Batch Mode Reactor for 3,5-Dinitrosalicylic Acid Degradation by Phanerochaete chrysosporium

Reviewer 1

Reviewer’s comments

Authors’ response

Revised text

 L. 17: “Three parameters chosen to optimize the process.” The process is investigated through variation of three parameters. However, this investigation does not constitute an optimization. As you point out and show then the mineral salt solution does play a role since the enzymes responsible for the downgrading of the 3,5-dinitrosalicylic acid do require a metal ion. In addition the concentrations of the minerals in the minerals salt solution also affects the biomass growth (as you write in line 180). Consequently one would expect that the composition of the mineral salt solution would require much more careful experimental investigation in order to optimize this process for industrial usage.

The purpose of our research was to identify the parameters that influenced the process efficiency. We focused on three parameters: pH, medium composition and immobilization matrices. We investigated the dependencies between them to find the answer for the process preparing with the highest degradation of the 3,5-DNS acid in the shortest time. In our conception it was the process optimization.

The presented results determine the initial study in a laboratory scale before their application in the industry.

We agree that our research requires much more careful investigation to optimize the process for industrial usage.  Of course, we will continue our research in this direction.

L. 44: You write: “The aim of the studies is to present a novelty process of the 3,5-dinitrosalicylic acid degradation by Phanerochaete chrysosporium in a continuous mode reactor working at a semi-industrial scale.”

The study is carried in a scale which to a process engineer is lab scale. The reactor vessel volume is 0.1 dm³, consequently it cannot be labeled a micro reactor either.

We know that micro is a unit prefix in the metric system SI and it is a million part. The word originated from Greek “μικρό” (mikró) which means small.

Following the word’s genesis we decided to label our reactor as the micro batch reactor.

L. 201 you also write: “The most effective results of the biodegradation in a continuous mode reactor …”. As pointed out in the introduction to this review then your reactor is in effect a (fed-)batch type reactor where you continuously recycle a mineral salt solution – which is depleted of some of its minerals and enriched with some of the breakdown products during the batch duration. Only the gaseous components are washed out continuously. Consequently, the phrase “continuous mode reactor” is misleading.

We agree that the term “continuous reactor” was used unfortunately. Only the exchange of solutions was carried out continuously with a volumetric rate of 1.4·10^-4 dm³/s. Indeed, the reactor was operated in a batch mode until the assumed reduction of the 3,5-dinitrosalicylic acid was achieved.

We changed the type reactor name in all texts.

L.166: In Table 1 it would be interesting to know when and in what amounts the compounds were detected. It would also be interesting to know if you could distinguish between, e.g. 3-amino-5-nitrosalicykic acid and 3-nitro-5-aminosalicykic acid?

The third column of Table 1 informs about time when the compounds appeared. Unfortunately we do not know the mass of by-products because we made only qualitative analysis. In our future research we will also make the quantitative analysis which allow to continue more interesting calculations.  

Throughout the paper you use the nomenclature: nitrate(V) and nitrate(III). In English language nitrate means NO3- whereas nitrite means NO2-

We would like to apologize for this big mistake. We changed the nomenclature in the manuscript.

L. 47: “Our studies allow to intensive the biodegradation efficiency as well as influence time of the process.” The meaning is uncertain, do you mean …to improve the biodegradation efficiency as well as influence its duration?

Thank you for the suggestion. Yes, we wanted to write that our research improved the biodegradation efficiency of the 3,5-DNS acid during the process conducted in a batch reactor.

We planned the experiments for four weeks of duration according to the scientific literature. After results analysis we noticed that time could be shorter if the experiments conduct in initial pH 6.5 with microorganisms immobilized on the sugar beet pulp and the polyurethane foam. 

Our research allows to improve the biodegradation efficiency as well as to reduce time of the process.

On L. 108-109 you write “… a mineral nutrients medium…” which probably is medium A or medium B and therefore can be described as“ …the mineral nutrients medium…”

Yes, lines 108-109 describe medium B which consist of medium A supplemented by Czapek’s modified medium. We changed the medium description in an indicated text.  

The samples of this experimental part were: B1 – medium B flowing the beech smoking chips; B2 – medium B flowing a sugar beet pulp; B3 –medium B flowing a polyurethane foam.

Reviewer 2

Reviewers comments

Authors’ response

Revised text

An English language check is required throughout the ms. It contains a few grammatical errors and bad wording.

English was checked by the proofreader.

The au-s report about “nitrate(V) and nitrate(III) ions”. Do they mean “nitrate(V) and nitrite(III) ions”? If yes, it should be corrected throughout the ms.

We would like to apologize for this big mistake. We changed the nomenclature in the manuscript.

A Scheme should be inserted about the different degradation processes and the degradation products.

Thank you for the suggestion. We prepared the scheme and inserted it in the manuscript as Figure 5.

The 2-4-week reaction period seems to be quite long. What are the cycle times in case of the known procedures for similar purpose?

We prepare the experiments according to the scientific literature. The researchers conducted only the stationary cultures from 3 up to 90 days. For example, Fernando et al. Investigated decomposition of 2,4,6-trinitrotoluene in both 18 and 90 days batches, for concentrations 1.3 and 100 mg/dm³, respectivelyⁱ. Bayman et al investigated Hexogen in periods 3 and 30 days (concentrations of 0.02 and 100 mg/dm³, respectively)ⁱⁱ, while Fournier et al. assessed CL-20 biodegradation during  3 and 46 day periods (concentrations 7 and 7.5 mg/dm³)ⁱⁱⁱ.

i) Fernando T, Bumpus JA, Aust SD. Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanerochaete chrysosporium. App Environ Microbiol 1990, 56:1666-1671.

ii) Bayman, P.; Ritchey, S. D.; Bennett, J. W. J. Ind. Microbiol. 1995, 15, 418-423.

iii) Fournier D, Monteil-Rivera F, Halasz A. Degradation of CL-20 by white-rot fungi. Chemosphere 2006, 63:175–181.

The au-s mention “continuous flow” in the Conclusions part. This is a continuous, but not a continuous flow technology.

Thank you for the suggestion. We apologize for this text. We rewrote this part of the conclusion.

The reactor was operated in a batch mode until the assumed reduction of the 3,5-dinitrosalicylic acid was achieved.

The Author Contributions part should be started with “Conceptualization, R.M.; E.S.; …”. The sentences before should be deleted.

Of course it is our mistake. We read this part carefully and changed the text to the editor’s requirements.