Vol. 14(6), pp. 489-499, 11 February, 2015 DOI: 10.5897/AJB2014.14311 Article Number: 8E5FCEB50352 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB Full Length Research Paper Exploitation of molecular genetics in microbial degradation and decolorization of industrial waste water effluent Shah M. P. Industrial Waste Water Research Laboratory, Division of Applied and Environmental Microbiology Environmental Technology Limited Gujarat, India. Received 11 November, 2014; Accepted 9 February, 2015 Activated sludge samples were used to analyse microbial community structures from the anoxic and aerobic zones of a laboratory-scale modified Ludzack-Ettinger system. Fluorescent in situ hybridization and denaturing gradient gel electrophoresis were applied for analysis. With the help of DNA specific fluorochrome DAPI, approximately 75 to 80% of total cells were detected, hybridised with a specific eubacterial probe for the anoxic and aerobic zones. Results corroborate the dominance of the alpha and gamma subclasses of the Proteobacteria in the anoxic zone whilst the aerobic zone was dominated with the beta subclass of the Proteobacteria. Genetic diversity of the microbial community present in each of the anoxic and aerobic zones was employed by the DGGE technique. Results were obtained from the application of fluorescent in situ hybridisation (FISH) and PCR-DGGE yields a more precise understanding of the microbial community structure and genetic diversity present in domestic wastewater of a laboratory scale treatment process. Nitrogen mass balances indicated an upset in the nitrogen levels for wastewater batches two and seven. The carbon mass balance fell in the range of 92.4 and 105.9% and the nitrogen mass balance fell in the range of 98.4 and 160.0%. Key words: Fluorescent in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), COD and nitrogen mass balances. INTRODUCTION The growth of the world population, the development of suspended solids and other constituents. These biodegra- various industries and the use of fertilizers and pesticides dable organic compounds are degraded by bacteria in an in modern agriculture has overloaded not only the water aerated reactor and the biomass is allowed to settle and resources but also the atmosphere and the soil with pollu- concentrate in a clarifier (Muyima et al., 1997). The tants (Shah et al., 2013). Treatment of activated sludge system is either a continuous or semi-continuous aerobic within the modified Ludzack-Ettinger (MLE) system involves method for wastewater treatment involving carbon oxidation the removal of biodegradable organics, unsettleable and nitrification. This process has been developed for the *Corresponding author. E-mail: [email protected]. Tel: +91-9099965504. Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 490 Afr. J. Biotechnol. removal of carbon, nitrogen and phosphate and it is well Table 1. Process parameters recorded at the time of known that prokaryotic micro-organisms catalyses these sampling. main biological processes in wastewater treatments (Juretschko et al., 2002); nutrients and oxygen present Parameters Value the microbial population in the wastewater achieves pH of the digester 7.1 optimal growth and respiration (Muyima et al., 1997). The Total Solid g/L 22 dynamics and diversity of the microbial populations in Organic loading rate applied kgCOD/m3 day 4.5 activated sludge have been analysed by culture-dependent Influent COD mg/L 41,300 methods, however many members of the natural bacterial VFA mg/L 517 communities are still unculturable (Wagner et al., 1994). Acetic acid mg/L 275 Hence, microscopic identification based on morphological COD removal efficiency % 93 characteristics was researched and developed in a Methane % v/v 56 culture-independent manner by direct rRNA sequence 3 Biogas rate m /day 1200 retrieval, where nucleic acid probes which are comple- mentary to the rRNA are used as tools to monitor popula- tion dynamics amongst bacteria (Amann, 1995). FISH makes use of rRNA targeted probes which are frequently between microbial communities (Kaewpipat and Grady, applied in order to quantify the composition of microbial 2002). In this study, a combination of molecular techniques, communities present. This procedure is based on the FISH and PCR-DGGE was used to monitor the microbial composition and examine the microbial community comparative analysis of macromolecules, mostly ribosomal population shifts within a steady state laboratory-scale RNA molecules and fluorescent derivatives of such probes. These probes have been applied successfully for in situ parent anoxic and aerobic activated sludge system. enumeration of defined groups of micro-organisms present in activated sludge (Manz et al., 1994). MATERIALS AND METHODS Fluorescently monolabelled, rRNA targeted oligonu- cleotide probes detect individual cells, allowing whole-cell Sampling and bioreactor set-up hybridisation with rRNA targeted probes to be a suitable Activated sludge was collected