Alkalinity, and Not the Oxidation State of the Organic Substrate, Is the Key Factor in Domestic Wastewater Treatment by Mixed Cultures of Purple Phototrophic Bacteria
, Natalia González-BenĂtez 2, Mari Carmen Molina 2, Raquel Simarro 2, Juan Antonio Melero 1, Fernando MartĂnez 1 and Daniel Puyol 1,*
Kenji Furukawa
Round 1
Reviewer 1 Report
This paper is interesting but I have some minor remarks concerning the final acceptance:
- line 44 - I suggest to explain ATP.
- Introduction - After the description of the aim of a research, I suggest to add one sentence concerning findings obtained.
- I suggest to write conclusions in points. It will be more clear for readers.
- I also suggests add more reference to scientific literature, especially in discussion part.
Author Response
This paper is interesting but I have some minor remarks concerning the final acceptance:
Response: I would like to express my sincere gratitude to the reviewer for the constructive comments made, which undoubtedly will help to improve the overall quality of the final version of the Manuscript. I hope that the changes made will make the paper suitable for acceptance.
- line 44 - I suggest to explain ATP.
Response: We have spelt out ATP in the revised manuscript as follows: “adenosine tri-phosphate (ATP)”
- Introduction - After the description of the aim of a research, I suggest to add one sentence concerning findings obtained.
Response: According to the reviewer’s suggestion, the following sentence has been added in the revised manuscript: “We conclude that bicarbonate alkalinity is a strong factor to check in the scale-up of the PPB technology for domestic wastewater treatment. The mixed cultures of PPB can evolve irrespective of the oxidation state of the organic source if there is enough bicarbonate alkalinity to allow for the release of the excess of electrons.”
- I suggest to write conclusions in points. It will be more clear for readers.
Response: we have modified the conclusions as follows:
P14, LL411-422:
“It has been demonstrated that the general performance of the PPB mixed cultures was not affected by the oxidation state of the external organic carbon source. This contrast with the strong effect of alkalinity availability shown in the preliminary experiment. Specific conclusions are:
- The similarity of growth parameters (µmax and biomass yield), and the COD, N and P removal efficiencies strongly suggest that the oxidation state of the external organic carbon source is not an impediment for the development of active PPB biomass in DWW treatment.
- High alkalinity supports the electron dissipation in PPB systems, allowing PPB to grow on reduced organics as butyrate and ethanol. Therefore, alkalinity, and not the oxidation state of the organic substrate, is the key limiting factor for PPB systems in DWW treatment.
- Rhodopseudomonas palustris prevails in reduced organics (butyrate and ethanol), whereas this species and Rhodobacter capsulatus co-dominate wastewater environments in oxidized organics (as acetate).”
- I also suggests add more reference to scientific literature, especially in discussion part.
Response: According to the reviewer’s suggestion, we have enlarge the discussion part and we have added four more references, where two of them are new references with respect to the previous version of the manuscript. The following text has been added in the revised manuscript:
P11, LL342-347: “An increase in phosphorus uptake is indicative of an efficient usage of energy, where excess of ATP produced during the exponential growth phase is used for poly-phosphate production when the culture enters into a stationary growth phase [18].”
P11, LL354-358: “Despite several works concluded that PPB behaves differently depending on the organic substrate [7,24], this work sustains that these metabolic-based differences do not suppose a big difference in the overall performance of the mixed culture. In a recent paper, it has been concluded that controlling the general control parameters in wastewater treatment (like pH, oxygen and alkalinity) is an efficient way to improve the performance of PPB-based wastewater treatment [25].”
Reviewer 2 Report
I could not find the novelty in your submitted paper. The level of your paper did not reach the acceptance level of this journal. Major changes are required before accepting your paper. The followings are the unclear and weak points of your paper. Please revise your paper carefully by taking into accounts the following points.
- English is poor except in Abstract and Conclusion. Your way of writing is too redundant. I have judged native check is required.
Line 62, 193 in origin?
Line 73 this axion
Line 89 purple phototrophic bacteria mixed culture
→　mixed culture of purple phototrophic bacteria
Line 101 exploratory experiment →　preliminary experiment
Line 121 central experiment →　main experiment
Line 135 9L of culture →　9L of culture broth or 9L of culture liquid
Line 146 were extracted →　were sampled
Line 151 Biomass evolution →　Biomass growth
Line 184 and Table 2 apparent biomass yield(Yx/s) → observed biomass yield(Yob)
Line 203 reduction of nutrient in origin →　initial reduction of nutrient
Line 229 to know →　to study
Figure caption of Figure 1 natural COD →　original COD
COD breakdown →　COD removal
Line 248 source added →　source addition
Line 250 in solid composition →　in suspended solid composition
Line 358-360, 360-364, 369-374
→　These sentences were long and unclear. Please re-write these.
