Insights in Waste Management Bioprocesses Using Genomic Tools.

Microbial capacities drive waste stabilization and resource recovery in environmental friendly processes. Depending on the composition of waste, a stress-mediated selection process ensures a scenario that generates a specific enrichment of microbial community. These communities dynamically change over a period of time while keeping the performance through the required utilization capacities. Depending on the environmental conditions, these communities select the appropriate partners so as to maintain the desired functional capacities. However, the complexities of these organizations are difficult to study. Individual member ratios and sharing of genetic intelligence collectively decide the enrichment and survival of these communities. The next-generation sequencing options with the depth of structure and function analysis have emerged as a tool that could provide the finer details of the underlying bioprocesses associated and shared in environmental niches. These tools can help in identification of the key biochemical events and monitoring of expression of associated phenotypes that will support the operation and maintenance of waste management systems. In this chapter, we link genomic tools with process optimization and/or management, which could be applied for decision making and/or upscaling. This review describes both, the aerobic and anaerobic, options of waste utilization process with the microbial community functioning as flocs, granules, or biofilms. There are a number of challenges involved in harnessing the microbial community intelligence with associated functional plasticity for efficient extension of microbial capacities for resource recycling and waste management. Mismanaged wastes could lead to undesired genotypes such as antibiotic/multidrug-resistant microbes.

Characterisation and optimisation of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste.

Eight aerobic bacterial strains were isolated from pulp paper mill effluent sludge. Out of eight through nutrient enrichment technique three potential aerobic bacterial strains ITRC S(6), ITRC S(7) and ITRC S(8) were found capable to effectively degrade the kraft lignin (KL), a major byproduct of the chemical pulping process and main contributor to the colour and toxicity of effluent. Further, these potential strains (ITRC S(6), ITRC S(7) and ITRC S(8)) were biochemically characterised as Gram variable small rod, Gram negative rod and Gram positive rod respectively. Subsequently, 16S rRNA sequencing showed 95% base sequence homology and it was identified as Paenibacillus sp. (AY952466), Aneurinibacillus aneurinilyticus (AY856831), Bacillus sp. (AY952465) for ITRC S(6), IITRC S(7) and ITRC S(8), respectively. In batch decolourization experiments Bacillus sp. ITRC S(8) reduced the colour of lignin amended mineral salt medium, pH 7.6 by 65% after 6th d, at 30 degrees C, A. aneurinilyticus ITRC S(7) by 56% and Paenibacillus ITRC S(6) 43%. Under these conditions the three strains degraded the KL by 37%, 33% and 30%, respectively while the mixed culture of these three bacteria reduced colour by 69%, lignin by 40% and total substrate by 50% under same conditions. Biodegradation of the KL was not affected by low (<0.2 mg l(-1)) dissolved oxygen content; thus oxygen inhibition is more likely to be a metabolism-dependent event. Initially with 48 h incubation the decolourization was slow with decreased pH. Further incubation there was rapid decolourization with slight increase in pH at 6d compared with initial pH by increasing culture optical density. The lignin analysis from medium with HPLC indicated complete degradation rather than biotransformation with complete loss of absorbance peak at 280 nm.

Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

BACKGROUND: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. RESULTS: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. CONCLUSIONS: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Tracking of phenol degrading genotype.

A simple and rapid protocol has been developed for monitoring of the phenol degrading population in the environmental samples. PCR protocol uses the total DNA prepared from the samples as a template in the amplification reaction. Primers have been designed from the sequence data for Dmp and Phe operon encoding multi-component and single component phenol oxidizing system, respectively. The phenol degrading genotype in various samples have been demonstrated using the developed PCR protocol.

Detection of etiological agent for cholera by PCR protocol.

BACKGROUND: PCR protocol for Vibrio cholerae, the causative agent of the diarrheal disease cholera has been described in this report. We report the detection of Vibrio species in drinking water samples in a duplex PCR reaction. The target loci used in the study were ctxA and tcpA. The sensitivity and efficiency of detection of this protocol can be applied in epidemic conditions, wherein monitoring of target organisms is very crucial. MATERIAL AND METHODS: Single step thermocycling programs have been reported for amplification of specific target loci. We have demonstrated that a gradient multi-step thermocycling program is more efficient in improving the sensitivity of detection by PCR. The method for preparation of template DNA from environmental sample uses filtration of water followed by harvesting the possible bacterial residue. This was further treated with proteinase K and heat to yield DNA compatible for PCR. The protocol was optimized for amplification of target loci from standard strains as well as from environmental water samples. RESULTS: The method can simultaneously detect two different loci of Vibrio cholerae in a single reaction. The sensitivity of detection achieved for the pathogen was 100 cells per reaction. The specificity of the primers was demonstrated by spiking the reaction with non-specific template. The developed protocol was successfully extended to environmental samples. CONCLUSIONS: The developed duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogen from water samples. This enhances the efficiency of detection and provides a sensitive tool for the rapid detection of the pathogen that can be useful in epidemic conditions where the time factor involved in the identification of target pathogen is very crucial.

Detection of enteropathogens by PCR.

The widespread problem of contaminated water by enteric microorganisms necessitates the need to develop a rapid protocol to detect pathogens in water bodies. Usual methods like plating, biochemical tests and use of DNA probes are time consuming which is a limiting factor especially in epidemic situations. Moreover, some cells can exist in a virulent viable but non-culturable state making detection very difficult by plating method. This paper describes a rapid method to detect the enteropathogens,E.coli, Salmonella andVibrio and enteric viruses from water samples by Polymerase Chain Reaction.E.coli was specially chosen as it is an indicator of fecal contamination. The sensitivity ofE.coli detection was improved to 10 cells to keep in tune with the WHO guidelines. The presence ofE.coli would also indicate the probable presence of other pathogens.