Production of Citric Acid by Aspergillus niger Cultivated in Olive Mill Wastewater Using a Two-Stage Packed Column Bioreactor

Round 1

Reviewer 1 Report

OMWs pose a serious problem in the Mediterranean Base, even though three-phase decanter olive mills tend to be replaced by two-phase systems which do not produce watewaters. This is an interesting m/s regarding OMWs valorization by a two-stage Packed Column Bioreactor.

Nevertheless it cannot be accepted prior revisions listed below (regarding text/spelling, additon of further information and addition of extra literature):

- In line 21: please replace "(YCit.A/BOD) of 38.5% and a productivity of 2.5 g/L/day", with (YCit.A/BOD) of 38.5% and a productivity of 2.5 g/L/day).

- In line 62: please cite the reference with the appropriate journal style.

- Lines 76-78: the authors should clarify whether the substrate (OMW) was used in the bioreactors crude (without any ohter prior actions) or after centrifugation/filtration to remove unwanted solids.

- The authors should clarify whether biomass was attached in loofa sponge. If yes, was the attached biomass been harvested? In which way? If it was not harvested the results regaring dry biomass are not safe at all.

- Even though interesting work is presented regarding OMWs valorisation, the authours should include a paragraph regarding OMW treatment. If measures have been performed regarding treatment (i.e. reduction of colour or/and total phenol concentration of the effluent) methods and results should be depicted. If not, it should be clarified that besides added-value compounds production (such as citric acid) by agro-industrial wastes (such as OMWs) it is really important to monitor the effluent of the process. We managed to use a waste stream as substrate (and recover i.e. citric acid) but what about the effluent of our process? Is this effluent safe to be disposed to the environment after our process?

- This m/s needs t obe fortified with extra literature. In line 65: please add the references listed below:

1. Tzirita, M. et al. (2019). Effect of salt addition upon the
production of metabolic compounds by Yarrowia lipolytica cultivated on biodiesel-derived glycerol diluted with olive-mill wastewaters.
Energies, 12(19), 3649.
2. Sarris, D. et al. (2017). Production of added‐value metabolites by
Yarrowia lipolytica growing in olive mill wastewater‐based media under aseptic and non‐aseptic conditions. Engineering in life sciences, 17(6), 695-709.

3. Sarris, D. et al. (2014). Aerated vs non-aerated conversions of
molasses and olive mill wastewaters blends into bioethanol by
Saccharomyces cerevisiae under non-aseptic conditions. Industrial Crops and Products, 56, 83-93.

Author Response

Dear Reviewer,

Thank you for  your time spent in evaluating our manuscript "Production of citric acid by Aspergillus niger cultivated on olive mill wastewater using a two-stage packed column bioreactor'. Attached is my response to your comments. Thank you 

 

Regards

 

Corresponding author

Muhannad I. Massadeh

 

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, Massadeh and co-workers reported the production of citric acid by Aspergillus niger cultivated in olive mill wastewater using two-stage packed column bioreactor. Main strengthens of this study are application of wastewater as substrate (olive mill wastewater (OMW) as well as continued technology using two-stage packed column bioreactor with entrapped Aspergillus niger cells. Despite the experiments in the work were well done, more additional experiments are needed such as optimisation of supplementation of cellulose and glucose, fermentation time etc., quality of OWM. The discussion also needs more attention because quality of this part is very low. Authors only referred the results from other studies without discussion of these. For instance, Authors should suggest some mechanisms to explain that why in the entrapped form, high production yield of citric acid was detected etc. Method for entrapment of mould cells was missing.

Author Response

Dear Reviewer,

Thank you for your time spent in evaluating our manuscript 'Production of citric acid by Aspergillus niger cultivated in olive mill wastewater using a two-stage packed column bioreactor'. Attached is our response to your valuable comments. Thank you

 

Regards

 

Corresponding author

Muhannad I. Massadeh

Author Response File: Author Response.pdf

Reviewer 3 Report

this manuscript describes production of citric acid by using a. niger and olive mill wastewater using a two-stage packed column bioreactor.

although the finding itself is beneficial

this manuscript has several problems

for example, indication of figures is not specific

fig2 a or b and so on

detailed method description is required

for example size of the reactor, loofa sponge and so on

a full  discussion by comparing with preceding researches on olive wastewater and a. niger should be described

Author Response

Dear Reviewer 3,

Thank you for your time spent in evaluating our manuscript " Production of citric acid by Aspergillus niger cultivated in olive mill wastewater using a two-stage packed column bioreactor". Attached is our response to your valuable comments. Thank you

 

Regards

 

Corresponding author

Muhannad I. Massadeh

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thnak you for taking into consideration the comments.

Author Response

Thank you for your valuable time

Reviewer 2 Report

Authors did some corrections in the revised version. The discussion was improved but it was still not enough. Some main points raised in previous comments are still untouch such as quality of OMW (Authors reffered to one published in 2008 and since then, 14 years were counted), method for immobilisation of A. niger cells, optimisation, statistical methods for data processing etc.  How did Authors adjuct the spore suspenssion to be 10^6 spores/mL?   

Author Response

Dear Respected reviewer,

Thank you for your time and comments. We have revised our manuscript. To some extent, the discussion was improved. Regarding the quality of OMW, as previously mentioned that we have used 50% diluted OMW collected from a 3 phase mill. We have conducted our experiments based on previous findings (please see reference 20, 21, and 23). The characteristics of our OMW samples were already published and we cannot reproduce the data (Reference 20 and 21). We have contacted Water Quality Center in our campus (The Hashemite University) for possible analysis of new OMW samples and accompanied our published work. I have been informed that the OMW characteristics of newly collected samples are within the range of our own data. However, we have improved the section of olive mill wastewater in the material and methods section (subheading 2.1). 

• Regarding the method of immobilization: the pre-inoculated medium was loaded into the packed reator at a subsequent doses to prevent close packing of  the sponge slices. After the germination stage, the cells/mycelia were entrapped in the lattice  matrix of the sponge slices as observed throughout sampling. The writing of this section was improved.
• Regarding optimization: as we have mentioned in our previous response, we have not performed full optimization procedures but in a preliminary experiments, we found that glucose presence showed inhibitory effects on cellulase production and citric acid formation in a fermentation medium of OMW. Increasing glucose or cellulose concentration did not enhance acid production. The parameters employed in our study i.e. temperature, airflowrate, pH,  etc. are based on previous optimization procedures (Reference 4 and 34). Papanikolaou et al. (2008) claimed that the reason behind enhanced citric acid production by Y. lipolytica yeast and the kinetics of the process in  general was the OMW presence (30% v/v) but not the high glucose concentration which is in agreement with our findings. Furthermore, supplementing the cultures with N sources or minerals did not affect the process significantly (References 4). Therefore, we chose to conduct the experiments according to these observations and findings. 
• Statistical analysis: all the values in Tables and figures represent the mean+/- SE. 
• spore suspension: adjustment to 10^6 was performed by counting the no. of spores using a hemocytometer. The writing of  this section was improved (2.2).  

Reviewer 3 Report

the manuscript is now improved

but it still has many problems

 

results should be divided into several sections and each should have short titles

Author Response

Dear Respected reiewer,

Thank you for your time. We have revised our manuscript again according to reviewers comments and added a new subheading:

3.2. Citric acid production by entrapped A. niger in a single stage packed column bioreactor. 

Thank you 

Corresponding author