- Line 81 and 82 The expression of “low COD-to-nutrient and high COD-to-nutrient” is not good. Please use “high COD/nutrient ratio” and “low COD/nutrient ratio”
- You must unify the usage of external carbon source. You have used various expression such as external COD, external organic source, extra organic source, added substrate and extra substrate
- You must unify the expression of round. You have used various expression such as round 1, Round 1 and R1
- I am strongly worry about the measurement of biomass concentration. I suppose your culture is the mixed with suspended biomass and non-settled biomass. You have measured the growth of phototrophic bacteria by OD. Did you apply the some pretreatment like sonication to solubilize the suspended biomass? Please show the conversion equation of OD to VSS concentration.
- You must mention the way of pretreatment like filtration for measuring SCOD, NH4-N and PO43-—P
- You have measured the specific growth rate(μ). Do not use μmax. μmax shows the maximum specific growth rate.
Author Response
I could not find the novelty in your submitted paper. The level of your paper did not reach the acceptance level of this journal. Major changes are required before accepting your paper. The followings are the unclear and weak points of your paper. Please revise your paper carefully by taking into accounts the following points.
Response: I would like to express my sincere gratitude to the reviewer for the constructive comments made, which undoubtedly will help to improve the overall quality of the final version of the Manuscript. I hope that the changes made will make the paper suitable for acceptance.
Regarding the novelty of the paper, we have indicated in the letter to the editor that the source of organic compounds to be supplemented to the raw domestic wastewater is an actual problem for the industrialization of the process. The treatment of domestic wastewater alone is not a viable application of purple phototrophic bacteria. We have observed that the mixed cultures of PPB are able to grow under a wide range of organic compounds, irrespective to the oxidation state, and therefore we concluded that other factor/s should be the key for understanding the mechanism that allows the PPB to growth on reduced organics in domestic wastewater. In this work we simplified this problem to a key parameter – alkalinity. This is a real problem in the upscaling of the process, and we are actually using wisely this information in demonstrative-scale reactors that are currently in operation. The information and conclusions of this paper are not evident since we work with mixed cultures (which can produce alkalinity “per se” under anaerobic conditions), and these conclusions have never been reported so far.
- English is poor except in Abstract and Conclusion. Your way of writing is too redundant. I have judged native check is required.
Response: we have carefully reviewed the quality of the English language with the help of a native speaker. All changes made are indicated with the “track changes” option in Word processor.
Line 62, 193 in origin?
Response: This has been changed to “from the original source”
Line 73 this axion
Response: we humbly consider that the correct spelling for this word is “axiom”, and not “axion” as pointed out by the reviewer.
Line 89 purple phototrophic bacteria mixed culture →　mixed culture of purple phototrophic bacteria
Response: Modified as per the reviewer’s suggestion
Line 101 exploratory experiment →　preliminary experiment
Response: Done
Line 121 central experiment →　main experiment
Response: we humbly consider that the word “central” in this context is a synonym for “main”, as it has a figurative meaning (according to Collins dictionary). Therefore, we consider that this modification is not necessary.
Line 135 9L of culture →　9L of culture broth or 9L of culture liquid
Response: Modified as suggested
Line 146 were extracted →　were sampled
Response: we changed “biomass samples were extracted” by “biomass was sampled” in the corrected manuscript.
Line 151 Biomass evolution →　Biomass growth
Response: Modified as suggested
Line 184 and Table 2 apparent biomass yield(Yx/s) → observed biomass yield(Yob)
Response: We changed “apparent biomass yield (YX/S)” by “observed biomass yield (YOB) along the manuscript.
Line 203 reduction of nutrient in origin →　initial reduction of nutrient
Response: Modified as suggested
Line 229 to know →　to study
Response: Modified as suggested
Figure caption of Figure 1 natural COD →　original COD
COD breakdown →　COD removal
Response: Modified as suggested
Line 248 source added →　source addition
Response: Modified as suggested
Line 250 in solid composition →　in suspended solid composition
Response: Modified as suggested
Line 358-360, 360-364, 369-374
→　These sentences were long and unclear. Please re-write these.
Response:
Lines 358-360. These sentences have been changed to “The oxidation state of the organic substrate affected the bacterial diversity. Indeed, some Rhodobacter ssp. species were displaced by Rhodopseudomonas spp. when they grew on reduced organic carbon sources such as ethanol and butyrate.”
