The mechanism of carbonate killing of Escherichia coli.

AIMS: To define the mechanism of carbonate killing in Escherichia coli. METHODS AND RESULTS: Sodium carbonate (150 mM) and ethylenediaminetetracetic acid (EDTA, 60 mM) both killed E. coli K-12 when the pH was 8.5, but ammonium chloride (150 mM) was ineffective. EDTA was a 5-fold more potent agent than carbonate, but some of this difference could be explained by ionization. At pH 8.5, only 1.6% of the carbonate is CO(-2), but nearly 100% of the EDTA is EDTA(-2). CONCLUSION: As carbonate and EDTA had similar effects on viability, cellular morphology, protein release and enzymatic activities, the antibacterial activity of carbonate seems to be mediated by divalent metal binding. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle manure is often used as a fertilizer, and E. coli from manure can migrate through the soil into water supplies. Previous methods of eradicating E. coli were either expensive or environmentally unsound. However, cattle manure can be treated with carbonate to eliminate E. coli, and the cost of this treatment is less than $0.03 per cow per day.

The effect of various carbonate sources on the survival of Escherichia coli in dairy cattle manure.

Manure slurries (n = 3) prepared from the feces and urine of lactating dairy cattle (1 part urine, 2.2 parts feces, and 6.8 parts distilled water) had an initial pH of 8.6 +/- 0.1; dissolved carbonate concentrations of 48 +/- 4 mm, and Escherichia coli counts of 5.9 +/- 0.7 logs per ml slurry. The pH of untreated slurries declined to pH 7.0 +/- 0.1 by the 10th day of incubation, and the E. coli count increased approximately 10-fold (P < 0.05). When slurries were treated with Na2CO3, K2CO3, NaHCO3 or Na2CO3.NaHCO3 (0 to 16 g/kg slurry), the dissolved carbonates increased in a linear fashion, but only Na2CO3 and K2CO3 (8 g/kg or greater) or Na2CO3.NaHCO3 (16 g/kg) ensured an alkaline pH. Even relatively low concentrations of Na2CO3 or K2CO3 (8 or 12 g/kg) caused a decrease in E. coli viability (P < 0.05), and E. coli could not be detected if 16 g/kg was added (day 5 or 10 of incubation). Na2CO3.NaHCO3 also caused a decrease in E. coli viability, (P < 0.05), but some E. coli (approximately 104 cells per g) were detected on day 10 even if the concentration was 16 g/kg. NaHCO3 did not prevent the decrease in pH or cause a decrease in E. coli numbers (P > 0.05). Calculations based on the Henderson-Hasselbalch equation (pH and dissolved carbonates) indicated that little E. coli killing was noted until the dissolved carbonate anion concentrations (CO3-2) were greater than 1 mm, but bicarbonate anion (HCO3-) concentrations as high as 180 mm did not affect E. coli viability. These results are consistent with the idea that carbonate anion has antimicrobial properties and can kill E. coli in dairy cattle manure.

Kinetics, stereospecificity, and expression of the malolactic enzyme.

Mass spectrometric measurement of carbon dioxide production was used to study malolactic fermentation (MLF) in Lactobacillus collinoides isolated from cider. The kinetics and stereospecificity of the malolactic enzyme (MLE) were studied, and the stoichiometry of the reaction sequence was investigated. The optimum pH for activity of the MLE was 4.9. MLF was more rapid (in both intact cells and cell extracts) when L-malic acid was used than when D-malic acid or the racemic mixture was added. The enzyme was found to be constitutively present in L. collinoides. Addition of L-malic acid (37 mM) to the growth medium resulted in increased MLE activity; addition of the D isomer alone or the racemic mixture resulted in lower activities. Addition of the main sugars in apple juice (fructose, sucrose, and glucose) to the growth medium in the presence of malic acid repressed production of MLE to similar extents in all three cases; in the absence of malic acid, instead of inhibiting MLF, addition of sugars to the growth medium somewhat increased the residual MLE activity.