CARBON SOURCE UTILIZATION OF SOIL EXTRACTED MICROORGANISMS AS A TOOL TO DETECT THE EFFECTS OF SOIL SUPPLEMENTED WITH GENETICALLY-ENGINEEREDAND NON-ENGINEERED CORYNEBACTERIUM-GLUTAMICUM AND A RECOMBINANT PEPTIDE AT THE COMMUNITY-LEVEL

Substrate utilization of microbial cells extracted from soil with a 0. 85% aqueous sodium chloride solution, was determined to estimate effec ts on soil microorganisms at the community level with microtiter plate s (Biolog GN)) containing 95 different sources of organic carbon. A co nsistent pattern of utilized substrates was obtained after 24 h of mic rotiter plate incubation at 28 degrees C. The absorbance values (OD590 ) obtained from a microtiter plate reader after background correction were transformed by using the average absorbance values of oxidized su bstrates as a threshold to distinguish between well utilized and poorl y or non-utilized substrates and thereby reduce variances between repl icates. Doubling times of the extracted soil microorganisms in the mic rotiter plates were tested with 12 substrates and ranged from 1.96 h t o 3.23 h, depending on the carbon source. The carbon source utilizatio n assay was used to assess the effects of soil inoculation with Coryne bacterium glutamicum with and without a genetically engineered plasmid (pUN1: 6.3 kb), which encoded for the synthesis of the mammalian prot ease inhibiting peptide, aprotinin. Additionally, aprotinin itself was added at two concentrations to soil samples. An identical decrease in the number of carbon sources utilized, especially carbohydrates,occur red upon soil inoculation with both C. glutamicum strains after inocul ation with 10(6) cells g(-1) soil. This effect was only detectable dur ing the first three weeks of incubation, as long as cell numbers of C. glutamicum (pUN1) were above 10(5) cfu g(-1). Soil amendment with apr otinin resulted in utilization of additional substrates, most of them carbohydrates. With 0.1 mg aprotinin g(-1) soil this stimulation laste d 2 days and with 10 mg g(-1) it lasted for 7 days.