Upgrading Major Waste Streams Derived from the Biodiesel Industry and Olive Mills via Microbial Bioprocessing with Non-Conventional Yarrowia lipolytica Strains

Round 1

Reviewer 1 Report

In the reviewed original research article, Sarris et al. investigate the effect of olive mill wastewater (OMW) on the formation of citric acid, polyols and microbial lipids by two strains of the non-conventional yeast Yarrowia lipolytica. On the one hand, OMW serves as a replacement for tap water for media formulation. On the other hand, the microbial cultivations are considered to decrease the high organic loads of OMW and, therefore, use the fermentations as an initial clearing step for resource-efficient waste management. To further improve the circular bioeconomy approach, raw glycerol from biodiesel production is used as the carbon source. 

The article starts with a rather broad introduction, putting the work into a more comprehensive framework to meet sustainable development goals. The introduction is followed by a detailed material and method section. After this, the results are presented. Those consist of a total of 8 cultivations conducted in shaking flasks with extensively sound analytics and calculations of molar yields and volumetric productivities. Subsequently, the results are discussed thoroughly. Finally, the article closes with a short conclusion, stating that Y. lipolytica strains could be considered a promising biorefinery-oriented cell factory.

As a general remark, the article is professionally written, and the overall appearance is entirely satisfactory. Also, the results were subject to triplicate measurements and statistical analysis, which is not always the case in microbial studies. The work fits the journal's scope well and might be of interest to a broad readership of the journal. This concerns two aspects: 1. that OMW can serve as a replacement for process water for microbial cultivations and 2. that by this, OMW might be treated what offers potential for integration into a bio-refinery concept. Nonetheless, some questions and unclarities arose while reviewing the manuscript, which should be addressed and/or answered by the authors before the article might be accepted (minor revision). I hope my comments below are helpful and will contribute to improving the quality of the manuscript. The manuscript lacks line numbers. Hence, the specific comments will name the section and the approximate location on the respective page.

General comments:

1. Even though the English language and general appearance of the manuscript are of overall sound quality, some (careless) mistakes require attention and correction. For example, this concerns the use of double spacing and a few other small mistakes. Some will be listed exemplarily in the specific comments, but not all.

 

Specific comments:

1. Section 1 (page 1 beginning): …”‘2030 Sustainable Development’ agenda for humans’…” Check if the two apostrophes are required.

2. Section 1 (page 2 middle): it is written that “The worldwide market of polyols is projected to reach from US 26 billion in 2019, to US 34 billion by 202413.” Please rephrase.

3. Section 1 (page 2 middle): In the context of industrial polyol production, the authors say that “….requires highly pure sugars as the initiating material and high-cost chromatographic purification steps“. Please add a reference for this.

4. Section 1 (page 2 middle): Replace “under shake flask batch fermentations” with “in shake flask batch fermentations”.

5. Section 2.1. (page 3 top): It is written that raw glycerol of 85% was obtained from a company. Did the authors obtain any further analytical data, for example, about impurities or the concentration of inhibitory compounds? Please comment and complement if possible.

6. Section 2.2. (page 3 top): remove the double spacing before “..at a final concentration..”.

7. Section 2.2. (page 3 top): generally, all media seems to have been supplemented with a nitrogen source and mineral salts. Therefore, OMW seems to have only replaced the water usually required for media formulation. Would it also be possible to supplement only with some specific salts since OMW already contains certain salts or minerals? Were experiments conducted in this regard, or is this planned for the future? Please comment. 

8. Section 2.2. (page 3 middle): The abbreviation “CA” occurs, which supposedly means citric acid. Please introduce the abbreviation and add it to the Nomenclature section or replace it with Cit, as in other manuscript parts.

9. Section 2.4. (page 4 top): was the GC run in split or splitless mode? What was the injection volume? Please add these information.

10. Section 2 in general: Check if sections 2.3. and 2.5. are missing or were skipped. Amend if necessary.

11. Section 2.6. (Nomenclature): Please briefly explain how the yields for biomass and products were obtained. By calculation from two values or linear regression of several data points? 

12. Section 3 (page 6 middle): it is written that “…X formation followed an exponential pattern up to 72 h of…” How was this verified? Was it indeed still exponential growth at this time? Please comment and weaken the statement if required.

13. Section 3 (page 7 top/middle): Two double spacing before numbers or units. Please correct.

14. Section 3: Growth is qualitatively described, and biomass yields on glycerol are given. However, it would be much appreciated if the authors could also add the maximally achieved growth rate µ for the conducted experiments. This might deliver additional information on the effect of inhibitory compounds, which were present at different levels.

15. Section 4 (page 10 bottom): The authors write that “The economic viability of the whole approach could be increased since crude glycerol was utilized as the sole carbon sources.“ In this context, what is the current price of crude glycerol, and is it indeed a promising long-term substrate for industrial bioprocesses? In addition to price, what is this compound's current and assumed future market availability? Please comment and rediscuss. 

16. Section 4 (page 11 middle): It is hypothesized that nitrogen limitation caused adownregulation of ICDH, which caused citrate accumulation in the cytosol and subsequent excretion into the medium. This mechanism is well-described for oleaginous microbes. In this context, two questions arise. The first is: can it be assumed that nitrogen was indeed limiting at the observed low cell densities? Was nitrogen or ammonium measured, or can this be assumed from earlier experiments or studies? The second question is: upon accumulation of Cit inside the mitochondrion, how can the compound be released into the cytosol? Is this only possible in exchange for malate, or does Yarrowia have additional mechanisms for this? Or might Cit formation also take place in the cytosol? Please comment and amend if required.

17. Section 4 (page 11 bottom): Aspergillus niger is named as the leading producer of Cit at the industrial scale. Please add some brief information about the current benchmark values for Cit concentration, yields and volumetric productivities for this strain to enable a comparison to the values achieved by Yarrowia.

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The present work investigates the valorization of glycerol from biodiesel industry and olive mill wastewater detoxification in a coupled bioprocess. The work is well structured, well written and scientifically sound, as well as of interest for the readers of the journal. In my opinion, it can be accepted after that the following points will be addressed

·       Some reference should be added in the introduction to provide some background on the main themes reported, such as the valorization of glycerol streams (see 10.1016/j.cattod.2021.06.002) and olive mill wastewater (see 10.3390/en15030920)

·       In the introduction, the novelty should be highlighted

·       In paragraph 2.1, the characterization of the crude glycerol should be provided, since they can affect the results: is the remaining water? If this is the case, it should be commented the possible influence of real glycerol

·       In paragraph 2.4, why the total dry biomass was not estimated at 105 °C, as commonly reported in literature?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf