Kinchel C. Doerner

To determine the source material and the responsible micro-organisms of 4-ethylphenol production in swine lagoon sediment slurries. Swine lagoon sediment was blended and incubated with tryptone-yeast extract broth containing 10 mmol l(-1) each of 4-hydroxycinnamic as well as other phenolics. 4-Ethylphenol was only produced from 4-hydroxycinnamic acid. Denaturing gradient gel electrophoresis analysis indicated that the microbial community was substantially altered from inclusion of 4-hydroxycinnamic acid. Serial dilutions and selective plating were employed to isolate a culture capable of 4-hydroxycinnamic acid conversion to 4-ethylphenol. Morphological and ribosomal gene analysis indicated the isolate to be a Lactobacillus sp., Lactobacillus sp. pep8 converted 4-vinylphenol, but not 4-hydroxy-3-phenylpropionate, to 4-ethylphenol and did not convert 4-hydroxy-3-methoxycinnamic acid to 4-ethyl-2-methoxyphenol. A p-coumaric acid decarboxylase (pdc) gene identical to Lact. plantarum pdc genes was cloned from Lactobacillus sp. pep8. Swine lagoon sediments produce 4-ethylphenol from 4-hydroxycinnamic acid due, in part, to the activity of Lactobacillus spp. 4-Ethylphenol, a malodourant of swine and beef wastes, is generated from plant materials by indigenous lactobacilli suggesting that altering the amount dietary of plant material may influence levels of 4-ethylphenol in the wastes.

To develop a defined medium for Clostridium scatologenes ATCC 25775, which produces the malodorants 3-methylindole (skatole) and 4-methylphenol (p-cresol). Clostridium scatologenes was cultured in anaerobic broth medium (pH 6.3) at 37 degrees C containing ammonia, minerals and a commercial vitamin solution. Data indicate alpha-ketoglutarate, l-glutamate or l-glutamine is a required nutrient that can also serve as a primary carbon and energy source. When cultured in defined medium containing glutamate; glucose, fructose and betaine served as primary carbon and energy sources. l-Tryptophan, l-tyrosine, sorbitol and indole acetic acid did not enhance growth. In the absence of tryptophan, cells produced indole when grown using glucose or fructose. 4-Methylphenol was produced when growing cells were supplied with tyrosine. When supplied with tryptophan, 3-methylindole was produced by glucose- or fructose-growing cells but not from glutamate-growing cells. Cells grown in the presence of pyruvate produced indole, 3-methylindole and 4-methylphenol. Clostridium scatologenes requires alpha-ketoglutarate, l-glutamate, or l-glutamine for growth in defined medium. Cells produce indole when glucose or fructose is included in defined medium. The development of a defined medium will assist in physiology studies and genetic analysis of this strain.

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 influencing 3-MI production in Clostridium scatologenes ATCC 25775 were investigated. Extracellular 3-MI levels in cells cultured in brain heart infusion (BHI) medium (pH 7.0) at 33 degrees C and 37 degrees C for 72 h were 907 +/- 38 and 834 +/- 121 micromol l(-1), respectively. Cells cultured in tryptone-yeast (TY) extract medium at 37 degrees C for 48 h produced 104 +/- 86 micromol l(-1) 3-MI; however, addition of 1 mmol l(-1) L-tryptophan failed to increase extracellular levels (113 +/- 50 micromol l(-1) 3-MI). Specific activity of indole acetic acid decarboxylase measured in BHI, TY and TY plus 1 mmol l(-1) tryptophan-grown cells displayed 35-, 33- and 76-fold higher levels than in semi-defined medium-grown cells. When cultured in rich medium, at 33 degrees C or 37 degrees C and pH 7.0, Cl. scatologenes ATCC 25775 optimally produced 3-MI. Addition of L-tryptophan to medium did not lead to significant increases in extracellular 3-MI levels. Whole cell assays indicate growth in rich medium significantly up-regulated 3-MI production. Information presented here may prove useful in understanding what factors influence 3-MI production in malodorous animal wastes.

