Control Efficiency of Biochar Loaded with Bacillus subtilis Tpb55 against Tobacco Black Shank

Round 1

Reviewer 1 Report

1. During the pyrolysis process which inert gas was used?

2. Why did the authors stop at 500^oC? In literature, one can find information that the best biochar is produced at 800^oC for full pyrolysis of the material.

3. The Celsius degree symbol sometimes is underlined - remove it.

4. Did authors prepare seeds of tobacco in any way like sterilization in chloramine etc?

5. How is it possible that BC300 has more %C than BC400 and BC500 ?

6. Authors did not show the quantification of the matrix components method - only other results

7. In table 3 The AN and Total nitrogen in TB300 sample are highlighted in yellow

8. Authors should provide information regarding pH of the soils before and after treatment as it is an important factor for the growth of plants.

9. What is the mechanism of cell immobilization in this particular phenomena

10. Authors can cite work which shows different possibilities for the use of immobilized microorganisms in bioremediation processes: https://doi.org/10.1016/j.colsurfa.2021.127735

Author Response

1. During the pyrolysis process which inert gas was used?

 

Biochar production was conducted by slow pyrolysis of the tamarisk powder in a muffle furnace at 300 ^oC (TB300), 400 ^oC (TB 400), and 500 ^oC (TB 500) for 2 h under a N² flow of 500 mL•min⁻¹

2. Why did the authors stop at 500^oC? In literature, one can find information that the best biochar is produced at 800^oC for full pyrolysis of the material.

Thank you for your useful comments. The pyrolysis temperature has a great influence on the production and characteristics of biochars that are suitable as soil fertilizer.  Zheng et al. investigated the effect of pyrolysis temperature on properties and nutrient valuesof biochars produced from giant reed (Arundo donax L.) at 300–600 °C. The results showed that low-temperatures (⩽ 400 °C) may be optimal for producing biochar from giant reed to improve the nutrient availability and soil fertility[1]. Zhang et al. and Meng et al. found that the elevation in pyrolytic temperature resulted in a notably lowering in the biochars yield produced from sewage sludge and aerobically composted swine manure. The high temperature (700 °C) also reduced the number of micropores, and decreased the specific surface area[2,3]. Therefore, the effect of pyrolysis temperature on the production and characteristics of biochars exhibited a significant correlation with the raw materials for producing biochar.

1. Zheng, H.; Wang, Z.; Deng, X.; Zhao, J.; Luo, Y.; Novak, J.; Xing, B. Characteristics and nutrient values of biochars produced from giant reed at different temperatures. Bioresource Technol. 2013, 130, 463-471.
2. Zhang, X.; Zhao, B.; Liu, H.; Zhao, Y.; Li, L. Effects of pyrolysis temperature on biochar’s characteristics and speciation and environmental risks of heavy metals in sewage sludge biochars. Technol. Innov. 2022, 26, 102288.
3. Meng, J.; Wang, L.; Liu, X.; Wu, J.; Brookes, P. C.; Xu, J. Physicochemical properties of biochar produced from aerobically composted swine manure and its potential use as an environmental amendment. Bioresource Technol. 2013, 142, 641-646.
4. The Celsius degree symbol sometimes is underlined - remove it.

Thanks. The underlined celsius degree symbol has been revised.

4. Did authors prepare seeds of tobacco in any way like sterilization in chloramine etc?

Thank you for your kind suggestions. The susceptible tobacco seeds (Zhongchuan 208) were surface-sterilized with 10% NaClO for 5 min, and then immediately rinsed with double distilled water three times.

5. How is it possible that BC300 has more %C than BC400 and BC500?

We appreciate this observation. We feel so sorry for the mistakes. The %C data have been modified.

6. Authors did not show the quantification of the matrix components method - only other results

Thank you for your useful comments.

The biofilm was harvested and suspended in 1 mL ultrapure water through ultrasonic oscillation. The supernatant solution was acquired from the centrifuged biofilm suspension (10 000 rpm, 2 minutes), and then used to quantify the total protein and polysaccharide content in EPS through Bradford assay [25] and phenol-sulphuric acid method [26], respectively. Amount of the polysaccharide and protein were both determined directly through the standard curve (Figure S1).

 

7. In table 3 The AN and Total nitrogen in TB300 sample are highlighted in yellow.

We appreciate this observation. The highlights in yellow have been deleted.

