Michael Krogsgaard Nielsen

Bacterium-based NO2- biosensor for environmental applications

ABSTRACT A sensitive NO2− biosensor that is based on bacterial reduction of NO2− to N2O and subsequent detection of the N2O by a built-in electrochemical N2O sensor was developed. Four different denitrifying organisms lacking NO3− reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO2− tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO3− and N2O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N2O reductase. The macroscale biosensors constructed exhibited a linear NO2− response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO2−, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO2−, and interference was observed only with NH2OH, NO, N2O, and H2S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO2− biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO2− biosensor were constructed and used to measure NO2− profiles in marine sediment.

Role of acetate in production of an autoinducible Class IIa Bacteriocin in Carnobacterium piscicola A9b

ABSTRACT Carnobacterium piscicola strain A9b isolated from cold smoked salmon inhibits growth of the food-borne pathogen Listeria monocytogenes partly due to the production of a proteinaceous compound (L. Nilsson, L. Gram, and H. H. Huss. J. Food Prot. 62:336-342, 1999). The purpose of the present study was to purify the compound and describe factors affecting its production, with particular emphasis on food-relevant factors. Amino acid sequencing showed that the compound is a class IIa bacteriocin with an N-terminal amino acid sequence identical to that of carnobacteriocin B2. The production of the bacteriocin was autoinducible, and the threshold level for induction was 9.6 × 10−10 M. We also report, for the first time, that acetate acts as an induction factor, with a threshold concentration of 0.3 to 12 mM. Acetate could not act as an inducer during the late exponential phase of C. piscicola A9b. The induction of bacteriocin production showed a dose-dependent relationship at acetate concentrations of up to 10 to 20 mM (depending on the growth medium) and at a concentration of 1.9 × 10−8 M for the bacteriocin itself; a saturation level of bacteriocin specific activity was reached at these concentrations of induction factors. The combined use of both inducers did not enhance the saturation level of bacteriocin production compared to that seen with the use of each inducer alone. Increasing NaCl and glucose concentrations negatively influenced the efficiency of acetate as an induction factor. Based on the results, carnobacteriocin B2 was used as an induction factor to manipulate the production of bacteriocin in cold smoked salmon juice and thus improve the ability to inhibit L. monocytogenes.

A sensitive trimethylamine-N-oxide aldolase assay in two steps without deproteinisation

A two-step assay for trimethylamine-N-oxide aldolase (the target enzyme) is described in which the second step, the indicator reaction, is of the enzymatic end-point type. This indicator reaction is carried out at a slightly alkaline pH, outside the target enzymes active pH range. An effective inactivation of the target enzyme is thus obtained solely by shifting the pH to that of the indicator reaction, thereby avoiding a deproteinisation step. By inserting a downward pH step as the stopping method, many samples may be collected and applied to the indicator reaction simultaneously. These features make the method very productive and easy to perform. © 2000 Society of Chemical Industry