Improved Delivery of Remedial Agents Using Surface Foam Spraying with Vertical Holes into Unsaturated Diesel-Contaminated Soil for Total Petroleum Hydrocarbon Removal

1) The study should mention as to where and how it can benefit in real life scenario at the end of the introduction.

⇒ Thanks for your valuable review comment. We have mentioned the application of this study as reviewer suggested.

Page 2 Line 80-83:

“The results of this study will be helpful in field-scale soil remediation to achieve an enhanced contaminant removal from entire contaminated soil by simple gravity driven foam spraying technology that uses lower cost, materials, energy and manpower.”

2) It study is conducted for very shallow depth, 25 cm that may not be suitable to use in real-life applications.

⇒  Thanks for your comment.

This study was carried out by taking 30-cm soil depth. We fully agree with the reviewer’ comment that this scale would be small. However, the tested scale is relatively higher than previous studies and batch experiments and this technology can be recommended for more deep soils in field scale remediation. This study was carried out under gravity driven natural infiltration of remedial agents without foam pressurized foam injection. Further study should be performed for more realistic soil conditions.

3) A sketch as to how the study can be transformed and applied into a real-life application needs to be included.

⇒ Thanks for your comment. The sketch of real site application of this method can be presented in next study. We think that presenting future plans (that may be confidential) would not be appropriate for present publication. We feel really sorry for this inconvenience. Please understand our situation. Thanks.

The outcomes of this study can be applied to remediation of real field contaminated site. For this, surfactant foams that carrying remedial agents can be delivered by means of small vertical holes. The depth and number of vertical holes can be calculated according to the depth of contaminated sites and effective radius of influence of foam spraying (horizontal & vertical movement of remedial agents).

4) Is there any future plan to extend this study with pilot scale project? Are there any limitations of the study? I would like to see these included in the manuscript.

⇒  Thanks for your comment.

This study successfully remediate the petroleum hydrocarbons in soil column having 30 cm height. In near future, we have plan to extend this study in real filed contaminated site with pilot scale projects. Furthermore, the limitations of this study were already mentioned in this manuscript (see section 3.3, last paragraph).

5) How the increased bacterial population (CFU) is handled once the treatment is completed. 

⇒ Thanks for your comment with valuable suggestion.

This study used oil-degrading bacterium (Acinatobacter sp. K-6), which was isolated from contaminated soil (Chaudhary et al., 2020). We fully agree with your comment regarding handling of increased bacterial population. However, it should be noted that this bacterium is not pathogenic which would not harm the human life and ecosystem. Thus, we think that the handling of bacterial population (CFU) after completing treatment is not required.

<Ref>

Chaudhary, D.K., Bajagain, R., Jeong, S.-W., Kim, J., 2020. Biodegradation of diesel oil and n-alkanes (C18, C20, and C22) by a novel strain Acinetobacter sp. K-6 in unsaturated soil. Environ. Eng. Res. 25 (3), 290-298. https://doi.org/10.4491/eer.2019.119

6) Any cost analysis that may help compare this method with available methods.

Þ Thanks for your valuable comment.

The cost analysis is not done yet and at that time it was not objective of this study. This is a laboratory based study, so an accurate cost analysis cannot be performed.

Review Comments

Response

1) I am wondering if the results of TPH analyses are correct. The maximum TPH values presented do not exceed 7000 mg/kg of soil, while the Authors claim that the soil was contaminated with 120 mL diesel per kg. Since diesel fuel usually has a density slightly below 0.85 kg/L, thus 120 mL of it should roughly weight 100 g which is equal to 100 000 mg. So this is not clear why the measured TPH values are an order of magnitude lower than expected even though some diesel components might have been lost due to evaporation.

⇒ Thanks for your valuable review comments.

We fully agree with your comment.

In this study, the concentration of selected hydrocarbons (n-alkanes from n-octane to n-tetracontane) within the carbon range from C8 to C40 (i.e. C8, C10, ……., C38, C40) were quantified as TPH with the help of a eight point calibration curve obtained by using standard TPH solution (TRPH Standard, Restek, USA). The TPH concentration was calculated by summing total peak area of total 17 n-alkanes at respective retention time with the help of TRPH standards (excluding peaks for other compounds) (https://www.restek.com/catalog/view/5980).

Thus, the TPH concentration seems lower than expected diesel oil concentration in soil (in total summation of all peaks would results higher TPH concentration).

We have revised the sentence in section 2.5.

Page 4 Line 166-169:

“The hydrocarbons (n-alkanes) within the range C₈–C₄₀ (i.e. n-octane to n-tetracontane) were quantified as TPHs [28] using an eight-point calibration curve obtained from the injection of standard TPH solution of n-alkanes (Florida TRPH Standard 500 μg/mL, Restek, USA).”