from a 500 m3 closed digester tank tool inferring phylogenetic evolution hence the cell (CDT) for the anaerobic treatment. The CDT was operated under morphology of an uncultured microbe and its abundance mesophilic condition (32 to 39°C) for 120 days. The system was can be determined in situ (Wagner and Amann, 1997). equipped with a closed digester tank, settling tank, pumps and flow meters for biogas and the effluent. There were three sampling ports Cell numbers of the bacteria can be obtained by enume- at the top, in the middle and at the bottom of the CDT. The sludge ration under an epi-fluorescent microscope. Enumeration sample was obtained from middle sampling port. The process procedures involve the use of a semi-automated digital parameters recorded at the time of sampling are shown in Table 1. image analysis tools in order to quantify the fluorescently labelled bacteria in samples (Daims et al., 2001). The Analyses molecular technique used for analysing the structure and providing a profile of the microbial population present in The laboratory-scale modified Ludzack-Ettinger process was fed wastewaters is the denaturing gradient gel electrophoresis with 30 ℓ of mainly domestic wastewater on a 10 day sludge age (DGGE) (Muyzer et al., 1993). DGGE that is performed process. After reaching steady state, the MLE system was run for duration of 18 batches. The wastewater was diluted with tap water on 16S rRNA genes has been used to produce a genetic to give an influent feed of approximately 500 COD mg /L from the fingerprint of mixed microbial communities (Kaewpipat and original 800 COD mg /L. The process was maintained by wasting Grady, 2002). The numbers of bands that are obtained 1.5 L/day of the mixed liquor from the aerobic reactor. Samples from DGGE profiles provide an estimate of the different from the influent, anoxic, aerobic and effluent were analysed daily microbial species present. The intensity of each band for the following: chemical oxygen demand (COD), total Kjeldahl provides a reflection of the relative abundance of each nitrogen (TKN), mixed liquor suspended solids (MLSS), volatile suspended solids (VSS) and nitrate analysis (nitrite tests were not species (Nasu, 2000) because the primers used to amplify performed because the nitrite concentration was 2% less than a fragment of the 16S rRNA produces a quantitative relation- nitrates). Microbial community analyses were performed on ship between the gene copy number and the PCR-DGGE samples obtained from the aerobic and anoxic reactors. The band intensity (Kaewpipat and Grady, 2002). However, oxygen utilisation rate (OUR) was monitored within the mixed liquor many factors can prevent the formation of the number with an online dissolved oxygen (DO) controller (hi-tech micro- and intensity of the bands in the DGGE gel, therefore system) according to Randall et al. (1991). The pH was kept constant at 7.5 and the temperature was kept constant at 20°C. representing the exact number and abundance of species in a microbial community can be difficult however DGGE is a sensitive and a rapid technique that detects most Total cell counts singe-base variations when a G-C clamp is added to one Membrane filtration was performed according to Hicks et al. (1992). of the primers in the PCR process. This provides a profile Fixed samples were sonicated and diluted (dilution factor of 200) of changes that occur within a microbial community or with phosphate-buffered saline (PBS) and 1% nonidet. Nucleopore Shah 491 filters with a pore size of 0.2 μm were pretreated with 0.3% Sudan The recombinant plasmids were extracted using the Qiagen black and placed on top of a 0.45 μm backing filter. The samples plasmid DNA extraction kit (Qiagen, Germany) according to the were stained with DAPI (4,6-diamidino-2-phenylindole) (Sigma, manufacturer's instructions. Approximately, 27 nucleotides were Deisenhofen, Germany) at an end concentration of 1.25 μg/mℓ. The sequenced using an ABI 3730 XL DNA Sequencer. Chromato- Zeiss Axiolab microscope (50 W high-pressure mercury bulb and grams were edited using Chromas software (Technelysium, Zeiss filter set 01) fitted for epifluorescence microscopy was used Australia), while CHECK_CHIMERA software (Maidak et al., 1997) with the Zeiss image analysis software in order to quantify the fluo- was used to scan for potential chimeric sequences. The sequences rescing cells. were compared to known 16S rRNA sequences in the GenBank™ database, using the basic logical alignment search tool (BLAST) to locate nearly exact matches in the GenBank database. DNA Hybridization and pretreatment sequences were aligned using the program CLUSTAL W (Thompson et al., 1994) and further edited manually. Phylogenetic The fixed and sonicated samples were immobilised onto pretreated analyses were performed using the neighbor-joining (NJ) method slides and dehydrated with 60, 80% and absolute ethanol to using the MEGA ver. 3.1 (Kumar et al., 2004). prepare for whole-cell hybridisation.