Study of the Effect of Endemic Microorganisms from a Copper Deposit on the Efficiency of Sulfuric Acid Leaching

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Manuscript is well written with vast methods description, which often seems to be chaotic and perspective reader may be lost in the first two paragraphs.

Response 1: Thank you for pointing this out. We agree with this comment. This difficulty arises because of the interdisciplinary connection of this article. Since bio-leaching affects the fields of microbiology, chemistry and metallurgy. In the process of writing the article, we tried not to include unnecessary details whenever possible.

Comments 2: Large heaps generate internal temperature increase, was this examined on the bacterial life?

Response 2: Agree. I/We have, accordingly, done/revised/changed/modified…..to emphasize this point The tests themselves were conducted in columns simulating heap leaching at room temperature. In practice, for northern regions with cold winters, it is known that during heap leaching in large dumps, the temperature is in the range of 3-5 degrees Celsius in winter. This is achieved through heating systems for process pipelines. For bacteria, such a temperature can significantly slow down their metabolism, but not lead to their complete death. Also, for the northern regions, it is recommended to start the stage of biological oxidation of the heap in the spring, because the optimal temperature for the growth of most crops is 20-25 degrees Celsius. To date, some development of prototypes of special wells is also underway in some parts on the surface of the heaps, which to some extent represent the implementation of borehole leaching on the surface of the heap. The point is to use specially equipped pipes with a thermal cable in some areas, which will ensure that the temperature is maintained in some areas of the heap layers, while the irrigation system is functioning. This will ensure the stable vital activity of bacteria in the thickness of the heap even at low ambient temperatures.

Comments 3: How the aeration of the heap was performed?

Response 3: Bio-oxidation and heap leaching does not involve special equipment. The oxygen in the air enters the ore layers naturally. If the leaching is carried out by the vat method, then air injection by a compressor is possible. However, as in the previous question, partially equipping some sections of the heap with perforated pipes in wells can provide not only a favorable temperature regime for bio-oxidation, but also create the possibility of aeration (systematic injection of air with a compressor). But this equipment is still at the stage of development and prototypes.

Comments 4: How the proper pH was kept to sustain high bacterial activity?

Response 4: According to the recommendations of microbiologists, the optimal pH level for bacterial growth is in the range of 1.8-2.2. To achieve this range, the ore is first treated with a weak solution of sulfuric acid (no more than 5 g /l). After the drains of the solution show stable pH values in the range of 1.8-2.2 after several circulations for 3-5 days, the supply of the bacterial solution begins.

Comments 5: Was there observed negative impact of iron content (1.5%)?

Response 5: This iron content in the range of 1-2% is quite acceptable and will serve as a source of Fe³⁺ during oxidation during the vital activity of iron-oxidizing bacteria. In practice, there are hydrometallurgical industries with ore containing much more iron. The main problem that most often occurs there is the accumulation of excess iron in cycled  solutions (PLS, raffinate, electrolyte), with a sufficiently long production process (about 3 years or more).

Comments 6: Methinks problem of elemental sulfur and / or jarosite formation was neglected.

Response 6: The solution to the problem of the formation of elemental sulfur is solved by a combination of microorganisms, which also includes sulfur-oxidizing bacteria. Experiments have shown that it is precisely the use of a complex of bacteria that allows achieving the most effective results than, for example, the separate use of iron-oxidizing bacteria. The problem of jarosite formation also exists in functioning hydrometallurgical copper plants. The resulting fragments of jarosite during the decomposition of iron sulfides are a precipitate capable of accumulating in an organic extractant solution upon contact with a productive solution. This leads to the accumulation of interfacial suspension at the stages of copper extraction. This problem is usually solved mechanically - by filtration of the solution before extraction and systematic purification the organic phase.

Comments 7: Please specify what is the increase in copper recovery? Authors claimed 4-5% while in abstract is value 5.4%?

Response 7: Recovery of copper on one of the samples using biological oxidation increased by 5.4% compared to conventional sulfuric acid leaching, while there was no increase in recovery on a more complex sample. However, the main effect of using biological oxidation was to reduce the consumption of sulfuric acid with relatively uniform recovery.

Comments 7a: What is referential value of copper recovery in sulphuric acid leaching. Authors mention only increment caused by bacteria?