<Ref>

[28] Jeong, S.W.; Jeong, J.; Kim, J. Simple surface foam application enhances bioremediation of oil-contaminated soil in cold conditions. J. Hazard. Mater. 2015, 286, 164–170.

2) No statistical analysis is presented.

⇒ Thank you for your comment.

We have added a subtopic (section 2.6) for the statistical analysis methodology.

We have described about statistical analysis.

Page 5 Line 186-192:

“2.6. Statistical analysis

The data were entered into an Excel file (Microsoft Office Excel 2016) to calculate descriptive statistics (mean; standard deviation, SD; and standard error, SE). These data were used in one-way ANOVA analysis, and the significant difference of the mean values of the various treatments at p<0.05 were determined through the Tukey (honestly significant difference) test using OriginPro 8.5 software.”

We have performed statistical analysis elsewhere required and the manuscript is revised by replacing new Figures (Figure 3 & 4) with figure captions.

Page 7 Line 258-259:

“The different letters (a-e) above the bars indicate significant differences (p<0.05, Tukey’s test).”

Page 8 Line 291-292:

“The different letters (a-h) above the bars indicate significant differences (p<0.05, Tukey’s test).”

3) It is not obvious if Acinetobacter sp. strain K6 tolerates lower pH values (please add such information). Otherwise one might suspect that all diesel degradation was due to the action of indigenous microorganisms. Control soil was supplemented only with water, therefore no mineral nutrients were provided to support the growth of indigenous hydrocarbon degraders (which could have been stimulated by nutrients present in the bacterial solution).

⇒ Thanks for your valuable review comment.

In section 3.5, we have mentioned that with soil pH decreasing after chemical oxidation, soil microbial population (CFU/g) decreased by one order of magnitude. However, during the oxidation period, the decreased bacteria were indigenous bacteria rather than subsequently added oil-degrading K-6 bacteria. In addition, the concentration of persulfate (PS) used in this experiment is very low (50 mN) and oxidant was used with surfactant (AOS solution, pH 9.5). The pH of PS when mixed with AOS was increased from 3.49 to 4.9. Also, the pH of soil was not significantly dropped (less than 0.5 unit, see Table S4) as expected for reducing tolerating power for bacteria. Thus, the decrease in CFU after PS foam is lower than expected reduction. Besides, the soil pH was improved after subsequent addition of bioaugmentation foam (bacteria & nutrients with AOS), which makes favorable conditions for bacterial growth. Our previous study also showed that subsequent application of bioaugmentation foam (periodically) exponentially increased the CFU level even after chemical oxidation treatment (Bajagain et al., 2018). Furthermore, the indigenous bacteria are not oil-degrading as TPH was not reduced by them.

<Ref>

Bajagain, R.; Lee, S.; Jeong, S.-W. Application of persulfate-oxidation foam spraying as a bioremediation pretreatment for diesel oil-contaminated soil. Chemosphere 2018, 207, 565–572.

4) What was the initial microbial population (at the beginning of experiment, after 60 days of diesel oil contamination)? Does the number of bacterial population equal 9.0×10⁰³ cfu/g (Table S1) refer to the population of diesel-contaminated soil or uncontaminated soil? Please, clarify.

⇒ Thanks for your query.

We have described in the Table S3 (caption) as reviewer suggested.

The bacterial population (CFU/g) reported in the manuscript (Table S1) refer to the population of microorganisms in diesel-contaminated soil (after 60 days of diesel contamination, at the beginning of experiment).

5) Some of the results are presented as the mean +/- standard deviation/standard error, while others are not. Examples: Figure 4. There is lacking some bars reflecting standard deviation for treatment (PS+Bio) with holes. Figure 5. there is lacking standard deviation for control treatment. Please, revise the all manuscript in the appropriate plots, tables etc.

⇒ Thanks for your valuable comment.

In Figure 4, the standard error is already presented for every results of all treatments. Since the obtained standard error for the treatment (PS+Bio) with holes (at 30 days) were of very small value, the error bars have not been displayed.

We have revised Figure 5 by adding error bars using standard error values obtained for control treatment.

We have added error bars in Figure 5 (for control). Page 10 Line 347. Thanks

Figure 6 is also revised with error bars from the data.

Page 11 Line 375. Thanks

Table S3 and S4 are also revised as reviewer’s comment.

6) Why did the Authors use AOS as a surfactant? Why not other compounds? Did the Authors have any experience with application of this compounds as surfactant in this kind of treatments?

⇒ Thanks for your query.

