Differences in the ratio of soil microbial biomass carbon (MBC) and soil organic carbon (SOC) at various altitudes of Hyperalic Alisol in the Amazon region of Ecuador

Protecting soil fertility represents a fundamental effort of sustainable development. In this study we investigate how different altitudes affect soil microbial biomass carbon (MBC) and soil organic carbon (SOC), and their ratio, MBC/SOC in Hyperalic Alisol. MBC and SOC are well established and widely accepted microbial quotients in soil science. Our work hypothesis was that a decrease in MBC and SOC should be observed at higher altitudes. This initial assumption has been verified by our measurements, being attributed to the increase in MBC and SOC at low altitudes. Our approach should contribute to the better understanding of MBC and SOC distribution in soil and changes in MBC/SOC at various altitudes in the region.


Introduction
Protection of soil quality is a pillar of sustainable development [1][2][3] . Recent studies considered effects of minerals 4-6 and soil cultivation techniques 7,8 on soil quality and soil microbial activity 9-12 . These studies reported interactions between soil minerals and decomposition of organic matter; positive correlations between soil microbial activity (measured by number of bacteria and fungi, soil respiration, and C and N cycle-related enzyme activity) and cultivation methods, and fertilizer usage. The undertaken efforts in agriculture practice, offer favorable conditions for soil-plant interactions. Additionally, these studies on soil respiration and enzymatic activity report correlation analyses between soil properties and microbial activity, documenting that soil quality is mainly determined by soil microbial activity, as verified by other authors' work [13][14][15] . A highly cited review paper on the topic 16 concludes that soil processes and properties are affected by climate change, which causes changes in soil organic matter (SOM) and microbial biomass carbon (MBC). Based on this, one can state that monitoring the soil carbon cycle is of increasing importance, given that microbial processes in soil may serve as an indicative of climate change. Among processes to be monitored, decomposition of organic matter is of primary significance 17 , considering that this process is strongly influenced by soil microbiological activity. Soil management is another research area of increasing interest 18 owing to its significance in soil microbial activity 19 that can be expressed by enzymatic activity, soil respiration, and MBC 11 . Local farmers have demonstrated willingness to start permaculture systems involving tropical orchards, like those previously operated successfully in another plant, Guayusa (Ilex guayusa) 20 , in the Amazon region. Because of the sensitivity of orchards to nutrient deficiency 21-23 we believe that measuring soil microbial activity is of priority when analyzing soil properties, as soil microbial activity is in strong correlation with nutrient uptake in plants 24 . For this reason, monitoring changes of soil MBC and soil organic carbon (SOC) allow to select favorable locations for orchard plantation in permaculture systems, contributing to sustainable agricultural practices. MBC is the living microbial component of soil organic matter 25 and is considered an indicator of microbial activity, owing to its rapid response (less than a year turnover time) to conditions that may alter soil organic matter 26 . SOC contributes positively to soil fertility and crop production 27 . Here we report variations with altitude of MBC, SOC, and MBC/SOC in Hyperalic Alisol.

Soil sampling
We were inspired by a prior study conducted in 2018 28 , in which soil properties at various altitudes were measured in the region. We collected samples at similar altitudes as in the prior study 28 (395, 1006, and 1554 m.a.s.l, meters above sea level) to gather data on soil properties at different altitudes. We collected 15 soil samples, 5 from each altitude, following the protocol proposed by Singla and co-workers 28 Fumigated is the produced CO 2 by fumigated soil samples, expressed in C µg/ml. Unfumigated is the produced CO 2 by not fumigated soil samples, expressed in C µg/ml.
To calculate SOC we determined humus content according to Székely et al. 33 . In this procedure, 1.0 g of air-dried soil was placed into a 300-mL Erlenmeyer flask. Then, 10. mL of 5% K 2 Cr 2 O 7 (Fisher Scientific, 7778-50-9) solution was added and mixed with the soil. Next, 20 mL of concentrated H 2 SO 4 (Fisher Scientific, 7664-93-9) were supplemented, and mixed with 100. mL distilled water. Solution was filtered with Grade 42 Whatman slow filter paper (pore size 2.5 µm) and analyzed with Model 240Z Atomic Absorption Furnace Spectrophotometer (Agilent), from 230 to 700 nm, and absorbances at 600 and 400 nm (E 600 and E 400 ), respectively, correspond to A,B, P, and Rp types of humic acids, relevant to humus content, according to Watanabe et al. 34

Statistical analysis
Linear regression (Z-test) was performed to reveal possible statistical differences (p<0,05) between parameters and altitudes, using SPSS (version 26). MBC/SOC increases with decreasing altitude (Figure 1 37 ), which means that MBC increases more rapidly with decreasing altitude than SOC. As MBC corresponds to microorganisms' weight (mainly bacteria and fungi), and is estimated at approx. 5% of the SOC 32 , the increased MBC value indicates faster microbial activity at lower laying sampling points. This finding yields three main observations: 1. Organic matter decomposition is accelerated by soil microbial activity 38,39 in lower-laying areas, because of more favorable conditions 40 offered to cellulose decomposing bacteria. Presence of these bacteria is confirmed by MBC/SOC increase at lower altitudes, reported here. Microbial carbon increases more significantly than organic carbon, which suggests increased microbial activity 40 .
2. Availability of SOC depends on soil properties, and soil depth 41 , rather than on altitude or coverage 42 . SOC may also depend on several other soil properties, therefore, enzymatic activities and physical-chemical soil properties should be measured. Total iron increases with decreasing altitude, as well 30 . With these findings in MBC/SOC value changes, general leaching can be documented.
3. Metabolic efficiency depends on the availability of substrate (organic matter). The greater the MBC, the greater the temporary immobilization of micro and macro-nutrients 43-46 . To confirm this, immobilization of nutrients should be measured in soil-plant interactions (mainly uptake), as planned for future work in which orchards will be investigated, given the local farmers' need to plantation in the area and the particular sensitivity of orchards to nutrient deficiency 21-23 .

Conclusions
Altitude affects SOC significantly. Decrease in MBC/SOC quotient is observed with increasing altitude. From obtained results we can conclude that lower-laying areas favor increased soil microbial activity. We recommend lower-laying areas for orchard plantations, considering that orchards are particularly sensitive to nutrient deficiency 47 , while soil microbial activity is in strong correlation with nutrient uptake in plants 24 .  Are the conclusions drawn adequately supported by the results?

Yes
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Soil ecology, soil carbon content I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The authors report changes in soil microbiological parameters and their ratio at different altitudinal levels of the Ecuadorian Amazon region. The study is well written and straightforward; anyways, in parts needed to be improved to increase readability, and lead readers to better understand the importance of the research findings. First, in the introduction, authors discuss the role of MBC and, then SOC in assessing soil microbiological activity, but what missing for me is the lack of explaining their ratio MBC/SOC as a microbial indicator. The importance of ratio is mentioned in the paper, but its role is unclear. Up to date and relevant literature is cited. Methods are well written, but missing some information. How many samples were measured for MBC and SOC, 15 (as sampled) in quadruplicated? It is not clear until we open the supporting material. Observation: generally in mobilization studies, apart from the altitudinal variant, sampling depths are also involved in the analysis. It is not clear to me why the authors disregarded different depths in the study that could provide a more complex image of examined changes. Results are presented logically, and succinctly. Conclusions are correct. Overall, it can be said that it is a well-designed study. In any case, I suggest making the following minor changes before article indexing: Expand introduction, and highlight the role of ratio MBC/SOC.

State number of samples used for biomass analyses.
Please clarify why depth was not involved in the analysis.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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