Functional Microbial Diversity and Bacterial Flora in Common Effluent Treatment Plants of South Gujarat and Their Application

Abstract

Background: Centralized industrial wastewater treatment processes encounter intense issues for removal of complex, toxic and refractory compounds present due to variety of industries providing effluents to them. Development of robust bioremediation process of such compounds at large working scale is a challenge. Conventionally developed organisms screened for good bioremediation potential in lab face major failures due to organism’s inability to survive in competitive environment of industrial effluents and inefficient scale up of bioprocess. As native microbial community is sustaining in effluent throughout treatment process of common industrial effluents it was hypothesized to look for bioremediation solution into them. Objective: This study was aimed to understand the metabolic capacities of wastewater microbial communities using substrate level physiological profiling. Using similarity analysis, it focused on isolating and screening of the most abundant bacteria having capability of hydrocarbon degradation and high survival rates. This study targeted statistical optimization of bioremediation process and scale up to achieve field level application. Methodology: Wastewater samples from 9 CETPs were collected in their operational conditions. Physico-chemical parameters of samples were analysed. Culturable fraction of microbial community belonging to functionally important groups of general microbial population, organisms involved in various nutrient cycling and filamentous bacteria and fungi were analysed. Community Level Physiological Profiling was used to calculate functional diversity indices and Principle Component Analysis was used to cluster the samples according to their microbial metabolic similarity. Isolation of most abundant bacteria was executed by similarity analysis using Amplified Ribosomal DNA Restriction analysis. Hydrocarbon degradation efficiency of screened organism were confirmed using spectrophotometric analysis and HPLC methods. Optimization of various factors for hydrocarbon degradation was performed using Plackett Burman and RSM like efficient statistical approach. The developed process was scaled up to 100 l capacity. Pathway of hydrocarbon degradation was analysed by gene amplification, SDS PAGE and estimation of enzyme activity from crude lysate. Observations and inferences High population of Nitrogen fixers (Azotobacter and Rhizobium), Sulphate reducing bacteria, Ferric reducing bacteria and Phosphate solubilizers found indicated prevalence of wide range of activity in effluents, whereas Fungi, Actinomycetes and Ammonia oxidizers were found in least abundance. The substrate utilization capacity of samples in Ecoplate® was found to be considerably high (30 out of 31 C sources) indicating high microbial diversity with even distribution of species. Sixty cultures were isolated on basis of abundancy and were compared using 16S rDNA restriction profiling (ARDRA). Finally, selected isolate designated as the most abundant bacteria was identified as Pseudomonas citronellolis, with 30.02% abundance in wastewater effluents. This bacterium was able to degrade 1 and 5 mM SB up to 97.05% and 98.6% respectively within 24 h. As this bacterium was able to degrade a model hydrocarbon, there was no need for genetic manipulation proposed as third objective. Using statistical tools for designing experiments, the bioprocess was optimized and later scaled up to 100 l of CETP wastewater. The pathway for benzoate degradation was found to be chromosomal and based on Benzoate 1,2- dioxygenase followed by catechol 2,3 dioxygenase leading to production of muconate or Hydroxy Muconic Semialdehyde (HMSA). Data obtained from trascriptome analysis of substrate induced organism is under progress.