Confirmation of E. coli among other thermotolerant coliform bacteria in paper mill effluents, wood chips screening rejects and paper sludges

Paper sludges are solid wastes material generated from the paper production, which have been characterized for their chemical contents. Some are rich in wood fiber and are a good carbon source, for example the primary and de-inking paper sludges. Others are made rich in nitrogen and phosphorus by pressing the activated sludge, resulting from the biological water treatments, with the primary sludge, yielding the combined paper sludge. Still, in the absence of sanitary effluents very few studies have addressed the characterization of their coliform microflora. Therefore, this study investigated the thermotolerant coliform population of one paper mill effluent and two paper mill sludges and wood chips screening rejects using chromogenic media. For the first series of analyses, the medium used was Colilert broth and positive tubes were selected to isolate bacteria in pure culture on MacConkey agar. In a second series of analyses, double selective media, based on ß-galactosidase and ß-glucuronidase activities, were used to isolate bacteria. First, the presence of thermotolerant coliforms was detected in low numbers in most water effluents, but showed that the entrance of the thermotolerant coliforms was early in the industrial process. Also, large numbers of thermotolerant coliforms, i.e., 7 000 000 MPN/g sludge (dry weight; d.w.), were found in combined sludges. From this first series of isolations, bacteria were purified on MacConkey medium and identified as Citrobacter freundii, Enterobacter sp, E. sakazakii, E. cloacae, Escherichia coli, K. pneumoniae, K. pneumoniae subsp. rhinoscleromatis, K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. pneumoniae, Pantoea sp, Raoultella terrigena, R. planticola. Second, the presence of thermotolerant coliforms was measured at more than 3700–6000 MPN/g (d.w) sludge, whereas E. coli was detected from 730 to more than 3300 MPN/g (d.w.) sludge. The presence of thermotolerant coliform bacteria and E. coli was sometimes detected from wood chips screening rejects in large quantities. Also, indigenous E. coli were able to multiply into the combined sludge, and inoculated E. coli isolates were often able to multiply in wood chips and combined sludge media. In this second series of isolations, API20E and Biolog identified most isolates as E. coli, but others remained unidentified. The sequences of the 16S rDNA confirmed that most isolates were likely E. coli, few Burkholderia spp, but 10% of the isolates remained unidentified. T