Bacterial community dynamics and associated genes in hydrocarbon contaminated soil during bioremediation using brewery spent grain
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Brewery spent grain (BSG) has previously been exploited in bioremediation. However, detailed knowledge of the associated bacterial community dynamics and changes in relevant metabolites and genes over time is limited. This study investigated the bioremediation of diesel contaminated soil amended with BSG. We observed complete degradation of three total petroleum hydrocarbon (TPH C10–C28) fractions in amended treatments as compared to one fraction in the unamended, natural attenuation treatments. The biodegradation rate constant ( k ) was higher in amended treatments (0.1021 k ) than in unamended (0.059 k ), and bacterial colony forming units increased significantly in amended treatments. The degradation compounds observed fitted into the elucidated diesel degradation pathways and quantitative PCR results showed that the gene copy numbers of all three associated degradation genes, alkB , catA and xylE, were significantly higher in amended treatments. High-throughput sequencing of 16S rRNA gene amplicons showed that amendment with BSG enriched autochthonous hydrocarbon degraders. Also, community shifts of the genera Acinetobacter and Pseudomonas correlated with the abundance of catabolic genes and degradation compounds observed. This study showed that these two genera are present in BSG and thus may be associated with the enhanced biodegradation observed in amended treatments. The results suggest that the combined evaluation of TPH, microbiological, metabolite and genetic analysis provides a useful holistic approach to assessing bioremediation.
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The original submission was reviewed thoroughly by two people with expertise in the field. The report from reviewer 1 was especially thorough. The authors have responded positively to every point made by both referees. More specifically, they have clarified points raised by the reviewers by adding extra text and added new references as requested. This paper is therefore now ready to be accepted for publication.
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Thank you for submitting your work for publication in Access Microbiology. From the attached files you will see that two expert referees see merit in your work and would like to see a revised version of the paper accepted for publication. However, it is essential that you first please respond fully to the recommendations made, especially the two major points made by referee 1. These are that several of the statements made require additional references; and all discussion in the Results section should please be removed and, if necessary, added to the Discussion section. As reviewer 1 has provided such a helpful report, the required changes can be completed quickly and amount to only a minor revision. I must stress, however, that a full response to all of the points must be made before the paper can be accepted for publication. If …
Thank you for submitting your work for publication in Access Microbiology. From the attached files you will see that two expert referees see merit in your work and would like to see a revised version of the paper accepted for publication. However, it is essential that you first please respond fully to the recommendations made, especially the two major points made by referee 1. These are that several of the statements made require additional references; and all discussion in the Results section should please be removed and, if necessary, added to the Discussion section. As reviewer 1 has provided such a helpful report, the required changes can be completed quickly and amount to only a minor revision. I must stress, however, that a full response to all of the points must be made before the paper can be accepted for publication. If you disagree with the referees, please explain why a recommended change has not been made.
