The soil “Ewiger Roggenbau Halle (Germany) FYM I” pH (CaCl 2 ) 6

1. Temperature 20 °C CO2 traps reactor 1 soil only reactor 2 soil + 12C E.coli RFM 443 reactor 3 soil + 13C E.coli RFM 443 Humid air with O2 Comparison between the amount of extracted DNA and the microbial biomass in soil The comparison of amount and residence time of extractable marker DNA with the amount and residence time of extractable and luminescent bacteria showed a smaller value and shorter detectability of E.coli resulting from the MPN method. There are 3 different explanations: 1. The bacteria are not extractable or culturable. 2. The bacteria exist as non-living compartiments. 3. The DNA is not associated to cells any more, but occurs in free state or bound to humic substances. Fate of microbial biomass in soil R. Kindler, A. Miltner and M. Kästner Department of Remediation Research, Centre for Environmental Research, UFZ Leipzig-Halle GmbH, Permoserstr. 15, 04318 Leipzig Tel. +49 341 235-2051; Fax.+49 341 235-2492; Email: rkindler@san.ufz.de • Introduction • Soil organic matter (SOM) consists of more carbon (C) than the sum of C of plants and microorganisms (MO) on and in soil [1]. However, the origin of SOM is the biomass. Especially the MO are of particular importance because they are involved in the formation of SOM by two processes. On the one hand the microbial biomass (MB) metabolizes plant residues and builds up the refractory soil organic matter and on the other hand they are the source material of SOM to a certain extent. • To investigate the fate of MB we incubated Escherichia coli cells with soil under constant conditions in a reactor. These cells are • isotopically labeled with 13C to trace each microbial C atom and • genetically labeled with the lux gene to trace the fate of nucleic acids in soil and the survival of the entire cells. ×× 1. The soil and the added biomass ØØ The soil “Ewiger Roggenbau Halle (Germany) FYM I” pH (CaCl2) 6 Corg 19 mg/g d13C -26,3 ‰ Ntot 1 mg/g d15N 19,9 ‰ microbial biomass 173 µg biomass C/g (Chloroform-fumigation-extraction) 148 µg biomass C/g (Substrate Induced Respiration) 7,5*106 cfu/g (R2A-Agar) The biomass E.coli RFM 443 TM 73 µg/g » 21 % of natural 13C in soil and natural microbial biomass (CFE) number of cells 1,3*108 cells/g ×× 2. The biomass in the soil ØØ Survival of E.coli carrying the lux marker gene Extraction of plasmidic marker-DNA from E.coli RFM 443 (lux-gene) The quantification of extracted lux-DNA (BIO101 FastDNA SPIN Kit for Soil, purified with polyvinylpolypyrrolidone [4]) based on PCRs with different primers [5, 6] points to a decreasing amount of microbial marker DNA in soil. This way of quantification does not allow to determine an exact value, but it allows to confirm the existence of the marker DNA after 15 weeks. For quantifi-cation, real-time PCR will be performed. UFZ CENTRE FOR ENVIRONMENTAL RESEARCH LEIPZIG-HALLE E.coli cells incubated in soil were extracted with sodium pyrophosphate [2] and quantified by the most propable number (MPN) method based on bioluminescence [3]. The amount of extractable and active E.coli cells decreased during incubation. After 15 weeks no more E.coli luminescence was detectable. No luminescent bacteria were extractable from the control soil without addition of E.coli. ×× 3. Mineralization of the biomass ØØ About 60 % of the added 13C incorporated in the E.coli were mineralized after 15 weeks of incubation. Thus 40 % of the added 13C corresponding to 40 % of the added microbial biomass remained in the soil. The remaining carbon may be bound in lifeless E.coli cells and residues or have been incorporated into soil microbial biomass or have been transformed to SOM compounds. • Conclusions • Microbial C added to soil is only temporarily bound in the original cells (if E.coli is representative for microbial biomass in general). • Viable (luminescent) E.coli cells declined rapidly and were not detectable after 15 weeks, whereas DNA could still be extracted after more than 23 weeks. Thus the genetic information seems to be preserved longer than the entire cells. The DNA may be • situated in non-living cells/compartiments, • transformed into other cells or • stabilized on humic substances. • The added C has a considerably longer residence time in soil than the cell components as indicated by the mineralization curve. The remaining C (ca. 40% of the added C) may be • assimilated by the natural or remaining in residual (E.coli) biomass (only temporarily) or • transformed into SOM and stabilized. References: [1] Haider, K., 1999, Z. Pflanzenernährung und Bodenkunde, 162, 363-371 [2] Näveke, R., Tepper, K.P., 1982, Einführung in die mikrobiologischen Methoden, Lehrstuhl für Mikrobiologie der TU Braunschweig [3] Rozen, Y., et. al., 1998, Chemosphere, 38(3), 633-641 [4] Berthelet, M., et. al., 1996, FEMS Microbiol. Lett. 138, 17-22 [5] Kästner, A., 1996, Forschungsbericht, Untersuchungen ökologischer und methodischer Faktoren beim Nachweis rekombinanter Mikroorganismen im Boden [6] Jacobsen, C.S., 1995, Applied and environmental microbiology, 61 (9) 3347-3352 Acknowledgements This study was supported by the DFG (SPP 1090). a