Using bioluminescent biosensors for hazard analysis and critical control point (HACCP) in wastewater control.

Starting from a new approach for water pollution control and wastewater treatment plant management, the hazard analysis and critical control point (HACCP) quality concept, the interest for the development of new rapid and sensitive methods such as bioluminescence-based methods is evident. After an introduction of the HACCP procedure, a bibliographic study of the bioluminescence potentiality is presented and discussed.

Bioluminescence-based assay used for toxicity monitoring.

A new ATP bioluminescence-based method is proposed in order to examine the effect of toxic shock on microbial communities in urban wastewater treatment plant (WWTP). As the efficiency of WWTP must be improved, the occurrence and toxic effect of hazardous substances have to be evaluated (Directive 2455/2001/EC). Among bioluminescence assay with firefly luciferase is commonly used for intracellular ATP monitoring. A multienzyme systems catalysed by three enzymes (adenylate kinase, pyruvate kinase and firefly luciferase) for adenine nucleotides (AN = ATP+ ADP+ AMP) estimation is used for monitoring metabolic changes in activated sludge. The AN method provides better sensitivity than ATP quantification alone. The pool of intracellular and extracellular AN is constant during growth and the rate of intracellular AN is correlated with the microbial growth. In the present investigation, the extraction of total adenylate assay is optimized (chemical and mechanical extraction) and applied for monitoring the effect of the toxic shock (eg. Naphthalene) on indigenous biomass activity (intracellular and extracellular AN rates). In the same time, the removal of naphthalene is addressed Laboratory-scale reactors (4 L) are inoculated with urban activated sludge is under continuous-flow aeration. The reactor, containing non acclimated sludge enriched with naphthalene. The other reactor containing sterilised activated sludge is used as abiotic and volatilisation control system. In all this batch cultures, ATP and AN (intracellular and extracellular) rates and naphthalene concentrations are controlled. One of the objectives of this research is to determine a relation between the ATP/AN ratio and the biodegradation of naphthalene. Evidence is given which demonstrates that the ATP/AN parameter is a possible alternative for monitoring very rapid metabolic changes in complex microbial community such as activated sludge.

Spiroplasmas of Group I: the Spiroplasma citri cluster.

We propose that Group I spiroplasmas be subdivided into seven, rather than four, subgroups. The seven subgroups showed remarkable homogeneity when several representative strains were compared. Hybridization reactions between DNAs of representative strains within subgroups were generally at least 90 percent, and usually at least 80 percent co-migrating cell proteins were found. In addition, when plasmid DNA was excluded, profiles of restricted DNA among strains within subgroups were very similar. In contrast, comparisons between Group I subgroups showed substantial heterogeneity. This heterogeneity was indicated by DNA-DNA hybridization reactions as low as 10-20 percent and only 10-15 percent co-migrating cell proteins. Spiroplasma citri (subgroup I-1), the honeybee spiroplasma (subgroup I-2), and the corn stunt spiroplasma (subgroup I-3) are all pathogenic organisms with more or less limited host ranges. Strains of these three subgroups have been repeatedly isolated from affected hosts. Since strains of subgroups I-2 and I-3 can be clearly differentiated from other Group I subgroups and all other spiroplasmas, the DNA-DNA hybridization reactions of the subgroups do not exceed 70 percent, and because they are important pathogens, we propose (subject to completion of standard requirements for species descriptions) that they be recognized as new species of the genus Spiroplasma.

Guanine-plus-cytosine content, hybridization percentages, and EcoRI restriction enzyme profiles of spiroplasmal DNA.

The guanine-plus-cytosine (G + C) content of spiroplasmal DNA was calculated from the melting temperature determined spectrophotometrically and the buoyant density determined by equilibrium density gradient centrifugation in CsCl. Only two ranges of G + C values were found: 25-27 mol% and 29-32 mol%. The DNA of the following spiroplasmas has 25-27 mol% G + C: Spiroplasma citri (serogroup I-1); the spiroplasmas pathogenic to the honeybee (KC3, BC3, and B63; serogroup I-2); the corn stunt strain (E275; serogroup I-3); the tick strain 277F (serogroup I-4); the drosophila strain (serogroup II); and one group of flower spiroplasmas (serogroup III). The DNA of a second group of flower spiroplasmas (serogroup IV) and the SMCA strain (serogroup V) has a G + C content of 29-31 mol/. The classification of flower spiroplasmas into two groups on the basis of G + C content agrees well with the groupings based on serologic and protein analysis. Spiroplasmas isolated from honeybees in Morocco (B13) or froghoppers in Corsica (L89) have 29-31 mol% G + C, a value that corroborates the relatedness of these strains and the flower spiroplasmas of serogroup IV found by serologic analysis. Reannealing experiments between the vivo-labeled DNA of S. citri and unlabeled DNA of other spiroplasmas gave the following percentages of hybridization: 64% with honeybee spiroplasma DNA, 49% with corn stunt spiroplasma DNA, and 19% with tick spiroplasma 277F DNA; no significant hybridization was observed with DNA of any other spiroplasma. The taxonomic position of the tick spiroplasma 277F within serogroup I was confirmed by hybridization experiments involving [3H]DNA of this strain. The value of polyacrylamide gel analysis of DNA fragments produced by the action of EcoRI restriction enzyme on DNAs from various spiroplasmas is also discussed.