|Doerner, Kinchel - WESTERN KY UNIVERSITY|
|Mason, Bryan - WESTERN KY UNIVERSITY|
Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 4, 2008
Publication Date: January 6, 2009
Repository URL: http://hdl.handle.net/10113/27521
Citation: Doerner, K., Cook, K.L., Mason, B. 2009. 3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775. Letters in Applied Microbiology. 48:125-132. Interpretive Summary: Skatole is on of the most malodorous compounds produced as a consequence of anaerobic degradation of livestock waste materials. However, little is know about how skatole is produced (i.e., the biochemical pathway used) or way it is produced. This study attempted to address those questions by looking at skatole production by one of the only organisms known to produce it in large quantities, Clostridium scatologenes. Results showed that C. scatologenes produced skatole optimally at pH7.0 incubated at 33 or 37ºC. Rich medium, such as BHI, produced more skatole than a minimal medium. Addition of greater amounts of the amino acid substrate (tryptophane) to medium did not lead to higher skatole production. Information from this study may prove useful in understanding what factors influence skatole production in natural systems, such as swine waste lagoons.
Technical Abstract: Aims: 3-Methylindole (3-MI) is a degradation product of L-tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions which influence 3-MI production in Clostridium scatologenes ATCC 25775 were investigated. Methods and Results: Cells cultured in anaerobic brain heart infusion (BHI) grew well at initial medium pH values of 5.0, 6.0, and 7.0 and also grew well at 24, 33, and 37ºC. Extracellular 3-MI levels in cells cultured in anaerobic brain heart infusion (BHI) medium (pH 7.0) at 33ºC and 37ºC for 48 hrs were 828 ±22 µM and 698 ±25 µM, respectively. Extracellular 3-MI levels in cells cultured in anaerobic BHI medium (pH 7.0) at 33ºC and 37ºC for 72 hrs were 907 ±38 and 834 ±121 µM, respectively. Cells cultured in anaerobic tryptone-yeast extract medium (TY) at 37ºC at 48 hrs produced 104 ±86 µM 3-MI however addition of 1mM L-tryptophan failed to increase extracellular levels (113 ±50 µM 3-MI). Similarly, peptone (1%)-supplemented semi-defined medium with or without 1 mM L-tryptophan exhibited culture supernatant 3-MI levels of 9.5 ±3.9 and 11.6 ±1.4 µM, respectively. In anaerobic whole cell assays, BHI-grown cells displayed significantly higher indole acetic acid decarboxylation rates versus semi-defined-grown cells. However, specific IAA decarboxylation rates for cells growth in TY and TY plus 1mM L-tryptophan were not significantly different. Conclusions: In anaerobic BHI medium, C. scatologenes ATCC 25775 produced 3-MI optimally at pH7.0 incubated at 33 or 37ºC. Rich medium, such as BHI, produced more 3-MI than austere medium when expressed on a cell density basis. Addition of L-tryptophan to medium did not lead to statistically higher extracellular 3-MI levels. However, specific activity of IAA decarboxylation to 3-MI is under metabolic regulation as BHI-grown cell displayed 500-fold higher levels that SD-grown cells. The role of L-tryptophan in this regulation is unclear. Significance and Impact of Study: 3-MI production is under metabolic regulation in C. scatologenes ATCC 25775. This information may prove useful in understanding what factors influence 3-MI production in natural systems, such as swine waste lagoons.