Unexpected results in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity

The number of studies investigating the effect of bio-fertilizers is increasing because of their importance in sustainable agriculture and environmental quality. In our experiments, we measured the effect of different fertilizers on soil respiration. In the present study, we were looking for the cause of unexpected changes in CO2 values while examining Chernozem soil samples. We concluded that CO2 oxidizing microbes or methanotrophs may be present in the soil that periodically consume CO2 . This is unusual for a sample taken from the upper layer of well-ventilated Chernozem soil with optimal moisture content.

Amendments from Version 1 We extended the Introduction chapter to clarify the importance of measuring physical and chemical soil properties when examining soil microbiological activities.

Introduction
The soil can be characterized by physical, chemical and microbiological properties [1][2][3][4] . The quantitative (microbial biomass, number of bacteria) 5,6 and qualitative (enzymatic activity, soil respiration) 7,8 microbiological properties of the soil greatly contribute to the impact analysis of land use [9][10][11] , nutrition 12 and soil management 13 . Research related to the benefits of microbes as biofertilizer has become increasingly important in the agricultural sector. This is due to the possibility of achieving higher crop yields while minimizing negative impact on the environment. It is well known that bio-fertilizers increase plant yield and improve soil fertility [14][15][16] . Soil respiration is an important indicator of soil microbial activity 17,18 . In our experiments, we measured the effect of different chemicals [19][20][21][22] and a bio-fertilizer on soil microbial activity, using both well-established and novel methods under laboratory conditions. We present some unexpected results from a setup in which Chernozem soil samples were examined.

Soil properties
Soil moisture content was determined gravimetrically, drying the soil at 105°C for 24 hours according to Klimes-Szmik's method (1970) 23 . Silt and clay fractions were measured by the settling method 24 . We measured the Arany-type plasticity index according to Stefanovits (1975) [25][26][27] , while the minimal water capacity and soil texture were determined by Klimes-Szmik's method 23 . To measure the chemical properties of the soil, the samples were sieved through 2mm mesh and pre-incubated at 25°C for 72 hours. Soil pH in distilled water and in 1M potassium chloride KCl (soil/ water, 1/2.5, w/w) were determined according to Buzás (1988) 24  where EC = organic C extracted from fumigated soils -organic C extracted from unfumigated soils ( Table 1).

Soil respiration
The experimental design was completely randomized, treatments were incubations (25°C). An OxiTop OC110 respirometer was used to quantify the release and capture of CO 2 that is automatically determined by the device after the biological oxygen demand (BOD) required for the degradation of organic matter has been measured. We used a 500 ml glass bottle system following the instruction manual (https://www.wtw.com/en/service/downloads/ operating-manuals.html). 10g of soil sample were placed into OxiTop flasks, and capped with the sensor heads according to Barrales-Brito et al. (2014) 33 . 2.5g of CO 2 absorber (sodalime) were then added to a tank to absorb the generated CO 2 33 . An induced method was also used, in which 0.1g glucose was added to the soil samples. Each treatment was replicated four times. As Figure 1 shows, four samples were always measured in parallel: Absolute control (does not contain fertilizer, nor added glucose), Induced control (contains added glucose), Treated (contains bio-fertilizer) and Induced treated (contains bio-fertilizer and glucose). The Oxitop automatically provides the values related to CO 2 production according to the pressure change measured by its sensor (there is no need to carry out titrations or any additional work).

Results
The treated samples produced more CO 2 than the controls, as expected (Dataset 1). Each repeat with the exception of one showed increasing CO 2 values (Figure 1), as the pressure continuously decreased in the bottle due to gas (oxygen) consumption. One sample produced unexpected results ( Figure 2). In the first 12 hours, the treated samples produced more CO 2 than the controls in each measurement. Following this, a fluctuation in the values was observed.   Discussion In a closed system where the pressure decreases due to oxygen consumption, the values of CO2 production must increase or stagnate with the passage of time, but this was not the case with one of the samples (Figure 2). Here, a decrease in CO 2 occurred (Dataset 2). The following possible explanations were excluded: • Presence of algae: there was no light in the incubator, so there was no photosynthesis.
• Changing pressure caused by changing temperature: the temperature was constant in the setup.
• Absorption by the water in the sample: all other samples that produced increasing amount of CO 2 had the same or comparable moisture content.
One reason that seemed more likely was that CO 2 oxidizing microbes or methanotrophs may have been present in the soil, using the produced CO 2 periodically. This is unusual, since most of the studies report the presence of these bacteria in seawater 34 , paddy fields 35 or industrial processes 36 and not in well-ventilated Chernozem soil. Further genomics research could detect the bacterial strains that consumed the CO 2 in this soil.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work. 8. How were soil microbial activities assessed? Methanogens and methanotroph's relative intensity were not found in this script, which are the major focus related to the current research topic.

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

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Referee Expertise: Soil GHGs flux measurement, soil microbes & environment I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

5% substrates application in soils?
This Research note discusses only unexpected results come from an experiment that was carried out using Oxitop devices. This is part of a project in which more methods are applied. In an other method (using liquid-alkaline absorption) is possible to setup the different levels, but that is not part of the discussion of the present paper. Using Oxitop bottles only one level is possible for the setup.
2. What were the initial content of organic carbon, total nitrogen, soil pH, redox status (Soil Eh) and microbial composition of the collected 24 soil samples?
In the Dataset 1: Average values for a number of different soil properties you can find the main phyical, chemical and microbial soil properties such as pH (H2O), pH (KCl), Organic carbon. We will extend the dataset with the Total Nitrogen in the second version of the paper.

How did the researchers control the pressure within the glass bottles during the experimental period?
The Oxitop automatically measures the changes in the bottles due to gas consumption by its sensor, there is no needed to apply external measurement.

How did the authors maintain moisture levels or water filled pore space uniformity in the 24 soil samples containing glass bottles?
The measurement was carried out in closed system (bootles), it is not possible to open the bottles during the measurement.

Why didn't you collect the gas samples evolved from the soils in glass bottles at varying time hours?
The Oxitop continously measures the changes. As Fig.1 and Fig.2 show during 168 hours the gas oxygen consumption/ CO2 production were measured.

Why didn't you follow the light/dark conditions in the Incubator where the glass bottles were kept?
In order to avoid the effect of the photosynthesis by algeas. We were interested in soil bacteria activities only.

All the Figures are not clear, no contrasting colors or bullets with lines used to differentiate the treatments.
We do not understand this question. In Both figures we used different colors and bullets.
Control (absolute): Blue Control + glucose: Red Treated: Green Treated + glucose: Purple 8. How were soil microbial activities assessed? Methanogens and methanotroph's relative intensity were not found in this script, which are the major focus related to the current research topic.
This paper was submitted as a Research note. Research notes are often , preliminary studies descriptions of unexpected and perhaps unexplained observations or lab protocols. We concluded that "Further genomics research could detect the bacterial strains that consumed the 2 in this CO soil."

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results?
Yes Are the conclusions drawn adequately supported by the results?

Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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