Paper Title
Detecting the Functional O-xylene Gene in a Novel Microbial Communities and Monocultures Isolated from Iraqi Wastewater Treatment Plant

Abstract
The efficacy of the biodegradation processes for organic compounds within sewage is a major concern for wastewater treatment plants, especially in third world countries such as Iraq. Consequently, there is a need for microbial quality assessment at the Iraqi WWTP to then integrate information on microbial ecology into full-scale operations and designs. For petroleum hydrocarbon removal, the biodegradation process is commonly used and globally popular. This is due to its ability to enhance the efficiency of the treatment processes and, as a result, improve the quality of the effluent wastewater. There is, however, still an important knowledge gap on the dynamic structure and composition of wastewater microbial communities with regard to the metabolism of different organic pollutants. Therefore, the current study aimed to detect the functional o-xylene gene in a novel microbial communities and monocultures isolated from Iraqi wastewater treatment plant. Bioaugmentation process could enhance or speed up biodegradation process in Iraqi WWTP. It provides findings on the ecology of o-xylene degradation using a novel bacterial community that were enriched and isolated from the four important treatment phases (combining phase, activated sludge, settlement tank and river discharge) of Iraq’s WWTP. Molecular biology analyses consisting of DNA extraction, PCR, and DGGE were employed to reveal how bacterial diversity changed under different concentrations of o-xylene 0.5, 5 and 50 mM, pH 6.5 and incubation temperatures of 35°C. The effects of o-xylene concentrations were measured after 14-day incubations of 10 ml triplicate microcosms. The develop 15-member of the synthetic consortium and sequencing analysis identified its components as Serratia ficaria strain NCTC12148, Serratia marcescens SM39, Echerichia coli strain ST540, Echerichia coli strain C8, Stenotrophomonas sp LM091 ,Stenotrophomonas rhizophila strain DSM14405, Bacillus subtilis strain MJ01, Bacillius subtilis strain GQJK2, Klebsiella variicola strain HKUOPIA, Pseudomonas aeruginosa S66938, Pseudomonas sp STFLB209, Pseudomonas aeruginosa S86968, and Streptomyces sp Strain RKND-187. The final degradation efficacy recorded as 87.23% at the highest concentration of 50 mM, 35°C and pH 6.5. Also, higher values were recorded for both richness and diversity showing a direct relationship between ecological indices, molecule concentration and incubation temperature. To determine XYLE and TOL gene presence for the synthetic bacterial consortium, DNA extracts of total communities from the four phases of the Iraqi WWTP and each individual isolate were studied with a range of PCR primers. The XYLE primers revealed positive results for the xylA and xylM genes, which suggested an o-xylene catabolic pathway that followed the dioxygenase enzyme route. In contrast to its absence, the addition of o-xylene at different concentrations resulted in an increase in bacterial diversity for all four phases of the Iraqi WWTP. Therefore, the current study delivers a preliminary understanding of the effects of one of the most recalcitrant organic compounds at Iraqi WWTP. It also proposes an application for bioaugmentation using indigenous microbial communities within the treatment plant, . Notwithstanding this, new questions come to the fore, from practical solutions to physiological mechanisms. For example, could the influent wastewater organic loading be reduced more sustainably by using a microbial community which employed a complex compound like o-xylene as its sole carbon source? In summary, degradation is used to propose that cooperation between process engineering and microbial ecology may be appropriate and viable for efficient biological wastewater treatment processes in Iraq. For a thorough assessment of the wastewater microbial community’s total and functional structure, it is important to apply high-throughput approaches such as next-generation sequencing. Identifying gene regulatory patterns and their functional populations at the translational and transcriptional levels is another important step forward. Keywords - DGGE: Denaturing Gradient Gel Electrophoresis, DNA: Deoxyribonucleic Acid, O-xylene: Ortho Xylene, PCR: Polymerase Chain Reaction, TOL: Toluene, WWTP: Wastewater Treatment Plant, XYLA: XyleneA, XYLE: XyleneE, XYLM: XyleneM.