8. Authors should provide information regarding pH of the soils before and after treatment as it is an important factor for the growth of plants.

Thanks for your good recommendations. The results regarding pH of the soils before and after treatment could be found in Figure 3 c.

9. What is the mechanism of cell immobilization in this particular phenomena

Thanks for your useful comments.

1. The porous structure and huge surface area of biochar provides a suitable habitat for microbial colonization and proliferation [1,2].
2. Biochars increase the concentrations of extracellular polymeric substances (EPS), which enhanced the biofilm formation. the bacterial cells appeared as cell aggregates, or biofilms, was adhered by and immobilized in the biochar surface. In addition, the surface hydrophobicity (CSH) of cells, as hydrophobic bacteria are favorably attached to abiotic/hydrophobic surfaces [3].
3. Zhang, H.; Tang, J.; Wang, L.; Liu, J.; Gurav, R.G.; Sun, K. A novel bioremediation strategy for petroleum hydrocarbon pollutants using salt tolerant Corynebacterium variabile HRJ4 and biochar. Environ. Sci. China 2016, 47, 7-13
4. Kwak, J.H.; Islam, M.S.; Wang, S.; Messele, S.A.; Naeth, M.A.; El-Din, M.G.; Chang, S.X. Biochar properties and lead(II) adsorption capacity depend on feedstock type, pyrolysis temperature, and steam activation. Chemosphere 2019, 231, 393-404
5. Xiong, B.; Zhang,Y.; Hou,Y.; Arp, H.P.H.; Reid, B.J.; Cai, C. Enhanced biodegradation of PAHs in historically contaminated soil by Gilvum inoculated biochar. Chemosphere 2017, 182, 316-324
6. Authors can cite work which shows different possibilities for the use of immobilized microorganisms in bioremediation processes: https://doi.org/10.1016/j.colsurfa.2021.127735

Thank you for your kind suggestions. The literature reference [54] has been added.

Author Response File: Author Response.docx

Reviewer 2 Report

Study has been presented very well

kindly check the script for minor spelling and grammatical mistakes

Comments for author File: Comments.pdf

Author Response

Study has been presented very well kindly check the script for minor spelling and grammatical mistakes

R: Thanks for your comments. We have thoroughly checked and corrected the grammatical errors and typos we found in our revised manuscript.

Reviewer 3 Report

I have deeply reveiwed the manuscript "Control Efficiency of biochar loaded with Bacillus subtilis Tpb55 against tobacco black shank", authors have niecely briefed their findings and concluded that biochar with biocontrol agents (Tpb55) could be a promising synergism for Tobacco Black shank. However, this is not for the first time someone described in a study, many studies have already investigated this type of synergism. Therefore, I suggest additional experiments and mechanisms for novelty evaluations and scientific soundness.

 

    

Author Response

I have deeply reviewed the manuscript "Control Efficiency of biochar loaded with Bacillus subtilis Tpb55 against tobacco black shank", authors have nicely briefed their findings and concluded that biochar with biocontrol agents (Tpb55) could be a promising synergism for Tobacco Black shank. However, this is not for the first time someone described in a study, many studies have already investigated this type of synergism. Therefore, I suggest additional experiments and mechanisms for novelty evaluations and scientific soundness.

R: We gratefully appreciate for your valuable suggestions. Many researchers investigated the application potential of biochar with a special focus on the synergistic and antagonistic effects on contaminant-degrading microorganisms in single and mixed-contaminated systems. Biochar amendment is acknowledged to favor plant resistance against oomycete pathogen in Phytophthora family [20,48], but the synery between biochar and antagonist bacteria against P. nicotianae is limitedly reported. Even though the synergistic effect of biochar combined with PGPR has been studied, there is scare data for the shelf life and EPS production of Bacillus subtilis Tpb55 with tamarisk biochar antagonist oomycete pathogens. Biochar promotes plants to recruit beneficial bacteria to the rhizosphere, and thus develop a disease suppressive rhizosphere microbiome. Therefore, future studies analyzing the composition of tobacco root exudates may improve our understanding of the detailed role of the plant in the biochar‐induced building up of soil suppressiveness against P. nicotianae.

Round 2

Reviewer 1 Report

Everything seems fine for me now.

Reviewer 3 Report

Authors have satisfactorily briefed the previously evaluated questions and the manuscript is much improved, no additional modifications are required, thus I recommend for possible publication.