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

Introduction

Research related to the benefits of microbes as biofertilizer has become increasingly important in the agricultural sector. This is due to their potential role in 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^1–3. Soil respiration is an important indicator of soil microbial activity^4–6. In our experiments, we measured the effect of different chemicals^7–9 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.

Methods

The phylazonit bio-fertilizer (produced by Phylazonit.Ltd, Hungary) was used for testing (15 l/ha). It has the following composition: Bacillus megaterium, Bacillus circulans, Pseudomonas putida, in an optimized ratio for soil injection. Number of bacteria: 10⁹ piece/cm³.

A total of 24 soil samples were collected near Debrecen, Hungary on the 19th April 2016, from an upper layer (0–20 cm) of Chernozem soil (47° 33’ 55.36” N; 21° 28’ 12.27” E). All samples had optimum moisture content with 19–21 percent. Soil moisture content was determined gravimetrically by drying the soil at 105 degrees C for 24 hours according to Klimes-Szmik, 1970¹⁰. The experimental design was completely randomized, treatments were applied at 25 °C. An OxiTop OC110 respirometer was used to quantify the release and capture of CO₂, 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 protocol in the manual (https://www.wtw.com/en/service/downloads/operating-manuals.html). 10g of soil sample were placed into OxiTop flasks, capped with the sensor heads according to Barrales-Brito et al., 2014¹¹. 2.5 g of CO₂ absorber (sodalime) were then added to a tank, to absorb the generated CO₂ according to Barrales-Brito et al., 2014¹¹. Induced samples were given 0.1g of glucose . 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 CO₂ production.

Figure 1. Differences in CO₂ production of treated and control soil samples.

The induced method was also applied in order to differentiate between the control and the treated soil samples to become detectable sooner. Glucose was applied as inducer. As expected the CO₂ values increase or stagnate.

Results

The treated samples produced more CO₂ than the controls, as expected. Each repeat with the exception of one showed growing CO₂ 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 CO₂ than the controls in each measurement. Following this, a fluctuation in the values was observed.

Figure 2. This sample shows CO₂ values periodically decreasing in all conditions.

After examining the Oxitop device’s operation, this pattern became more interesting for us, as the device quantifies CO₂ production by measuring BOD required for the degradation of organic matter. From the decreasing CO₂ values, we conclude that there was oxygen production and/or CO₂ consumption in the Oxitop bottles.

Discussion

In a closed system where the pressure decreases due to oxygen consumption, the values 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 CO₂ occurred. The following possible reasons were excluded:

One reason that seemed more likely was that CO₂ oxidizing microbes or methanotrophs may have been present in the soil, periodically using the produced CO₂. This is unusual since most of studies report the presence of these bacteria in seawater¹², paddy fields¹³ or industrial processes¹⁴ and not in well-ventilated Chernozem soil. Further genomics research could detect the bacterial strains that consumed the CO₂ in this soil.

Data availability

Dataset 1: Average values for a number of different soil properties. DOI, 10.5256/f1000research.12936.d182655¹⁵.

Dataset 2: Average values of produced CO₂ (ml/l) with different treatments. ’Control’ does not contain fertilizer, nor added glucose. ’Control+Glucose’ contains 0,1 g of added glucose. ’Biofertilizer’ contains Phylazonit bio-fertilizer. ’Biofertilizer+Glucose’ contains Phylazonit bio-fertilizer and 0,1 g of added glucose. DOI, 10.5256/f1000research.12936.d182663¹⁶.

Dataset 3: Comparison of produced CO₂ (ml/l) in the sample in which unexpected (periodically decreasing CO₂) values can be observed. ’Control’ does not contain fertilizer, nor added glucose. ’Control+Glucose’ contains 0,1 g of added glucose. ’Biofertilizer’ contains Phylazonit bio-fertilizer. ’Biofertilizer+Glucose’ contains Phylazonit bio-fertilizer and 0,1 g of added glucose. DOI, 10.5256/f1000research.12936.d182664¹⁷.