Response 7a: For a relatively simple ore sample, copper recovery by conventional sulfuric acid leaching was 52.87%, and for a complex ore sample it was 47.69%. With the preliminary biological oxidation of the first sample of ore of a simple composition, the recovery increased to 58.31%. There was no extraction growth in the second complex ore sample. However, in both ore samples, a decrease in sulfuric acid consumption was observed when using biological oxidation. This effect is relevant for ore deposits of mixed complex composition with a high degree of sulfuric acid absorption. In fact, the main effect of biological oxidation for such copper ores is to increase the efficiency of the chemical work of sulfuric acid, which leads to a decrease in its consumption.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Role of Non-Chemolithotrophic Microorganisms

Response 1: Yes, we agree that the role of these bacteria is not the main one in the process of biological oxidation. The discovered effect was observed precisely when using a complete bacterial complex. By itself, the culture of Skermanella aerolata and microfungi are not able to have an effect on the biological decomposition of minerals. However, their combination with iron- and sulfur-oxidizing bacteria contributed to more efficient oxidation than each culture individually. The idea of the article is not to state the main role of Skermanella aerolata and microfungi in the oxidative process. The main goal was to determine which microorganisms are found during the development of endemic iron- and sulfur-oxidizing bacteria selected from ore deposits.

Comments 2: Microbial Interactions

Response 2: Yes, we agree, a deeper study of the microbiological consortium will certainly be useful. Such results may be of the nature of biological research. This task will be assigned to microbiologists, and they will be able to refer to this article. The presented article discusses the results of copper hydrometallurgy, in particular, the effect of reducing sulfuric acid consumption using pre-biological oxidation.

Comments 3: When bacterial species are mentioned for the first time in the manuscript, the full name should be written (e.g., Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans). In subsequent mentions, the genus name should be abbreviated, while species name should be consistently formatted in lowercase and italic (e.g., A. ferrooxidans, A. thiooxidans). It should be corrected throughout the manuscript

Response 3: Abbreviations of microorganisms have been corrected.

Comments 4:

Study of the Material Composition of Ore Raw Materials

-The first paragraph appears to describe the objective of the study; therefore, it would be

more appropriate to move it to the Introduction section or another relevant part of the

manuscript.

- In Table 1 and Table 2, the authors should specify whether the ore samples are oxidized

or sulfide in nature.

- Table 3: The terms “absolute” and “relative” distribution of copper require clarification.

Units for absolute distribution must be specified. 

-  The statement “This is because the sulfide lattice strongly binds copper…” requires a

more detailed and scientifically grounded explanation.

Response 4: - This paragraph has been moved to the end of the introduction.

- «According to the technical data provided by the geological service, copper ore samples from two different deposit sites are sulfide in nature. However, before the start of the experiments, the composition of the ore samples presented was additionally determined» This text was added before Tables 1 and 2.

-« The content of copper forms in the table is expressed as absolute and relative. The absolute value reflects the content of a specific form of copper in the ore, and the relative value shows how much of this form of copper is from its total content» This text was added before Table 3. The table itself shows the unit of measurement – percentages %.

 - «Copper sulfide minerals such as chalcopyrite (CuFeS) are difficult to decompose during leaching due to their chemical stability and resistance to acid dissolution. This is due to the fact that the sulfide lattice strongly binds copper, and special methods and reagents are required for its release.» I'm sorry, I didn't notice this paragraph when submitting the translated article. This is a translation error, apparently one of my colleagues made a fragment from a series of experiments on gold related not to chalcopyrite, but arsenopyrite. I deleted this fragment.

Comments 5: Microbiological Studies of Ore Samples

- While enrichment of acidophilic iron-oxidizing microorganisms using 9K medium is appropriate, the methodology for isolating sulfur-oxidizing bacteria and heterotrophs is not described. The type of solid media used should be specified.

- Procedures for obtaining pure cultures of both chemolithotrophs and heterotrophs should

be described in detail. 

- Key cultivation conditions (temperature, pH, aeration, nutrients) for all microbial groups,

including bacteria and fungi, must be clearly stated. 

- Figure 1: Images of fungal isolates are unclear and of insufficient quality.

- Figure 2: The origin and observation method for Skermanella aerolata should be clarified. Since these bacteria are not chemolithotrophic, their physiological group should be identified. Basic characterization (Gram reaction, metabolic type, growth conditions) is required.

-It should be clearly stated whether the bacteria were isolated as pure cultures.

Clarify whether identification was performed using 16S rRNA gene sequencing of pure cultures or mixed/enriched cultures.

-The claim regarding correspondence to Skermanella aerolata lacks supporting biological characterization data.

Response 5:

- «In the present study, a 9K medium was used to isolate and purify acidophilic iron-oxidizing microorganisms.

The 9K liquid medium consists of two solutions, A and B. Solution A includes: (NH₄)₂SO₄ — 3,00 g/l; KCl — 0,10 g/l; K₂HPO₄ · 3H₂O — 0,655; g/l; MgSO₄ · 7H₂O — 0.50 g/l; Ca(NO₃)₂·4H₂O — 0.01 g/l.