To determine the effects of anaerobic electron acceptors on 3-methylindole (3-MI) and 4-methylphenol (4-MP) production in swine lagoon enrichments and Clostridium scatologenes ATCC 25775. Swine lagoon sediment was incubated anaerobically in tryptone-yeast extract medium with (10 mmol l(-1)) Na(2)SO(4), KNO(3), dimethyl sulfoxide or Fe(III). With Fe(III), 3-MI and 4-MP levels increased significantly to 138 +/- 15.8 and 187 +/- 14.0 micromol l(-1), respectively. Clostridium scatologenes cultured in brain-heart infusion medium amended with (10 mmol l(-1)) Na(2)SO(4), KNO(3), MnO(2) or Fe(III), resulted in only Fe(III) significantly increasing 3-MI (1308 micromol l(-1)) and 4-MP (367 micromol l(-1)) levels. In semi-defined medium, Fe(III) alone and Fe(III) + L-tryptophan (1 mmol l(-1)) resulted in a 1.85-fold and 15.6-fold increase in 3-MI levels over L-tryptophan alone, respectively. Fe(III) alone and Fe(III) + L-tyrosine (1 mmol l(-1)) caused a 4.4-fold and 22.9-fold increase in 4-MP levels over tyrosine alone, respectively. Fe(III) did not increase growth of Cl. scatologenes. Fe(III) increases 3-MI and 4-MP in swine lagoon enrichments and Cl. scatologenes broth cultures. Previous studies suggest Fe(III) addition to swine lagoons could remediate malodorous volatile fatty acids; however, here data suggest Fe(III) could increase malodorous indolic and phenolic levels.

To isolate and characterize micro-organisms from poultry litter capable of growing under phosphate concentrations typical of poultry litter. Poultry litter extracts were plated onto brain-heart infusion medium (BHI) containing an additional 0.75 mol l(-1) phosphate (BHI-P). Colonies were screened for the presence of inclusion granules with five being selected for further study. All strains displayed identical biochemical characteristics consistent with Staphylococcus spp. and grouped with Staphylococcus spp. by comparative 16S rDNA analysis. Thus all five strains were identified as such. All strains displayed elevated intracellular phosphate levels when cultured in BHI-P broth (0.417-0.600 microg phosphate mg(-1) protein) vs BHI broth (0.075-0.093 microg phosphate mg(-1) protein). When grown using an austere semi-defined medium or BHI-P, Staph. sp. #7 displayed similar elevated intracellular phosphate levels compared with growth in BHI. Poultry litter contains novel Staphylococcus spp. capable of robust growth when exposed to phosphate levels comparable with that typically found in poultry litter. Data suggest intracellular phosphate levels in these strains increase in response to increasing phosphate in the medium or austere medium conditions. Intracellular phosphate did not reach levels comparable with known hyper-accumulating micro-organisms. These data suggest poultry litter possesses a resident microflora that thrives and accumulates intracellular phosphate in response to high phosphate conditions.

To determine if mixed microflora from poultry litter accumulates phosphate when deprived of carbon and energy or nitrogen sources. Microbial enrichments from poultry litter capable of metabolizing ammonia, amino acids, and glucose were subjected to nutritional deprivation and the effects on intracellular phosphate levels were determined. Results indicate deprivation of glucose yields a 38 and 50% increase in intracellular phosphate and polyphosphate levels, respectively. Deprivation of nitrogen sources did not result in significant intracellular phosphate accumulation. Micro-organisms normally present in poultry litter respond to carbohydrate deprivation by accumulating intracellular phosphate. Poultry litter typically contains significant levels of phosphate which contribute to environmental pollution when applied to land. Phosphate is highly mobile in soils and often drains into local watersheds following rain events. This study raises the possibility that poultry litter micro-organisms may have the capacity to sequester phosphate, which could delay or diminish phosphate run-off.