Structural And Functional Analysis Of Thauera Genus In Wastewater Treatment Plants

It has been well recognized that the performance of a wastewater treatment plant (WWTP) is mainly determined by the structure and activity of its microbial community. Thauera genus has been known as one of the functionally important groups, which has been widely found in WWTPs and mostly shown high versatile organic substrate degrading capacity. However, our understanding of the structure and function of Thauera genus in WWTPs was still limited, due to its resistance to isolation and lack of group-specific analysis method.Therefore, a Thauera-specific nested-PCR denaturing gradient gel electrophoresis (DGGE) method was firstly developed, and its usefulness was demonstrated by monitoring the structural shifts of Thauera spp. in an anaerobic-anoxic-oxic fixed-biofilm coking wastewater treatment plant (WWTP) responding to operational perturbations. The specificity of the PCR method was demonstrated by the fact that all 16S rRNA gene sequences, which were cloned from the amplicons of a biofilm sample, belonged to Thauera genus. 16S rRNA gene V3 region was then amplified from the first round Thauera-specific PCR product and applied for DGGE analysis. Different amplified fragments of Thauera clones, with 13 different V3 regions, migrated into 10 positions on DGGE gel, which demonstrated the high resolution of this DGGE method.When the WWTP experienced a gradual deterioration in chemical oxygen demand (COD) removal function due to a mechanical failure of the recirculation pump, biofilm samples were collected from the reactor and analyzed by this method. Principal component analysis (PCA) of the DGGE fingerprinting data showed that the composition of Thauera group exhibited a time related trajectory when the plant's COD removal rate decreased from 84.1±2.7% in the first 4 weeks to less than 75% at week 5 and 6, suggesting the structural shift of Thuaera genus was closely related with the system's COD removal function.A new isolation method that was guided by Thauera-specific PCR and DGGE fingerprinting was developed to get purified cultures of the Thauera spp. from the WWTP. According to the physiological characteristics of known Thauera strains and the living environment of the target bacteria, six types of media were designed. The biofilm from the denitrifying bioreactor of the coking WWTP was inoculated to these media and cultured under both aerobic and anaerobic conditions, the diversity of Thauera spp. that grew under different conditions were analyzed by Thauera-specific PCR-DGGE. Two media (1/10 NB and MMQ) which recovered higher diversity of Thauera spp. and lower number of colonies were used to isolate Thauera sp. under aerobic condition. The colonies were screened by Thauera-specific PCR. The homogeneity of colonies showing positive PCR signals was then checked by DGGE. The colonies with multiple species were further purified by being streaked on different selective media. The positive colonies were tracked by Thauera-specific PCR, and their homogeneity analyzed by DGGE. Finally, three Thauera strains (Q4, Q20-C and 3-35) were isolated to pure cultures. Guided with group-specific PCR and DGGE method, the efficiency and sensitivity of bacterial isolation can thus be significantly improved.The functional genes and pollutants-degrading capacity of these Thauera isolates were then characterized. Although sequencing analysis showed they had identical 16S rRNA genes, their ERIC-PCR fingerprinting patterns were significantly different (similarity <65%), indicating wide variation of genomic structures. The degradation of organic pollutants in coking wastewater by these strains was studied with gas chromatography-mass spectrometry (GC/MS). All the main organic pollutants (phenol, methylphenol, quinoline and indole) in the coking wastewater, except quinoline, were degraded by them under aerobic condition. Their phenol degradation rates were different (Q4> 3-35> Q20-C). However, within all the eight Thauera species, only T. phenylacetia has ability to degrade one of these aromatic compounds (phenol) under aerobic condition. Nitrite reductase gene (nirS) was detected in Q20-C, but none of these strains showed denitrification capacity. However, all the known Thauera species were denitrifiers. These results suggested the Thauera strains that isolated from coking WWTP may represent a new Thauera species, and have high versatile aromatic compounds degrading capacity.To understand the roles of Thauera spp. played in nitrogen removal in WWTPs, the denitrification genes and functions of 8 Thauera strains (T. aminoaromatica, T. linaloolentis, T. phenylacetica, T. terpenica, Thauera sp. DNT-1, Thauera sp. 27, Thauera sp. 28 and Thauera sp. 63) that originated from different environments were studied. All the strains emitted little NO (<50 nmol per flask; < 32.9 nM in liquid) during the denitrification process. All of them were able to transform nitrate to N2, except T. phenylacetica, which produced N2O as the final product and was found to be lack of N2O reductase (nos) gene. Analysis of the denitrification of T. aminoaromatica under different pH,O2 and NOx condition, indicated that in its activity pH range (pH 7-9), less intermediates (NO and N2O) were produced at higher pH level. In comparison to nitrate, the N2O production was significantly increased (6-40 times) under nitrite condition; the O2 level that denitrification started was also increased from < 0.8μM (in nitrate) to > 4μM (in nitrite), showed the induction effect of nitrite on denitrification of T. aminoaromatica. The activity of N2O reductase was inhibited by O2, but the other denitrification enzymes were not affected. It improved our knowledge on the regulation mechanism of O2 on denitrification.In conclusion, a Thauera specific PCR-DGGE method was developed for analyzing the structure of this functionally important population in WWTPs. The Thauera strains in the coking WWTP, which were known as difficult to be isolated by conventional methods, were isolated under the guidance of this group-specific method. This study demonstrated the important roles of Thauera spp. in degradation of toxic organic pollutants and nitrogen removal in WWTPs, illustrated the impacts of different conditions on the denitrification of Thauera, and provided new insights, strains and methods for optimizing the function of WWTPs.