An Assessment of Variability of Pulp Mill Wastewater Treatment System Bacterial Communities using Molecular Methods

Abstract

The DNA fingerprinting techniques, 16S-restriction fragment length polymorphism (16S-RFLP), ribosomal intergenic spacer analysis (RISA) and repetitive extragenic palindrome PCR (Rep-PCR), were used for analyzing the bacterial communities of seven pulp and paper wastewater treatment systems. All three methods generate DNA fingerprints that can be compared using the computer-assisted program, Gelcompar©. Community similarity coefficients were based on quantitative determinations of both the positions of the DNA bands and the band intensities in order to compare the relative differences in the populations. Unique 16S-RFLP DNA fingerprints were observed for each mill suggesting that individual mills contained phylogenetically different communities. However this method was not sensitive enough to detect differences within a mill treatment system from different locations or from different sampling times. The RISA method, which generated more complex fingerprints than 16S-RFLP, could, for some mills, discern differences between samples. The Rep-PCR technique, however, showed the highest degree of resolution and produced not only distinct patterns for each mill but also distinct fingerprints for the temporal and spatial samples from some of the treatment systems. The sensitivity of this method might potentially be used to monitor the stability of the bacterial community within a secondary treatment system.

40-year long-term study of microbial parameters near Helgoland (German Bight, North Sea): historical view and future perspectives

“…DGGE, which has already been applied to our long-term studies of bacterial populations in the Helgoland Roads, was presented here. For future analysis of marine bacterial populations, we suggest a step-by-step approach beginning with PCR fingerprinting techniques based on native electrophoresis, such as ribosomal intergenic spacer analysis (RISA; Ranjard et al 2001) or the analysis of repetitive extragenic palindromic sequences (repPCR; de Bruijn et al 1996;Baker et al 2003), which can be analyzed by automated image analysis software. With a PCR-DGGE approach, resolving larger 16S-rDNA fragments (Seibold et al 2001;Schäfer et al 2002;Wichels et al 2004), it should be possible to identify OTUs by DGGE band sequencing and thus obtain a closer look at the bacterial community structure.…”

Review of Molecular Techniques for the Identification of Bacterial Communities in Biological Effluent Treatment Facilities at Pulp and Paper Mills

“…Similarities with and between mills were derived from this dendrogram, i.e., the similarity value of the node where all samples from one mill converge and defines similarity within-mill. Similarity between 16S-RFLP profiles from mills was partially correlated to wood furnish (Baker et al 2003). This method was not sensitive enough to detect differences within a mill treatment system from different locations or from different sampling times.…”

“…There are kits in the market such as DiversiLab, which allows for automated detection, standardization, and analysis using a software (Healy et al 2005). Baker et al (2003) analyzed the bacterial communities of seven treatment systems of pulp and paper wastewater. They compared RISA, RFLP, and REP-PCR methods and reported that the REP-PCR was the one that showed a higher degree of resolution, with different patterns for each sample at different times and sampling points.…”

“…In this study individual members of the communities were not identified (Rademaker and de Bruijn 1997). The sensitivity of this method might potentially be used to monitor the stability of the bacterial community within a secondary treatment system (Baker et al 2003). However, REP-PCR test is not an accurate tool for identifying organisms to the subspecies level (Mougari et al 2014).…”

“…RISA provides a community-specific profile, with each band corresponding at least to one organism of the original community. The RISA method can be used to generate more complex fingerprints than 16S-RFLP and discern differences between samples (Baker et al 2003).…”