Biodegradation of Olive Mill Effluent by White-Rot Fungi
Round 1
Reviewer 1 Report
This paper deals with interesting and important question – biological treatment of olive mill water – focusing on waste from the two-phase extraction process.
The authors choose the white rot fungus Phanerochaete chrysosporium, known for its ability to degrade recalcitrant compounds. The introduction section is comprehensive and describes well the need for treatment.
Looking at the results I think that the authors have to be more careful with the conclusion that: "The biotreatment with the white-rot fungus Phanerochaete chrysosporium is useful to degrade AL waste".
Few Comments:
The differences between the non-inoculated control and the treatment are minor, the authors must conduct better statistical analyses to demonstrate id differences exist.
The authors did not follow any activity of the fungus: biomass accumulation, enzymatic activity (carbohydrate degradation, phenols oxidation, toxicity at the end of the experiment. It could be helpful if the bacterial community development was followed in both treatment and control.
The optimum pH and temperature range for enzyme production and growth by the fungus is around 4.2 and 32 -34 °C and higher. The authors choose 26°C, thus the conditions were probably not optimal for the fungus. (for example: Couto, S.R., Moldes, D. & Sanromán, M.A. Optimum stability conditions of pH and temperature for ligninase and manganese-dependent peroxidase from Phanerochaete chrysosporium. Application to in vitro decolorization of Poly R-478 by MnP. World J Microbiol Biotechnol 22, 607–612 (2006). https://doi.org/10.1007/s11274-005-9078-0
Author Response
Response to Reviewer 1 Comments
This paper deals with interesting and important question – biological treatment of olive mill water – focusing on waste from the two-phase extraction process.
The authors choose the white rot fungus Phanerochaete chrysosporium, known for its ability to degrade recalcitrant compounds. The introduction section is comprehensive and describes well the need for treatment.
Looking at the results I think that the authors have to be more careful with the conclusion that: "The biotreatment with the white-rot fungus Phanerochaete chrysosporium is useful to degrade AL waste".
Point 1: The differences between the non-inoculated control and the treatment are minor, the authors must conduct better statistical analyses to demonstrate id differences exist.
Response 1: Please, see Figure 1. Dashed lines shown the data for the non-inoculated test C1 and C2, used as controls. The solid lines shown the inoculated tests E1 and E2. C1 must be compared with E1 and, C2 must be compared with C2. As you can see, differences are not minor, as commented in the text.
Point 2: The authors did not follow any activity of the fungus: biomass accumulation, enzymatic activity (carbohydrate degradation, phenols oxidation, toxicity at the end of the experiment. It could be helpful if the bacterial community development was followed in both treatment and control
Response 2: Although the enzymatic activity of the fungus has not been measured in this work, the evolution of main parameters that indicate the effect of fungus inoculation were followed. In addition to sCOD, sBOD5 and colour, the evolution of the concentration of reducing carbohydrates and phenolics compounds are shown in Figure 3 and Figure 4, respectively.
Regarding biomass accumulation, to estimate the microorganisms growth, the total solids of the AL were measured at the initial and final times control and fungal treatments. The increase in the amount of solids was associated with the growth of microorganisms. In the case of the control tests (non-inoculated), these data were related to the growth of the endogenous microbiota of AL, while for the inoculated tests (E1 and E2) this increase was related to the growth of the endogenous microbiota and P. chrysosporium fungus. This information has been added in Table 2 and commented in the text (see Pag 6, line 210-222).
Point 3: The optimum pH and temperature range for enzyme production and growth by the fungus is around 4.2 and 32 -34 °C and higher. The authors choose 26°C, thus the conditions were probably not optimal for the fungus. (for example: Couto, S.R., Moldes, D. & Sanromán, M.A. Optimum stability conditions of pH and temperature for ligninase and manganese-dependent peroxidase from Phanerochaete chrysosporium. Application to in vitro decolorization of Poly R-478 by MnP. World J Microbiol Biotechnol 22, 607–612 (2006). https://doi.org/10.1007/s11274-005-9078-0
Response 3: A temperature of 26ºC was used for the recovery and growth of the freeze-dried strain CECT 2798, following the technical instructions provided by the supplier (Spanish Collection of Type Cultures) of the strain. Besides, the operating conditions of pH (6.00) and temperature (26ºC) used for the fungal treatment of AL are in agreement with previous works that treated similar effluents. These investigations were performed at temperatures between 25-30ºC and pH 5-6. See for example:
- Ntougias, S., Baldrian, P., Ehaliotis, C., Nerud, F., Antoniou, T., Merhautová, V., Zervakis, G.I., 2012. Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus. Chemosphere 88, 620–626. https://doi.org/10.1016/J.CHEMOSPHERE.2012.03.042
- Zerva, A., Zervakis, G.I., Christakopoulos, P., Topakas, E., 2017. Degradation of olive mill wastewater by the induced extracellular ligninolytic enzymes of two wood-rot fungi. J. Environ. Manage. 203, 791–798. https://doi.org/10.1016/J.JENVMAN.2016.02.042
Furthermore, it must be taken into account that the use of higher temperatures for the treatment would not be convenient from a practical point of view because the operating cost of the process would be considerably increased.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Recalcitrant components of lignocellulosic waste resulted in low rate direct biodegradability. Lignocellulosic enzymes from white-rot fungi can provide possible solutions for this problem. Authors focused on the application of Phanerochaete chrysosporium for enhanced biodegradability of olive industry waste investigating of the total phenolic compound degradation; COD and colour removal efficiency. Therefore, the topic of manuscript can be considered as interesting for the readers. The manuscript is generally well structured. Introduction section summarizes well the background of the research and research motivations, it reflects well to the gap in knowledge. Applied analytical methods are adequate to the sample characteristics. Materials and methods are given clearly and in details. The manuscript contains valuable results and establishments. Experimental results are discussed with relevant references.