This study used an anionic surfactant, sodium C₁₄-C₁₆ alpha olefin sulfonate (AOS) as a foaming agent, which is biodegradable and this surfactant is environmentally compatible. AOS surfactants are usually reported to have high biodegradation, foaming, detergency and mildness properties (https://www.stepan.com).

In addition, we have experience with application of AOS with similar studies (Jeong et al., 2015; Bajagain et al., 2018), so this surfactant was selected for current study.

<Ref>

Jeong, S.W.; Jeong, J.; Kim, J. Simple surface foam application enhances bioremediation of oil-contaminated soil in cold conditions. J. Hazard. Mater. 2015, 286, 164–170.

Bajagain, R.; Lee, S.; Jeong, S.-W. Application of persulfate-oxidation foam spraying as a bioremediation pretreatment for diesel oil-contaminated soil. Chemosphere 2018, 207, 565–572.

7) How dense was the bacterial solution used to prepare bio-foam? What was the quantities of bacterial solution used to prepare the bio-foam? What was the number of bacteria introduced to the soil?

⇒ Thanks for your comment.

The Bio-foam was prepared by using a 1:1 mixture of 0.1% AOS (surfactant) solution and bacterial solution (which was inoculated in sterile R2A liquid medium). The nutrients were added during preparation of AOS solution. The bacterial density in inoculated bacterial solution was about (7.0±1.1)E+06 CFU/mL. The number of bacteria introduced in soil were about (4.3±0.8)E+06 CFU/mL, which was present in the bio-foam.

We have revised the sentence. Page 3 Line 113-115:

“The bioaugmentation foam was generated by injecting air at the same flow rate into the mixture of bacterial solution inoculated in R2A, 0.1% AOS and inorganic nutrients (1:1 ratio of bacterial solution with the AOS solution).”

8) Line 139: The Authors provided that equal volume of biodegradation foam was sprayed every 2^nd Please, clarify what was the quantity of foam sprayed in a single application.

⇒ Thanks for your valuable comment.

To spray bioaugmentation foam, the volume of bioaugmentation solution used in each single injection (every second day, total 14 times) was 0.585 L (total volume for 28 days = 8.19 L). The foam volume at each injection was about 19.625 L. The foam was continuously sprayed such that the succeeding foam sprayed after each preceding foam was broken, until 0.585 L of solution was used within one-day.

9) How did the Authors monitor bacterial populations (lines 171-172). Did the Authors monitor the heterotrophic populations or population of hydrocarbon-degraders? Please, clarify. Please, provide the information about the used medium/media, the incubation conditions (temperature, time of incubation)? To compare the microbial population, the number of bacteria should be given in CFU/ dry weight of soil. In this manuscript, the Authors used CFU/g of soil. Please, revise it. Once again, the numbers presented in Table S3 mean that only a single enumeration was done. Maybe, these numbers are the mean values of replications.  Please, correct this table.

Þ Thanks for your valuable comment.

We have monitored the population of heterotrophic bacteria in soil during experimental period. In addition, we have added a brief description at the end of section 2.5.

We have revised and added brief description. Thanks.

Page 4 & 5 Line 181-184:

“The bacterial population (CFU/g of dry soil) was monitored throughout the experimental period by the dilution and plating method [24]. Briefly, 1 g soil sample was serially diluted in water and 100 µL of each diluted soil suspensions were sprayed over the surface of solid agar in petri plates (Agar A + nutrient, R₂A). Then the petri plates were incubated in an incubator at 28℃ for 48-72 h.”

<Ref>

Bajagain, R.; Lee, S.; Jeong, S.-W. Application of persulfate-oxidation foam spraying as a bioremediation pretreatment for diesel oil-contaminated soil. Chemosphere 2018, 207, 565–572.

The bacterial population was measured in terms of colony forming unit (CFU) per gram of dry soil.

Also, Table S3 is also corrected as reviewer suggested. Thanks

10) My other remark is about the Paragraph 3.5. The Authors described the changes of soil bacterial populations in the analyzed soil treatments. It is correct. But in my opinion, the lines 350-352: “However, there was no apparent change in the  bacterial population due to the surfactant (AOS). Separate analysis using a bacterial solution (prepared in R2A medium) has indicated that 6.1×10⁹ CFU/L and 5.2 × 10⁹ CFU/L was observed before and after adding AOS, respectively.” refer the influence of AOS on the Acintobacter K-6. In my opinion, the Authors wanted to indicate that application of AOS as a surfactant did not have a negative impact on the Acinetobacter sp.  Please, clarify. Maybe, I am wrong. But if I am right, these sentences should be mentioned in the Section Materials and Methods.

⇒ Thanks for your comment.

The application of AOS as a surfactant did not show a negative impact on the Acinetobacter sp K-6.