Lines 360-364. These sentences have been changed to: “A previous work reported that a Rubisco-mutant of R. palustris was unable to grow on acetate, whereas a Rubisco-mutant of Rhodobacter sphaeroides (R. sphaeroides) was able by using the ethylmalonyl-CoA pathway as a way to reduce CO2 [26,27].”
Lines 369-374. These sentences, and the previous one, have been changed to: “Other ancillary species non-related with PPB were detected in low proportions and evolved mainly in the last feed rounds, especially in R2 and R3. The presence of some anaerobic fermentative bacteria belonging to genera Proteiniclasticum, Desulfomicrobium and family Porphyromonadaceae indicated a transition of dominance in reactors fed with reduced substrates (R2 and R3). This might indicate bacterial decay that may become substrate for these ancillary fermentative communities.”
- Line 81 and 82 The expression of “low COD-to-nutrient and high COD-to-nutrient” is not good. Please use “high COD/nutrient ratio” and “low COD/nutrient ratio”
Response: The expressions “COD-to-nutrients” and “carbon-to-nutrients” have been changed to “COD/nutrients” and “carbon/nutrients” along the revised Manuscript.
- You must unify the usage of external carbon source. You have used various expression such as external COD, external organic source, extra organic source, added substrate and extra substrate
Response: according to the reviewer’s comment, we have unified the terminology to refer to the external carbon source. We have use the term “external organic carbon source” along the paper
- You must unify the expression of round. You have used various expression such as round 1, Round 1 and R1
Response: In order to clarify the manuscript, when we refer to a specific round, first letter is in capital letter (e.g. “Round 1”), but when we refer to the concept of round, first letter is in lower case (e.g. “along the different rounds”). The acronyms R1, R2 and R3 are used for the description of Reactor 1, Reactor 2 and Reactor 3, and are not related to the feed round. We have spelt out these acronyms the first time that they appeared, in Page 4, Lines143-144, as follows:
“Organic substrates tested were Acetate (Reactor 1, R1), Ethanol (Reactor 2, R2) and Butyrate (Reactor 3, R3).”
- I am strongly worry about the measurement of biomass concentration. I suppose your culture is the mixed with suspended biomass and non-settled biomass. You have measured the growth of phototrophic bacteria by OD. Did you apply the some pretreatment like sonication to solubilize the suspended biomass? Please show the conversion equation of OD to VSS concentration.
Response: According to our experience, we have set that the measurement of the OD at 660 nm is a good predictor of the biomass concentration (expressed as VSS) if the culture is growing in planktonic form. In order to evidence this, we have included the information about the linear correlation between the OD 660 and VSS as follows:
P4, LL158-160: “A linear response between the absorbance at 660 nm and the VSS values was ensured due to the planktonic nature of the enrichment cultures, where the calibration curve was determined to be Abs660 = 0.0021·VSS (mg/L) (R2 = 0.989).”
- You must mention the way of pretreatment like filtration for measuring SCOD, NH4-N and PO43—P
Response: The following text has been added into the revised manuscript:
P4, LL155-156: “Samples were filtered by using a 0.45 µm syringe filters in order to determine soluble components (SCOD, NH4-N PO43—P and VFAs).”
- You have measured the specific growth rate (μ). Do not use μmax. μmax shows the maximum specific growth rate.
Response: We humbly consider that the term µmax is the correct one in this case, as is the term we have measured. This term has been calculated by linear regression between the n-Log of the OD-660 and the time. The biomass growth can be represented by the following differential equation:
Eq. [1]
Where µ is the specific growth rate, X is the biomass concentration (in this case, expressed as OD660 due to linear relationship with VSS concentration), and t is the growth time. In the exponential phase (non-limiting stage),µ = µmax = constant, and Eq. 1 is solved as:
Taking logarithms at both sides of the equation, we have:
|
|
Where Xe is the first value of OD660 in the exponential growth phase at t = te. Using the data at the exponential phase, µmax can be determined by linear regression of lnX versus t, where the slope of the linear curve corresponds to the µmax value.
This method is depicted in detail in most of the biotechnology text books, and we humbly consider that it is not necessary to describe in this paper. In any case, the reviewer is right in that the term was wrong, and we have modified the manuscript as follows:
P4, LL186: “). The maximum specific growth rate (µmax, 1/d) was calculated…”
(PLEASE CHECK THE WORD ATTACHED FOR THE DESCRIPTION OF THE EQUATIONS)
Round 2
Reviewer 2 Report
You have revised your paper properly by taking into account the reviewer’s comments.
I have judged your paper reached to the acceptance level of this journal.
