Investigation About The Response Of Abundance And Activity Of Autotrophic Microorganisms In Purple Soil To Environmental Factors

Soil is the main carbon reservoir in the terrestrial ecosystem participating in the global carbon cycle,which is closely related to global climate change.Autotrophic microorganisms in soil can assimilate carbon dioxide?CO₂?in the atmosphere and play an important role in soil carbon fixation,which can be divided into the photoautotrophic type and chemoautotrophic type according to their requirement for energy sources.Although there are several pathways for soil autotrophic microorganisms to fix CO₂,the Calvin-Bussham cycle with the highest CO₂ fixation efficiency is widely found in most autotrophic microorganisms.The rate-limiting step of the Calvin cycle is the process of CO₂ assimilation catalyzed by 1,5-bisphosphate ribulose carboxylase?RubisCO enzyme?.Therefore,RubisCO enzyme activity and the cbbL gene encoding the large subunit of this enzyme have been widely used in the ecological study of soil autotrophic microorganism.At present,studies using cbbL gene and RubisCO enzyme activity as molecular markers have shown that the ecological characteristics of soil autotrophic microorganisms responded significantly to the disturbance of the external environmental factors.However,few studies have reported the response of autotrophic microorganisms to the environmental factors in purple soil.In this study,a representative neutral purple soil?entisol in the USDA soil taxonomy?endured 26-year?1991–2017?of continuous fertilization was selected and combined the Illumina Miseq sequencing to explore the effect of long-term chemical fertilization on the activity and community composition of soil autotrophic bacteria and find out the dominant factors controlling the ecological features of these microorganisms.Subsequently,to confirm the effects of those important environmental factors governing the activity and abundance of soil autotrophic bacteria,the effects of some important environmental factors such as soil water content,pH,and light on the autotrophic microorganisms in purple soil were further explored through indoor microcosm incubation experiments.The result of the investigation based on the purple soil with long-term fertilization suggested that the sulfur-oxidizing microorganism?Sulfuricaulis limicola?might be an important autotrophic bacterial genus in this soil.Thus finally,through ¹³C-CO₂ stable isotope-labeled soil culture experiments with addition of reduced inorganic sulfur?Na₂S?,we used the DNA-SIP technology to explore the effect of energy substance?Na₂S-energy for the sulfur-oxidizing bacteria?on the activity and abundance of autotrophic microorganisms in the neutral purple soil.The results of this study should provide some theoretical basis for the formulation of reasonable farmland management measures to increase the soil carbon reservoir content,and lay a foundation for the further exploration of the ecological characteristics of soil autotrophic microorganisms.The main results were as follows:?1?In this study,the cbbL gene and RubisCO enzyme were used as indicators to study the abundance and composition of autotrophic bacteria in an entisol with 26 years of continuous fertilization.In this process,five chemical fertilization regimes were examined:without fertilization control?CT?,nitrogen and phosphorus fertilization?NP?,N and potassium fertilization?NK?,PK fertilization?PK?,and NPK fertilization?NPK?.The results showed that after the 26-year period,the CT with a neutral pH?7.55?had a higher RubisCO activity than did the chemical fertilizer treatments,but the cbbL abundance was the lowest under CT.Soil RubisCO activity positively correlated with soil pH?R²=0.67,P<0.001?;soil cbbL abundance significantly positively correlated with soil available N?R²=0.54,P<0.01?and significantly negatively correlated with pH?R2=0.30,P<0.05?.Meanwhile,the soil RubisCO activity significantly negatively correlated with cbbL abundance?R²=0.67,P<0.001?.In addition,the alpha-diversity of cbbL was lowest under CT,indicating a positive effect of fertilization on the soil cbbL diversity.Soil pH,available nitrogen,total nitrogen,and organic matter are the key factors that determine the soil cbbL community structure.Besides,Sulfuricaulis limicola was found to be the most abundant known-bacterial genus in this purple soil.In conclusion,a 26-year long-term chemical fertilization changed the community structure of soil cbbL-carrying bacteria,and soil pH and available N played crucial roles in controlling the ecological properties of soil cbbL-carrying bacteria.?2?Using the cbbL and RubisCO enzyme as markers,the effect of soil water content on the abundance and activity of soil autotrophic microbes was investigated through an indoor incubation experiment of 28 days.Four treatments with varied water content?mass percentage,ratio of water to dry soil?were conducted for the water contents of 10%(W₁₀),15%(W₁₅),20%(W₂₀),and water-logged?WL?soils.Soil samples were harvested after 3,7,14,and 28 days of incubation,and then the soil organic carbon content,cbbL abundance,and RubisCO enzyme activity were detected.The results indicated that the soil cbbL abundance showed a tendency of increasing with the increase of soil water content during the first 14 days,and the WL treatment had the highest value of cbbL abundance during the whole period of incubation.At the initial stage of incubation?day 3?,no significant difference was detected about the soil RubisCO enzyme activity among these