Soil microbial respiration is an important factor in regulating carbon (C) exchange between the soil and atmosphere. Thermal adaptation of soil microorganisms will lead to a weakening of the positive feedback between climate warming and soil respiration. The thermal adaptations of microbial communities and fungal species has been proven. However, studies on the thermal adaptation of bacterial species, the most important decomposers in the soil, are still lacking. Here, we isolated six species of widely distributed dominant bacteria and studied the effects of constant warming and temperature fluctuations on those species. The results showed that constant warming caused a downregulation of respiratory temperature sensitivity (Q₁₀) of the bacterial species, accompanied by an elevation of the minimum temperature (T_min) required for growth. Similar results were seen with the addition of temperature fluctuations, suggesting that both scenarios caused a significant thermal adaptation among the bacterial species. Fluctuating and increasing temperatures are considered an important component of future warming. Therefore, the inclusion of physiological responses of bacteria to these changes is essential to understand relationships between microbiota and temperature and enhance the prediction of global soil-atmosphere C feedbacks.

2.2 Respirations and Q₁₀

The bacteria were transferred to liquid LB medium upon completion of the secondary culture and cultured in respiration flasks with sealed rubber stoppers and three-way valves. The test temperatures for bacterial respiration were set between the extreme high and low temperatures for incubations, i.e. 5°C, 15°C, 25°C and 35°C. After the bacteria were cultured at the test temperatures for approximately 18 hours, the air over the bottles was replaced with pure CO₂-free air (the initial CO₂ concentration was 0). The bacteria were then cultured at the test temperatures for one additional hour, and then the incubations were finished. The air over the bottles was extracted, and the CO₂ concentrations were measured by gas chromatography (Agilent 6890; Agilent Corp, USA), while the concentrations of the bacteria in the bottles were measured by an enzyme-labeled instrument (Synergy™ HTX, BioTek, USA). The mass-specific respiration rates (R_mass) were calculated as:

R_mass = R / (m ∙ V)

where R is the variation in the concentration of CO₂ (Δppm) measured in equal volume over the respiration bottle, m is the concentration of the bacterial liquid measured in the bottles at 600 nm wavelength, and V is the total volume of the bacterial liquid in the bottles (L).

After obtaining the R_mass of the bacteria, the respiratory temperature sensitivity (Q₁₀) was calculated:

Q₁₀ = (R_mass2/R_mass1)^[10/(T₂-T₁)]

where R_mass1 and R_mass2 are the per unit bacterial respiration rates measured at temperatures T₁ and T₂ (°C), respectively (where T₁ < T₂), with identical units of R_mass1 and R_mass2. The temperature gap between T₁ and T₂ was not required to be 10°C.

2.3 Bacterial growth and T_min

To fit the curve of bacterial growth with respect to temperature, bacteria cultured entirely at the incubation temperatures were transferred to liquid LB medium, and each species was grown at seven test temperatures (10°C, 15°C, 20°C, 25°C, 30°C, 35°C and 40°C). Biomass was sampled in 96-well plates to estimate the optical density at 600 nm. The specific turbidity of each species in the stationary phase was recorded in advance as a 100% value, and the time taken to reach 35% specific turbidity for each bacteria at each test temperature was recorded to calculate T_min according to the Ratkowsky square root relationship (Ratkowsky et al. 1983):

√r = b (T - T_min)                                                               (1)

where r is the growth rate constant of the bacteria, expressed in the experiment as the reciprocal of the time taken for the bacteria to reach 35% turbidity, b is the regression coefficient, and T is the test temperature. However, when the temperature exceeds the optimum temperature for bacterial growth, the bacterial activity decreases, accompanied by a decrease in the growth curve due to, for example, changes in protein structure. Hence, the equation is extended to describe the relationship more completely (Ratkowsky et al. 1983):

√r  = b (T - T_min) {1 – exp [c (T – T_max)]}                                             (2)

where T_min and T_max are the minimum and maximum temperatures for bacterial growth, respectively, i.e., bacteria stop growing when the temperatures reach T_min and T_max, b is a parameter in equation (1), and c is an additional parameter. When T is much lower than T_max, √r is linear with T, allowing a more accurate estimation of bacterial T_min.