TY - JOUR
T1 - Separating cellular metabolism from exoenzyme activity in soil organic matter decomposition
AU - Blankinship, Joseph C.
AU - Becerra, Caryl A.
AU - Schaeffer, Sean M.
AU - Schimel, Joshua P.
N1 - Funding Information:
We thank Robert Brown and Jim Casto in the Radiation Safety Program at University of California Santa Barbara for their help with gamma safety considerations and dose calculations. We thank Eugene Elmore for his critical help with accessing and operating the gamma irradiator in the Department of Radiation Oncology at University of California Irvine. We thank Kenneth Marchus for his help with laboratory preparation related to chloroform fumigation, autoclaving, and substrate-induced respiration. We thank Seeta Sistla and Kate Buckeridge for their help with enzyme assays. We thank Daniel Berg for his help with sterility and mineral catalysis tests. We thank Michael Weintraub for discussions about abiotic oxidative activities. We acknowledge the use of the NRI-MCDB Microscopy Facility and the instrumentation supported by the Office of The Director, National Institutes of Health under Award #S10OD010610. This work was supported by National Science Foundation grant DEB-1145875 .
PY - 2014/4
Y1 - 2014/4
N2 - Soil organic matter (SOM) decomposes both inside and outside of cells. Cellular metabolism and extracellular depolymerization normally operate simultaneously in soil but are difficult to separate in practice. To learn more about the extracellular component of SOM decomposition, we sterilized a semiarid annual grassland soil to inhibit cellular metabolism, and then assayed cell viability, exoenzyme activities, and pathways of carbon dioxide (CO2) emission. Chloroform (CHCl3) fumigation was intended to disrupt cellular activities while leaving biochemical processes intact. Gamma (γ) irradiation and autoclaving were intended to disrupt both cellular and extracellular biochemical processes while leaving abiotic processes intact. We measured the potential activities of eight enzymes (six hydrolytic, two oxidative) and CO2 emission induced by seven substrates (glucose, three amino acids, three tricarboxylic acid [TCA] cycle intermediates). We found that all three sterilization techniques clearly disrupted cellular metabolism. Chloroform and irradiation decreased cultivable cell counts by 2-3 orders of magnitude, inhibited CO2 emission pathways associated with glucose and amino acids, and decreased the hydrolytic activities of α-glucosidase and xylosidase by 72-82%. The other hydrolytic enzymes (β-glucosidase, cellobiohydrolase, NAGase, phosphatase) were less sensitive to both CHCl3 and irradiation. All hydrolytic activities that we assayed were inhibited by autoclaving, indicating that biochemical reactions and other extracellular processes drive hydrolytic SOM decomposition. Oxidative activities, on the other hand, did not stop after autoclaving or even combusting at 500°C. This supports other studies which have found that mineral catalysts partly drive oxidative SOM decomposition. Unexpectedly, CO2 emission from TCA intermediates decreased by only 26-47% after sterilization suggesting that the required dehydrogenase enzymes for decarboxylation are still active when cells are dead but relatively intact. Because CHCl3 had slightly smaller effects on exoenzyme activities compared to irradiation, and because it may be continuously applied, limiting the potential for recolonization and regrowth (unlike irradiation), we suggest it is an adequate and more accessible method for separating the activity of exoenzymes from cellular metabolism under realistic soil conditions.
AB - Soil organic matter (SOM) decomposes both inside and outside of cells. Cellular metabolism and extracellular depolymerization normally operate simultaneously in soil but are difficult to separate in practice. To learn more about the extracellular component of SOM decomposition, we sterilized a semiarid annual grassland soil to inhibit cellular metabolism, and then assayed cell viability, exoenzyme activities, and pathways of carbon dioxide (CO2) emission. Chloroform (CHCl3) fumigation was intended to disrupt cellular activities while leaving biochemical processes intact. Gamma (γ) irradiation and autoclaving were intended to disrupt both cellular and extracellular biochemical processes while leaving abiotic processes intact. We measured the potential activities of eight enzymes (six hydrolytic, two oxidative) and CO2 emission induced by seven substrates (glucose, three amino acids, three tricarboxylic acid [TCA] cycle intermediates). We found that all three sterilization techniques clearly disrupted cellular metabolism. Chloroform and irradiation decreased cultivable cell counts by 2-3 orders of magnitude, inhibited CO2 emission pathways associated with glucose and amino acids, and decreased the hydrolytic activities of α-glucosidase and xylosidase by 72-82%. The other hydrolytic enzymes (β-glucosidase, cellobiohydrolase, NAGase, phosphatase) were less sensitive to both CHCl3 and irradiation. All hydrolytic activities that we assayed were inhibited by autoclaving, indicating that biochemical reactions and other extracellular processes drive hydrolytic SOM decomposition. Oxidative activities, on the other hand, did not stop after autoclaving or even combusting at 500°C. This supports other studies which have found that mineral catalysts partly drive oxidative SOM decomposition. Unexpectedly, CO2 emission from TCA intermediates decreased by only 26-47% after sterilization suggesting that the required dehydrogenase enzymes for decarboxylation are still active when cells are dead but relatively intact. Because CHCl3 had slightly smaller effects on exoenzyme activities compared to irradiation, and because it may be continuously applied, limiting the potential for recolonization and regrowth (unlike irradiation), we suggest it is an adequate and more accessible method for separating the activity of exoenzymes from cellular metabolism under realistic soil conditions.
KW - Autoclave
KW - Chloroform fumigation
KW - Extracellular enzymes
KW - Gamma irradiation
KW - Soil sterilization
KW - Substrate induced respiration
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U2 - 10.1016/j.soilbio.2014.01.010
DO - 10.1016/j.soilbio.2014.01.010
M3 - Article
AN - SCOPUS:84893441602
SN - 0038-0717
VL - 71
SP - 68
EP - 75
JO - Soil Biology and Biochemistry
JF - Soil Biology and Biochemistry
ER -