Microbial biomass and community composition of a tallgrass prairie soil subjected to simulated global warming and clipping A. Belay-Tedla, M. Elshahed, L. Krumholz, Y. Luo Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA Abstract Introduction Soil microorganisms are the driving force behind most belowground processes and may considerably influence ecosystem responses to global warming. However, the consequences of warming on the microbial community, both in terms of total biomass and species composition, are not known for certain. The present study assessed the effects of experimental warming and clipping on soil microbial biomass contents and examined warming-induced alterations in the microbial community structure and diversity using the 16S ribosomal ribonucleic acid (16S rRNA) sequence analysis. Materials and methods Site descriptions and experimental design: The study was conducted in the Great Plain Apiaries of Central Oklahoma (34058’54’’ N, 97031’14’’ W). The design of the experiment was a paired factorial with warming as the main factor and clipping as the secondary factor, replicated six times. The plot size was 2mX2m each divided into four 1mX1m subplots. Two diagonal subplots in each main plot were clipped 10 cm above the ground once every year. The other two diagonal subplots were the unclipped control. An infrared radiator (Kalglo Electronics, Bethlehem, Pennsylvania) suspended at 1.5 m above the ground in each warmed plot was used as the heating device. The plots have been warmed continuously since November 21, The four treatments in the experiment were clipped control (CC), unclipped control (UC), clipped warmed (CW) and unclipped warmed (UW). Sampling and analysis: soil samples were collected at 0- 20cm depth in the Winter and Spring seasons between 2002 and Soil microbial biomass: Chloroform-fumigation - Community composition: 16S rRNA The present study examined the effects of simulated global warming and clipping on soil microbial biomass and community composition of tallgrass prairie soil in Central Oklahoma. Soil microbial biomass C and N contents were significantly increased (>50%) by the warming but remained unaffected by the clipping treatment. Warming also enhanced the microbes’ C and N use efficiency by more than 48%. The 16S ribosomal RNA analysis revealed seasonal and differential effects of warming on bacterial diversity and community composition. Assays conducted in the present study have provided both metabolic and molecular evidence that soil microorganisms may adjust their activity rate, physiology, and/or community composition in response to global warming. Fig. 2. Soil microbial biomass C (SMB-C) and N (SMB-N) on two different seasons. SMB-C in unclipped plots increased by >50% as a result of the warming treatment. SMB-N was also higher (with average increase of more than 20 mg/kg over other treatments) in the UW plots but differed significantly only from CC plots. Results Infrared heater “dummy” heater clip Unclip Soil microbial biomass C and N Fig. 3. Soil microbial biomass C (SMB-C) and N (SMB-N) to labile C (LPC) and N (LPN) ratio. SMB-C:LP-C rose by 48, 31, and 22% in UW, CW, and CC. SMB- N:LPN ratio also increased by warming Soil sample (0.5 g) Bulk DNA Amplified 16S rRNA clones 16S rRNA sequences FastDNA® SPIN Kit for Soil (Qbiogene, Inc.) Gene Amp PCR system 9700 thermocycler TOPO-TA Advanced Center for Genome Technology DNA extraction PCR Cloning Sequencing Fig. 1. Overview of the 16S rRNA procedure LibrarySeasonClone #OTU # Control Warmed Spring ’03 Winter ’04 Spring ’03 Winter ‘ Bacterial community diversity and composition Table 1. Total number of clones sequenced and OTUs identified. Clone number in UC higher by 10% ; OTU richness was similar for both. Fig. 4. Shannon-Weiner index and species even- ness (Sp03; Win04). Fig. 5. Collectors’ curve Analysis (Sp03; Win04) Fig. 7. Bacterial clone distribution. There was a persistent relatively higher proportion of bacterial groups belonging to Actinobacteria, CFB, beta- Proteobacteria, Chloroflexi and comparative decline of those belonging to Alpha-proteobacteria, Gamma-proteobacteria, and Gametoides. Conclusion The study has demonstrated consistent and significant increases of soil microbial biomass indicating enhanced microbial activity in response to warming. An alteration in C and N use efficiency of the microbes was also observed, suggesting warming-induced physiological changes. The observed differences in the relative proportions of different bacterial groups indicate that the microbial community structure or specific components of the population could have been changed as a result of the imposed warming.