to maintain the same biomass of anammox cultures but at times it was very difficult.

L229, mg/L.

It has changed as suggested.

L277, technique.

It has changed as suggested.

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper should be revised extensively before consideration of its publication.

Title: Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (anammox) Activities

The authors have investigated the effect of caffeine and COD 66 from coffee wastewater on specific anammox activity (SAA). The presented work is good, but it needs to modify before for its acceptance. Here are the detailed comments to considered.

1) The language of the abstract should be corrected. For example, The results from this work suggested 21 that bCOD rather than TCOD should be used for calculating the COD:TN ratio during……….. should be the part of discussion not the abstract part.

2) In the introduction section, last 10 years literature should be reported rather than very old data (i.e. 1995, 1998 etc..)

3) The last paragraph of the Introduction section should be revised.

4) Table 1, reported in Material section should be move to results and discussion section, also it should be discussed.

5) In the methodology section, why statistical analysis methods were not reported? It should be mentioned.

6) The formatting of the equations from 2-4 should be checked properly and correct it with the same format.

7) The standard deviation (S.D.) of all the values from Fig. 1-9 should be added. Without S.D. there is no reliability of the data.

8) The authors should compare the presented results with the published work and explain it.

9) In general, English usage is poor and would require proofreading of a native English speaker.

Author Response

The paper should be revised extensively before consideration of its publication.

Title: Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (anammox) Activities

The language of the abstract should be corrected. For example, The results from this work suggested 21 that bCOD rather than TCOD should be used for calculating the COD:TN ratio during……….. should be the part of discussion not the abstract part.

It has been edited as suggested.

The results from this work suggested that bCOD rather than TCOD should be used for calculating the COD:TN ratio during the study of the effect of nitrogen removal using anammox process when real wastewaters have been implemented.

In the introduction section, last 10 years literature should be reported rather than very old data (i.e. 1995, 1998 etc..)

It has been edited and changed as suggested.

The last paragraph of the Introduction section should be revised.

It has been edited and changed as suggested.

The main goal of this study was to investigate the effect of caffeine and COD from coffee wastewater on anammox process in both acute and long-term periods by observing specific anammox activity (SAA) of anammox suspended growth system. A simple Monod-based model would be used to distinguish the mechanisms of caffeine inhibition on anammox activity. The results from this work could lay a foundation to apply the anammox process for nitrogen removal from coffee wastewater.

Table 1, reported in Material section should be move to results and discussion section, also it should be discussed.

Table 1 has been moved as suggested.

In the methodology section, why statistical analysis methods were not reported? It should be mentioned.

It has been added as suggested.

2.5.4. Statistical analysis

Statistical analyses (one-way ANOVA) were used to compare the presentation of all effluent samples during experimental works and including performance of the different biomasses (total bacteria, AOB, NOB, and DNB). Statistical significance tested at p-values ≤ 0.05 were applied by using Excel program.

The formatting of the equations from 2-4 should be checked properly and correct it with the same format.

It has been checked as suggested.

The standard deviation (S.D.) of all the values from Fig. 1-9 should be added. Without S.D. there is no reliability of the data.

It has been added in the Figs 1-9 as suggested.

The authors should compare the presented results with the published work and explain it.

Published works which studied the effect of coffee wastewater with caffeine on anammox activities are very limited. However, we still tried to compare our results with other researchers, for example:

Esimone et al. [19] reported that caffeine concentrations of 5 and 10 mg/mL, mixed with an antibiotic (amoxicillin concentrations of 7.81, 15, 15.23, 31.25, 125, 250, and 500 mg/mL) could increase the inhibition of the activity of Staphylococcus aureus. In this re-search work, it was found that anammox bacteria are more sensitive to caffeine con-centration than other microorganisms (E. Coli, Staphylococcus aureus, and Vibrio).

In our work, the Monod-base not-competitive model was used to describe caffeine inhibition. As indicated in Figure 3, the not-competitive model could fit the data with SAAmax of 0.594 mg-N/mg-VSS/d and Ki of 480.97 mg/L. More comparison caffeine effect on anammox activity with Chen et al.’s and Li et al.’s researches.

Chen et al. (2019) studied effect of quinoline on anammox activity. Quinoline is one type of nitrogen heterocyclic compounds that are found in refractory coking wastewater. They found that only 13.1 mg/L of quinoline concentration would affect anammox activity. A non-competitive model was able to be used in this case. Quinoline is much more toxic than caffeine on anammox activity. Li et al (2021) investigated the effect of pyridine (that is also found in refractory coking wastewater) on anammox activity. They used a non-competitive model to explain anammox activity and reported the K_i of pyridine at 135.19 mg/L.