Comments, suggestions:
Please give the standard deviation for the data presented in Table 1 and figures.
Authors used the soluble COD to calculate the biodegradability index, but not provide the sCOD values.
How ‘fit’ the temperature of effluent to the optimum temperature of fungal treatments?
I suggest to discuss the efficiency of white-rot fungus treatment vs. conventional treatments in more details (in discussion parts).
Author Response
Response to Reviewer 2 Comments
Recalcitrant components of lignocellulosic waste resulted in low rate direct biodegradability. Lignocellulosic enzymes from white-rot fungi can provide possible solutions for this problem. Authors focused on the application of Phanerochaete chrysosporium for enhanced biodegradability of olive industry waste investigating of the total phenolic compound degradation; COD and colour removal efficiency. Therefore, the topic of manuscript can be considered as interesting for the readers. The manuscript is generally well structured. Introduction section summarizes well the background of the research and research motivations, it reflects well to the gap in knowledge. Applied analytical methods are adequate to the sample characteristics. Materials and methods are given clearly and in details. The manuscript contains valuable results and establishments. Experimental results are discussed with relevant references.
Point 1: Please give the standard deviation for the data presented in Table 1 and figures.
Response 1: The standard deviation for the data presented in Table 1 has been added. In the case of the figures, the percentages of the standard deviation with respect to the mean values have been provided in the figure legend to facilitate the visualization of the data in the graph.
Point 2: Authors used the soluble COD to calculate the biodegradability index, but not provide the sCOD values.
Response 2: In this work, only sCOD values were measured. These data are shown in Figure 1. According to the reviewer's comment, the manuscript has been revised and modified to clarify this misunderstanding. Please, see the following sections: Material and Methods (Page 4, lines 145-151), Results and Discussion (Section 3.1, Pages 5-7, lines 182-273). In addition, Figures 1 and 2, as well as their respective legends, have been corrected. Please see Page 5, lines 182-187 and Page 6, lines 233-238.
Point 3: How ‘fit’ the temperature of effluent to the optimum temperature of fungal treatments?
Response 3: The fungal treatment was performed at 26ºC taking into account the technical instructions provided for the Spanish Type Culture Collection for an optimal recovery and growth of the freeze-dried strain. In addition, the operational conditions used in previous works focused on the treatment of olive mill wastewaters and similar effluents were considered. These investigations were performed at temperatures between 25-30ºC. See for example:
[1] A. Zerva, G.I. Zervakis, P. Christakopoulos, E. Topakas, Degradation of olive mill wastewater by the induced extracellular ligninolytic enzymes of two wood-rot fungi, J. Environ. Manage. 203 (2017) 791–798. https://doi.org/10.1016/J.JENVMAN.2016.02.042.
[2] S. Ntougias, P. Baldrian, C. Ehaliotis, F. Nerud, T. Antoniou, V. Merhautová, G.I. Zervakis, Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus, Chemosphere. 88 (2012) 620–626. https://doi.org/10.1016/J.CHEMOSPHERE.2012.03.042.
[3] L. Hu, G. Zeng, G. Chen, H. Dong, Y. Liu, J. Wan, A. Chen, Z. Guo, M. Yan, H. Wu, Z. Yu, Treatment of landfill leachate using immobilized Phanerochaete chrysosporium loaded with nitrogen-doped TiO 2 nanoparticles, J. Hazard. Mater. 301 (2016) 106–118. https://doi.org/10.1016/j.jhazmat.2015.08.060.
[4] F.E. Ergül, S. Sargın, G. Öngen, F.V. Sukan, Dephenolisation of olive mill wastewater using adapted Trametes versicolor, Int. Biodeterior. Biodegradation. 63 (2009) 1–6. https://doi.org/10.1016/J.IBIOD.2008.01.018.
Point 4: I suggest to discuss the efficiency of white-rot fungus treatment vs. conventional treatments in more details (in discussion parts).
Response 4: New information has been added in the discussion section considering the editor's comment. Please see Page 9, line 337-347.
Author Response File: Author Response.docx
Reviewer 3 Report
Very interesting project relating to the study of the application of fungi in the preliminary treatment of wastes from the olive oil industry.
Overall, the paper is well written and contains important information about the subject addressed gathering information from a large number of relevant bibliographic sources. The analysis is clearly presented and all the sections (including the Discussion and Conclusion sections) are rather interesting and seem to be quite thorough. In my opinion the paper is essentially ready for publication.
Author Response
Dear reviewer,
Thank you for considering our work suitable and ready for its publication in Applied Sciences Journal.
Best regards,
Prof. Dra. Adriana Laca
Lecturer
Department of Chemical and Environmental Engineering
University of Oviedo
Round 2
Reviewer 1 Report
The authors responded to some comments.
I still think that the conditions were not optimal and the 2 references given by the authors deal with different fungi, thus different conditions.