Solution B contains FeSO₄ · 7H₂O and is sterilized separately from solution A. After cooling to room temperature, solutions A and B were evenly mixed before use.

The 9K solid medium includes solutions A, B (as described above), as well as solution C. Solution C is an agarose solution, 7.5 g/l is standard, however, in our case, a con-centration of 20 g/l was used. Alkali was added to the agar and the pH was adjusted to 9-10 to ensure a final pH of 3.0 when mixed with an acidic solution and solidification of the agarized medium.» I added this text to the article. Unfortunately, our microbiologist has gone abroad at the moment. If you include a full description of bacterial growth and breeding, it can take many pages. When experiments were conducted to simulate heap leaching in columns, the microbiologist was given the task that bio-oxidation should be as close as possible to the conditions of large-scale production. That is, for laboratory-scale small experiments, it is possible to use a sterile culture, whereas in conditions of real heap leaching of copper, the growth of associated microorganisms will be observed. Therefore, the task of the microbiologist was to determine which other endemic microorganisms from the ore deposit will develop when conditions are created for iron-oxidizing bacteria, which are the main biological agent during pre-oxidation.

- In the process of performing these experiments, I conducted the hydrometallurgical part of the work - leaching and extraction (SX-EW), because I am a metallurgical chemist. I carried out the activation of biological oxidation in the comparative test columns of the bio-variant with conventional leaching after reaching the pH range of 1.8-2.2 according to the conditions that the microbiologist set for me. Therefore, unfortunately, I cannot comment in detail on the biological data.

- Figure 1: I tried to improve the quality of the drawing

- During the identification process, the following conclusion was issued from the Institute of Microbiology: The results of strain identification by analyzing a fragment of the 16SrRNA gene can be used as a molecular biological characteristic of the strains. The type of bacteria is sulfur-oxidizing chemolithotrophic bacteria. It is possible that they work in association with other microorganisms, contributing to the oxidation of iron- and sulfur-containing minerals. Also, these bacteria are independently capable of oxidizing sulfur in the absence of an organic source.

Comments 6: Experimental Part.

- The experimental conditions for both bioleaching and chemical leaching are

insufficiently described. It should be clarified whether 9K medium, raffinate, or another

solution was used. 

-The experimental setup (e.g., percolation columns vs. stirred reactors) must be clearly

specified.

Response 6:

- Based on the required properties and composition of the biological solution, the raffinate was diluted to achieve the following parameters: pH 1.8–2.2 (for acidophilic cultures, pH may be as low as 1.0), Fe²⁺ concentration of approximately 10 g/L, Cu content not exceed-ing 0.1 g/L, and H₂SO₄ concentration of 3–5 g/L (depending on the target pH). The water balance of the bacterial oxidation process depends on the initial properties of the spent raffinate. Thus, for oxidative treatment with acidophilic cultures (pH 1.0–1.8), undiluted raffinate may be used. In contrast, for bacterial cultures requiring a pH range of 1.8–2.2, dilution of the raffinate with water is necessary. Water was added to achieve the required raffinate parameters at a ratio of 1:5, followed by inoculation with A. Ferrooxidans and A. Thiooxidans cultures grown on nutrient media at a ratio of 1:100.

- The main experiments were carried out using column percolation leaching, simulating heap leaching conditions over a period of 125 days. Two types of copper ore samples (Type 1 and Type 2) were used in the percolation experiments. The column tests included comparative evaluation of conventional sulfuric acid leaching and a method involving preliminary bacterial treatment followed by leaching.

Comments 7: Daily Measurements

- A reference for the titrimetric method used to determine copper concentration is missing. 

- The paragraph describing Eh values should be rewritten for clarity, as it currently presents

apparent inconsistencies between bulk solution and interfacial measurements. 

Response 7:

- Titrimetric analysis of copper is a well-known technique for a long time (W. F. Hillebrand, G.E.F. Lundell, APPLIED INORGANIC ANALYSIS, 1953, John Wiley & Sons, Inc., p 256)

- Corrected

Comments 8: What is referential value of copper recovery in sulphuric acid leaching. Authors mention only increment caused by bacteria?

- In Table 5 and Figure 4, the term “Standard” should be replaced with “Conventional” to

maintain consistency with the text. 

- Figure 3: The results indicate minimal differences in copper extraction among individual

microbial cultures (including chemolithotrophs, heterotrophs, and fungi), which reduces

confidence in the conclusions. The authors should provide a clear explanation for this

observation. 

- Figure 3 legends are not clearly visible and should be improved for readability.

Response 8: The minimal differences in copper recovery during bio-leaching are explained by the insufficient duration of contact of the bio-solution with the ore mass during mixing, which is more achievable during subsequent testing on a column.