9) In general, English usage is poor and would require proofreading of a native English speaker.

All text in the manuscript has been carefully rechecked by experts fluent in idiomatic English.

Author Response File: Author Response.pdf

Reviewer 4 Report

The content of your submitted paper is unique and interesting information were obtained. Obtained results will contribute the application of anammox process to the nitrogen removal from coffee wastewater. But, there are some unclear points in your submitted paper. I have judged minor revision is required before accepting your paper.

1. For the proper application of anammox process to the nitrogen removal from coffee wastewater, removal of high organic matters using anaerobic treatment, ammonification of TKN and partial nitritation(PN) treatment are required. Among these reaction, PN treatment will be the key technology. You must define the PN treatment characteristics for coffee wastewater such as reactor type, loading rate, operational condition etc.

2. You need to provide the essential information on the anammox culture from which you obtained the seed sludge in detail.(operational temperature, applied wastewater, loading rate, operational temperature, granular size etc.)

3. Your applied glass vial reactor is too small. It is not easy to get the reliable result using small size reactor. You must describe the way of keeping oxygen free condition.

4. Also, you must describe the way of keeping oxygen free condition for cylindrical acrylic vessel. You have used the magnetic stirrer for mixing the mixed liquor. I suppose the size of anammox granule should decrease by share stress cased by magnetic stirrer. This size reduction will give much influence on the physiological properties of anammox sludge. You must discuss this point in your text.

5. Please check the following English.

ü Line 79 ammonia-nitrogen →　ammonium-nitrogen

ü Line 105, 124　 mMol →　mM

ü Line 229 was 252 mgN/L →　was set to 252mg-N/L

ü Line 274 that when ??

ü Line 291 when using ??

Author Response

Comments and Suggestions for Authors

For the proper application of anammox process to the nitrogen removal from coffee wastewater, removal of high organic matters using anaerobic treatment, ammonification of TKN and partial nitritation (PN) treatment are required. Among these reaction, PN treatment will be the key technology. You must define the PN treatment characteristics for coffee wastewater such as reactor type, loading rate, operational condition etc.

We have addressed this comment below:

The reviewer’s understanding on partial nitritation (PN) process is correct. However, in our research we were not directly focused on PN technology. For this reason, we controlled the optimum PN process by using a synthetic wastewater having an exact ammonium (NH₄⁺): nitrite (NO₂^-) ratio (1:1.32), sufficient alkalinity, optimum pH (7.8-8.4) and other trace elements as substrates requirement for anammox cultures growth. Caffeine concentration of 5 mg/L would be added into synthetic wastewater. A sequencing batch reactor was used as a reactor, loading rate of nitrogen was 0.12 Kg N/m³ d. NH₄⁺:NO₂^- ratio is 1:1.32. Room temperature was maintained through operational condition.

You need to provide the essential information on the anammox culture from which you obtained the seed sludge in detail. (operational temperature, applied wastewater, loading rate, operational temperature, granular size etc.)

We have addressed this comment below:

The operational conditions for partial nitritation and the anammox process that were requested were as follows: NH₄⁺:NO₂^- ratio of 1:1.32, room temperature (25°C), high alkalinity and other trace elements as substrates for anammox cultures. A sequencing batch reactor was used as a reactor and growth media were provided (attached-growth or granular media known to be better than suspended-growth media).

Your applied glass vial reactor is too small. It is not easy to get the reliable result using small size reactor. You must describe the way of keeping oxygen free condition.

We have addressed this comment below:

Although we used small reactor (working volume 80 mL), into each small reactor was bubbled an inert gas mixture (95% Ar and 5% CO₂) for about 2 min in order to remove and limit dissolved oxygen (DO). Reactors were then completely sealed with a cap before beginning an experiment.

Also, you must describe the way of keeping oxygen free condition for cylindrical acrylic vessel. You have used the magnetic stirrer for mixing the mixed liquor. I suppose the size of anammox granule should decrease by share stress cased by magnetic stirrer. This size reduction will give much influence on the physiological properties of anammox sludge. You must discuss this point in your text.

The reviewer’s understanding is correct. We were concerned with this issue also. For this reason, impeller speed by magnetic stirrer was fixed at only 40 rpm through experimental work. We explained this issue into the Materials and Methods part, see detail information below.

Impeller speed by magnetic stirrer is fixed only 40 rpm through experimental work because high share stress could affect the size of anammox granule.

Please check the following English.

All text in the manuscript has been carefully rechecked by experts fluent in idiomatic English.

ü Line 79 ammonia-nitrogen →ammonium-nitrogen