Studies On Microbial Community Diversity And Activities In The Integrated Vertical-Flow Constructed Wetland

Microbial population and activities, enzyme were studied in the Integrated Vertical-flow Constructed Wetlands (IVCWs) with down-flow chamber and up-flow chamber, in this paper. The nitrification-denitrification and respiration in different sites of IVCWs were investigated and the spatial characteristics of the biochemistry activities of the microbe were acquired. The biodegradation of the most frequently identified PAE, di-n-butyl phthalate (DBP) were investigated by using the mediums of IVCWs, and the effects of DBP on enzyme were also performed. These revealed the responses of the constructed wetlands to the special pollutant. The DGGE, RAPD and PLFA were successfully employed in the study on the microbial ecology of the constructed wetland, and the research methods were well developed. The main results are shown as follows: The studies on the microorganisms in the IVCWs showed that bacteria, fungi and actinomycetes were distributed widely in the medium, and the microorganism amounts were different in different layers. The aerobic microorganisms of the 0-10 cm layer in both of the up-flow chamber and the down-flow chamber were 1-2 magnitudes higher than that of the 30-55 cm layer. And the amounts of denitrifying bacteria were up to 107/g (dry soil) in the medium except of the 0-10 cm layer in the up-flow chamber. The enzymes activities and the amounts of bacteria physiological groups in the medium of the IVCWs were measured. The results showed significant temporal differences for enzyme activities during the year (p<0.05). Compared with the temporal characteristics, the spatial characteristics of all the six enzymes were identical: the enzyme activities in the down-flow chamber were significantly higher than those in the up-flow chamber (p<0.05), and all of the enzyme activities in both chambers were relatively lower with the increase of the depths. The amounts of bacteria physiological groups reached a peak in June and September, and their spatial characteristics were the same with the enzyme activities. The studies of biochemistry activities in the medium showed that nitrification-denitrification existed prevalently in the IVCWs. The nitrification in different layers of medium was different, and the nitrification rate in the 0-10 cm layer was 20 percents higher than that in the 40-55 cm layer. But the spatial difference of denitrification rate was little in the whole system and the lowest value amounted to above 85.5%. The respiration, to some extent, was positive correlation with the microorganism amounts. The effects of the DBP and its biodegradation were examined by comparison of two small scale plots (SSP) of IVCWs. The results showed the responses of different enzymes to the DBP were different. Compared the results of the treatment plot with the control plot, DBP enhanced the activities of dehydrogenase and catalase and inhibited the activities of urease, cellulase and β-glucosidase. And urease, cellulase, β-glucosidase activities were even unrestored one month following the final DBP addition. Degradation of DBP was greater in the surface soil and was reduced in sterile soil, indicating that this process may be mediated by aerobic microorganism. DBP degradation fitted a first-order model, and the kinetic equation showed that the rate constant was 0.0994 and 0.0345 d-1, the half-life was 6.97 and 20.09 d, and the r2 was 0.99 and 0.98, in the surface and subsurface soil, respectively. These results indicated that constructed wetlands are able to biodegrade organic PAEs such as DBP.DGGE and RAPD, two molecular biological methods based on the Polymerase Chain Reaction, were used to study microbial community diversity especial the bacteria diversity of different sites in IVCWs. The results of the two methods were similar: The amplification bands of the upper layers of medium were more than those of the sub-layers in the both of up-flow chamber and down-flow chamber, which in the up-flow chamber were more than those in the down-flow chamber compared with the same depths. The genetic similarities were 0.571-1.000 and 0.492-0.967, Shannon-Viener Index were 0.3772 and 0.3031, revealed by DGGE and RAPD, respectively. The results showed there was higher genetic diversity of bacteria in the constructed wetland. The cluster analysis indicated the amplification bands in the same depth layers of two chambers clustered together firstly. The phospholipid fatty acids of the medium in the IVCWs included saturated, unsaturated, branched, cyclopropane and hydroxy fatty acids, and the contents of the different fatty acids were distinct. In both chambers, the contents of bactPLFA, G-PLFA and G+PLFA were negative correlation with the depths, but the contents of fungiPLFA were extremely significantly lower than that of bactPLFA, and which decreased with the increase of the depth. The principal component analysis results showed the relation of two surface layers in both chambers were the nearest, the subsurface layer in the down-flow chamber were in the next place, and the other sites clustered together by themselves.