These sentences were mentioned in the Materials and Methods section.

We have revised as reviewer suggested. Thanks

Page 3 Line 115-118:

“It was found that the surfactant (AOS) did not show a negative impact on the population of bacteria (Acinetobacter sp K-6). An analysis using bacterial solution (prepared in R₂A medium) found that the colony forming units (CFU) were 6.1×10⁹ CFU/L and 5.2 × 10⁹ CFU/L before and after adding AOS, respectively.”

11) Two different units for biodegradation rate constant (k) were applied, day^-1 (Figure 5) and d^-1 (text). Please, revise it.

⇒ Thanks for your comment.

We have revised the unit for biodegradation rate constant (k) in Figure 5 (d^-1).

Page 10 Line 346-347:

“Figure 5. Comparison of the first order biodegradation rates (k, d^-1) as a function of soil depth.”

12) Reference 25. Acinetobacter should be in italics.

⇒ Thanks for your comment.

We have revised the reference with writing the name of bacterium in italics.

Page 13 Line 474:

“25. Chaudhary, D.K.; Bajagain, R.; Jeong, S.-W.; Kim, J. Biodegradation of diesel oil and n-alkanes (C18, C20, and C22) by a novel strain Acinetobacter sp. K-6 in unsaturated soil. Environ. Eng. Res. 2020, 25, 290–298.”

13) Please, unify the names of treatments since there are at least 3 version of each treatment. Example: Figure 1: Na2S2O8 (PS)-foam + bio-foam (with holes); Figure 3: (Ps+Bio) with holes, Figure S3: Na2S2O8+K-6 (with holes).

Þ Thanks for your comment.

As reviewer suggested, the treatment names are revised in Figures 2, S2 and S3. We have also added explanation in Figure caption.

We have revised the name of treatments to make uniformity.

Page 6 Line 210 (Figure 2) & 214:

“Control indicates the treatment of water in soil columns with holes.”

1) I recommend changing the notation of units from e.g. mg/kg to mg kg^-1.

⇒ Thanks for your valuable review comment.

We have revised the notation of units as reviewer suggested.

Please see Page 8 & 9, section 3.2 & 3.3.

2) Line 101: control

⇒ Thanks for your comment. We have revised the control treatment.

Page 3 Line 104:

“For the control treatment, deionized water from a water purification system (Human Power Corporation, Korea) was used.”

3) Line 162: What type of indicator electrode was used in this ORD electrode?

⇒ Thanks for your comment.

The ORP electrode (Part # 5779601-010B) contains Ag/AgCl as internal reference element.

<Ref>

http://www.istek.co.kr/shop/02_03_eng.php?part_idx=170

4) Line 244: should there be “(PS+Bio) w/ holes” or “(PS+Bio) with holes”?

⇒ Thanks for your query.

The notations and Figure captions are accurately placed as they are in the manuscript. In this line there is (PS+Bio) without holes (“(PS+Bio) w/o holes”) which is correct and defines for the results obtained in soil columns without using vertical holes.

5) Line 322: How were the biodegradation rates calculated? There is no information on this in the methodology.

⇒ Thanks for the valuable comment.

We have mentioned in section 3.4 that the biodegradation is assumed to follow the first-order degradation kinetics and biodegradation rates are calculated from the TPH removal results to reflect only biodegradation after the oxidation pretreatment was completed.

The biodegradation rates were calculated after the oxidation period of 2 days (from 3^rd day to 30^th day of bioaugmentation period).

The diesel degradation was supposed to follow the first order kinetics. So, the first order kinetic rate constant was calculated using eq (1)

Rate = -d[TPH]t/dt = k [TPH]t  (1)

where, [TPH]₀ and [TPH]_t are represented for TPH just after chemical oxidation (considered as initial TPH) and final TPH (at time t, after biodegradation) concentrations respectively. Also,‘t’ is the elapsed biodegradation time and k is the first order rate constant of TPH degradation kinetics.

6) Lines 350-351: It should be emphasized that it is only about quantitative changes, because only such changes can be inferred from these results.

⇒ Thanks for your comment.

We have mentioned these sentences in section 2.3.

Page 3 Line 115-118:

“It was found that the surfactant (AOS) did not show a negative impact on the population of bacteria (Acinetobacter sp K-6). An analysis using bacterial solution (prepared in R₂A medium) found that the colony forming units (CFU) were 6.1×10⁹ CFU/L and 5.2 × 10⁹ CFU/L before and after adding AOS, respectively.”

A very simple, laboratory, but quite interesting study. It opens up the prospect of further works in this field with using natural, non-artificial soils with various properties.

⇒ Thank you very much for your good compliment. We are feeling delighted to receive your positive review comment for our manuscript.