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Comments to Author
In this study, the authors examined the bioremediation of diesel-contaminated soil amended with BSG. They monitored for degradation of petroleum hydrocarbons and for recovery of CFUs. They then determined the bacterial pathways involved in degradation of petroleum hydrocarbons and determined that the bacterial community shifts towards Acinetobacter and Pseudomonas. Overall, the authors found that amendment with BSG resulted in an improved reduction in TPH, and that the bacterial community shifts to enrich towards bacteria that are able to break down the hydrocarbons. The introduction was well-written and made the topic accessible to microbiologists outside of the bioremediation field. Some minor points to address/areas to improve: Figure 2: The scale of the y-axes should be the same in Fig 2A and 2B
Comments to Author
In this study, the authors examined the bioremediation of diesel-contaminated soil amended with BSG. They monitored for degradation of petroleum hydrocarbons and for recovery of CFUs. They then determined the bacterial pathways involved in degradation of petroleum hydrocarbons and determined that the bacterial community shifts towards Acinetobacter and Pseudomonas. Overall, the authors found that amendment with BSG resulted in an improved reduction in TPH, and that the bacterial community shifts to enrich towards bacteria that are able to break down the hydrocarbons. The introduction was well-written and made the topic accessible to microbiologists outside of the bioremediation field. Some minor points to address/areas to improve: Figure 2: The scale of the y-axes should be the same in Fig 2A and 2B What is the cause of the sudden increase in CFUs of hydrocarbon degrading bacteria at Day 5 in the control soil? Figure 3: The scale of the y-axes should be the same in Fig 3A-C In Fig. 3, are gene copy numbers in the soil+diesel and soil+diesel+BSG shown relative to gene copy numbers in control soil? If so, this should be made clear. If not, how do they compare to the gene copy numbers of the control soil? Does diesel impact the recovery of genomic DNA from the soil?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
General comments This paper presents an interesting study dealing with the enhanced degradation of total petroleum hydrocarbons through the addition of brewery spent grain. This is of interest to researchers in the field of environmental bioremediation. The results are clear and convincing. However, in some cases the results were also discussed in the results section, which is not appropriate. In addition, a number of claims lack supporting references. I recommend moving all discussions to the "Discussion" section unless the authors want to combine both sections. Specific comments that address these and other changes are provided below. Introduction Line 82-84: "The efficiency…degradation pathways." Add one or two more refences after this sentence. One relevant reference is the recent study:
Comments to Author
General comments This paper presents an interesting study dealing with the enhanced degradation of total petroleum hydrocarbons through the addition of brewery spent grain. This is of interest to researchers in the field of environmental bioremediation. The results are clear and convincing. However, in some cases the results were also discussed in the results section, which is not appropriate. In addition, a number of claims lack supporting references. I recommend moving all discussions to the "Discussion" section unless the authors want to combine both sections. Specific comments that address these and other changes are provided below. Introduction Line 82-84: "The efficiency…degradation pathways." Add one or two more refences after this sentence. One relevant reference is the recent study: https://www.nature.com/articles/s43247-022-00526-2 Materials and Methods Line 140: Add the word "in" between the words "used" and "this". Line 171-175: How many times was extraction performed per sample? Granted, the effectiveness of n-hexane over other solvents such as DCM for the extraction of diesel fuel hydrocarbons was clearly demonstrated in the study http://dx.doi.org/10.1039/C9RA10919F. However, using non-standardized instruments like shakers (without sonication) once may not lead to a complete extraction of residual diesel fuel. The standard methods of extraction include Soxhlet extraction (USEPA Method 3540C), ultrasonic extraction (USEPA Method 3550C), accelerated solvent extraction (ASE) and microwave-based extraction method. Line 186-187: Use the formula function in MS Word to express %TPH reduction in order to avoid any ambiguity associated with subtraction, division and multiplication here. Line 188-190: "The concentration…in the samples." How? Did you make use of an internal standard that have the same chemical characteristics as your target analyte? Or did you prepare calibration curves for all target compounds and then determined the concentration of each analyte using the parameters of its calibration curve? Please provide more details of the approach you used. Results The results are clearly presented. In some cases, some of the results were also discussed in this section, which is not appropriate, unless the authors decide to combine "results and discussion". I recommend moving discussions to the "Discussion" section. See specific comments below. Line 325-333: Move this part to "Discussion" section and provide sufficient references as mentioned below. Line 328-329: "The degradation pathways…to catechol". Provide supporting references from published articles. For example, a detailed analysis of these pathways including the mechanisms leading to catechol as well as meta/ortho-cleavages of catechol can be found in doi.org/10.3390/genes12010098 and doi.org/10.1007/BF00186968. Line 335-337: "Aldehydes being…pathways of aromatics." Provide supporting references. Line 341-342: "However, carboxylic acids…resulting from aldehyde oxidation," Provide supporting references just after the word "oxidation" and before the continuation of the sentence.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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