Comparison on the measurement method of soil live bacterial number as part of the biogeochemical site characteristics

다국어 초록 (Multilingual Abstract)

Soils are rich in bacteria (1 × 10 2–2 × 109 cells g-1), with live bacterial abundance dependent on a number of physical and chemical properties, such as organic matter, temperature, particle distribution, moisture, and pH. Soil bacteria play a significant role in global nutrient cycling as well as soil biogeochemistry. There are a number of different methods that have been used to quantify live bacteria in soil. To date, an evaluation of these methods has not been undertaken to identify the most precise and accurate method(s). This study evaluates the conditions of appropriate storage temperature conditions (i.e., -20, 4, 24, 30 °C) and pretreatment methods (i.e., sonication, centrifugation, filtration) for measuring live soil bacterial populations. We also investigated culture-dependent methods (CDMs; i.e., colony forming unit (CFU), spotting, and most probable number (MPN)) and culture-independent/direct counting methods (CIMs; i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA extraction). Each method was tested using 72 soil samples collected from a local farm site at three different depths (i.e., 20, 100, and 180 cm). As the storage temperature at 4 °C showed the lowest variation of live bacterial cell number, storage at 4 °C was most appropriate, and the highest number of live bacterial populations was measured with the pretreatment of 3 minutes of sonication time (300 W), 1400 × g of centrifugation speed. Among all CDMs, MPN was found to be rapid, simple, and reliable (low variability amongst triplet). However, the number of bacteria quantified by MPN was 1–2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA extraction method appeared to overestimate soil bacterial numbers, which may be attributed to DNA extraction from dead bacteria and free DNA in the soil
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matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Bacterial numbers obtained using CDMs and CIMs were also compared based on soil pH, soil water content, and clay/silt content. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead), and their spatial link to soil physical and chemical properties.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Material and Method 4
• 2.1. Soil sampling 4
• 2.2. Soil sample preparation 6
• 2.2.1. Soil storage temperature test 6
• 2.2.2. Soil pretreatment methods test 6
• 2.3. Bacterial cell number quantification 7
• 2.3.1. Pretreatment 7
• 2.3.2. Culture media and condition 7
• 2.3.3. Culture-dependent methods 7
• 2.3.4. Staining and culture-independent methods 8
• 2.4. Analyses 12
• 2.4.1 Soil physical and chemical properties 12
• 2.4.2 Statistical analyses 13
• 3. Results 14
• 3.1. Soil sample preparation 14
• 3.1.1. Soil bacterial cell numbers according to storage temperature and time 14
• 3.1.2. Soil bacterial cell numbers according to pretreatment 16
• 3.2. Bacterial cell number quantification 19
• 3.2.1. Abundance of soil bacteria determined by culture-dependent methods 19
• 3.2.2. Abundance of soil bacteria determined by culture-independent methods 22
• 3.3. Analyses
• 3.3.1. Soil physical and chemical properties and bacterial numbers 25
• 4. Discussion 29
• 4.1. Optimal soil storage temperature and pretreatment method 29
• 4.1.1. Effect of storage temperature and time on soil bacterial numbers 29
• 4.1.2. Effect of soil pretreatment methods on soil bacterial numbers 30
• 4.2. Bacterial cell number quantification 32
• 4.2.1. Differences in bacterial numbers among culture-dependent methods 32
• 4.2.2. Differences between bacterial numbers among culture-independent methods 35
• 4.2.3. Comparison of soil bacterial numbers by CDMs and CIMs 37
• 4.3. Soil physical and chemical factors affecting bacterial numbers 41
• 4.4. Other soil bacterial quantification methods 43
• 5. Conclusion 44
• 6. Reference 45