treatments?P<0.05?.However,at end of incubation?day 28?,the RubisCO enzyme activity in the treatments with lower water content(W₁₀0 and W₁₅)was significantly lower than those treatments(W₂₀0 and WL)with higher water content?P<0.05?.The result of one-way repeated ANOVA indicated that soil water content significantly affected soil organic carbon content?F=11.91,P=0.03?,cbbL abundance?F=42.33,P<0.01?,and RubisCO enzyme activity?F=51.55,P<0.01?.Moreover,there was a significantly positive relationship between soil cbbL abundance and RubisCO enzyme activity?R²=0.23,P<0.01?,which all were negatively correlated with soil organic carbon content.Taken together,our results indicate that soil water content can play an important role in the determination of the abundance and activity of soil autotrophic microbes,which should be valuable for further understanding of the response mechanism of soil carbon transformation to soil moisture variation.?3?The CO₂ assimilation in the soil can be conducted by both photoautotrophic and chemoautotrophic microbes,while these two kinds of microbes can assimilate CO₂by different pathways,using the sunlight and oxidation of inorganic compounds as their power sources,respectively.Presently,the key role of CO₂ assimilation in the soil CO₂fixation process has been confirmed,but the relative contribution of photoautotrophic and chemoautotrophic microbes to soil CO₂ fixation remains uncertain.In this study,to investigate the relative contribution of photoautotrophic and chemoautotrophic microbes to the CO₂ assimilation in purple soil,three purple soils with acidic?ACI?,neutral?NEU?,and alkaline?ALK?pH values were collected,and then an indoor microcosm incubation experiment was conducted at room temperature.For each soil,two treatments with different light conditions?in natural light or dark?were carried out and incubated for 45 days,and then the soil samples were collected at day 7 and 45.Subsequently,the RubisCO enzyme activity,cbbL gene abundance,soil respiration rate and organic carbon content were measured to investigate the effects of light on carbon assimilation activity of purple soil with different pH values.The results showed that throughout incubation,the RubisCO enzyme activity and cbbL gene abundance of each treatment under light conditions were significantly higher than under dark conditions?P<0.01?.Under light conditions,RubisCO enzyme activity and cbbL abundance in NEU treatment were significantly higher than those in ACI and ALK treatment?P<0.01?.There was no significant difference in RubisCO enzyme activity in soil with different pH throughout the incubation under dark conditions.Correlation analysis showed that the activity of RubisCO enzyme in purple soil was positively correlated with cbbL gene abundance?R²=0.39,P<0.01?,negatively correlated with respiration rate?R²=0.40,P<0.01?and organic carbon content?R²=0.08,P<0.01?.In summary,this experiment again confirmed that pH is an important factor affecting the abundance and activity of autotrophic microorganisms.Compared with chemoautotrophic microbes,the growth of photoautotrophic microbes in soil is more sensitive to pH value,and its potential for assimilating CO₂ in neutral environments is higher than those in acidic and alkaline environments.This result can provide some basic information for further exploration of the relative contribution of photoautotrophic and chemoautotrophic microbes to soil CO₂ fixation in varied pH environments.?4?The result of part?1?showed that the sulfur-oxidizing bacterial genus?Sulfuricaulis limicola?was the most abundant cbbL-carrying known-bacteria in the target purple soil,indicating that sulfur might be an important energy source for the autotrophic microbes in this soil.Therefore,in this part,the effect of energy source?reduced inorganic sulfur,Na₂S?on the activity and abundance of autotrophic microbes in the purple was investigated through the ¹³C-CO₂ labeled stable isotope nucleic acid probe?DNA-SIP?technology.Three kinds of treatments were set up:no Na2S?CT?,5mg·kg^-11 of Na₂S?LS?and 10mg·kg^-11 of Na₂S?HS?.The treatments were incubated at room temperature and labeled by ¹²CO₂ and ¹³CO₂ respectively,and then were sampled at 7 and 28 days for the isolation of ¹³C-labeled DNA and determination of soil cbbL gene abundance,Rubis CO enzyme activity,respiration rate,and organic carbon content.The results showed that,13C was partly successfully labeled on the soil DNA.At day 7of the incubation,the RubisCO enzyme activity in the ¹²CO₂-HS treatment was significantly higher than that in the ¹²CO₂-CT treatment?P<0.05?,and the buoyancy density at the peak of relative abundance of cbbL genes in ¹³CO₂-HS treatment was significantly higher than that in ¹³CO₂-CT and ¹³CO₂-LS treatment.After the incubation of 28 days,there was no significant difference in the RubisCO enzyme activity among these treatments,and the buoyancy density at the peak of relative abundance of cbbL genes in ¹³CO₂-CT treatment was significantly higher than that in ¹³CO₂-LS and¹³CO₂-HS treatment.There was no significant difference in cbbL abundance,soil organic carbon content and respiration rate among these treatments during the whole incubation.The result of this study indicated that soil sulfur content might not be the key factor affecting the abundance of soil autotrophic microorganisms,but high soil sulfur content may affect the activity of autotrophic microorganisms under short-term culture conditions,and the addition of sulfur may positively stimulate the growth of the soil sulfur-oxidizing autotrophic microorganisms.