Estimation of Microbial Diversity in Poultry Litter Using Terminal Restriction Fragment Length Polymorphism and Isolation of Phosphate Accumulating Bacteria from Poultry Litter

The contamination of fresh water by phosphates in poultry litter results in substantial eutrophication of fresh water causing fish kills and other types of environmental damage. The poultry indus try in Kentucky is expanding rapidly. The number of broilers is increasing as more poultry farms are established in the state producing waste that needs disposal. Investigations were made to study the possibility of using microorganisms normally found in poultry litter to sequester phosphate, thereby delaying phosphate runoff after litter is applied to croplands. Little is known, however, about the microflora of poultry litter. Terminal restriction fragment length polymorphism of 16S rDNA from bacteria was used to investigate the bacterial diversity of poultry litter. Poultry litter was collected from a local producer. DNA was isolated using commercial kits and amplified using the polymerase chain reaction with primers specific for bacterial 16S rDNA. The amplified fragments were digested using HhaI restriction endonuclease and the DNA fragment lengths were determined. To determine the sensitivity of this method, known quantities of Escherichia coli cells were spiked into litter prior to DNA extraction. Successful amplification of the bacterial rDNA was highly variable but could be improved by passing the purified DNA through two purification columns in lieu of only one column. The detection threshold for E. coli was 10 cells, however, the results also varied widely. Bacteria capable of hyper-accumulating intracellular phosphate were isolated from poultry litter as possible tools for phosphate remediation in poultry litter. Five strains of phosphate accumulating bacteria were successfully isolated from poultry litter. Poultry litter was suspended in sterile nanopure water and 100μl was plated on BHI plates containing an addtional 750mM K2HPO4. Isolated colonies were screened for intracellular metachromatic granules using the Nile blue stain, a presumptive test for polyphosphate. Positive colonies were cultured in BHI and BHI with supplementation of K2HPO4 and free intracellular phosphate concentrations were determined in cell extracts. Total phosphates were measured in cell extracts subjected to hydrolysis by addition of 12N HCl and heating at 100°C for 60 min. Polyphosphate was determined by subtraction of free phosphates from total phosphates. Results showed five isolates of gram-positive bacteria were obtained from poultry litter. All isolates were cocci arranged in chains or clusters and were catalase positive. All isolates showed considerable levels of intracellular phosphate accumulation, which were comparable to Microlunatus phosphovorus, a bacterium known to hyper-accumulate phosphate. Biolo g analysis indicated four of the five strains isolated were Staphylococcus sp. and one strain was unidentified