ABSTRACT
2,2-Dibromo-3-nitrilopropionamide (DBNPA) has been used as a biocide in industrial water applications due to its instantaneous antimicrobial activity and rapid chemical breakdown. In this study, DBNPA is considered a potential alternative for antibiotics used for bacterial control during corn-to-ethanol fermentation. A method using LC/MS/MS was developed to accurately quantify DBNPA in water. When this method was applied to quantify DBNPA concentration in a fermentation matrix, DBNPA was found to be unstable and to decay rapidly, preventing validation of the method or quantitation. This method was then used to evaluate the degradation rate of DBNPA in whole stillage, which is the nonvolatile residue produced by removal of ethanol from corn-based fermentation beer by distillation through the relative decrease in measured signal. In addition, a method was developed and validated to quantify bromide, one of the degradation products of DBNPA, in whole stillage using LC/MS/MS. The degradation rate of DBNPA in whole stillage was found to display first-order kinetics with a calculated half-life of 85 min. Laboratory analytical chemistry results on DBNPA degradation were confirmed based on a bacterial viability assay in field trials.
Subject(s)
Disinfectants , Tandem Mass Spectrometry , Fermentation , NitrilesABSTRACT
The DNA of Bacillus subtilis bacteriophage SP10 is partially resistant to cleavage and methylation in vitro by restriction enzyme R . BsuRI and its cognate methylase even though greater than 20 copies of the target sequence, 5' ... GGCC ... 3', are present on the phage genome. YThy, a hypermodified oxopyrimidine that replaces a fraction of the thymine residues in SP10 DNA, was responsible for this protection, since YThy-free DNA was no longer resistant. Sites that were normally resistant could nevertheless be cleaved or methylated in vitro if the salt concentration was reduced or dimethyl sulfoxide was added to the reaction buffer. Analysis of the termini produced by cleavage suggested that resistant sites occurred in the sequence 5' ... GGCC-YThy ... 3', whereas sensitive sites, of which there were only two per genome, occurred in the sequence 5' ... GGCCG ... 3'. These in vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. They also suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation. YThy also strongly protected 5' ... GCNGC ... 3' (R . Fnu4HI) sequences on SP10 DNA, but the biological relevance of this protection is unclear.
