In constructed wetlands, microorganisms associated with plants are assumed to play a major role. A one-year survey was conducted in five vertical flow constructed wetland systems that had been operating from 2 months to 8 years in small French villages (100–500 People Equivalent) to provide a better understanding of microbiological activity. The objective of our study was to highlight the most important factor generating variability between microorganisms communities compared to treatment performances. Results of community level physiological profiling using Biolog Ecoplates were analyzed using principal component analysis. The greatest microbial activity was observed in the oldest wetland during summer. Profiles of fed and rest bed were differentiated by the nature of the main carbon source metabolized. Whereas carbohydrates and carboxylic acids appeared to be better assimilated with fed beds, it seemed that phosphate compounds as well as amines allowed better growth in the plates inoculated with samples of rest beds. In all fed beds, the most important parameters affecting the diversity were the season and the age of the wetlands. There were only slight profile differences between surface and subsurface samples and between the first and second stage samples.
In constructed wetlands (CWs), it is widely recognized that microorganisms associated with macrophytes play a major role in pollutant removal. Over the last ten years a greater focus has been put on understanding microorganisms diversity and spatial distribution, on characterizing bacterial communities, or on monitoring microbial biomass. A large number of biochemical tools were successfully adapted to monitor microorganisms in CWs, such as epifluorescence, enzymatic activities, protein concentration, flow cytometry, fish probes, PCR, and DGGE [
In vertical flow constructed wetlands (VFCWs), pollutant removal efficiency (especially TKN removal) can be affected by several factors including season, CW age, and feeding mode (batch loads, periods of rest, etc.) Our study aimed at verifying whether treatment performance was linked or not to microbial diversity by applying the community-level physiological profiles (CLPPs) method using Biolog EcoPlate for constructed wetlands microorganism communities. The CLPP method using microtiterplates with multiple sole-carbon sources has become a popular tool for the comparison of microbial communities with respect to their functional potential. CLPP was successfully adapted to the study of complex communities [
We analysed microbial diversity in five VFCWs designed to treat raw wastewater. We inoculated a total of 46 plates during two sampling campaigns. The first campaign (25 plates) was realized in February, April, June, and October 2002 in 5 different sites and enabled to highlight season influence on bacteria community profile. The second campaign (21 plates) was performed in November 2003 in two sites to determine the vertical distribution of microbial activity in relation to CW age and feeding mode (fed and rest rotation).
The studied two-stage VFCWs, constructed by the Société d'Ingénieries Nature et Techniques, were designed to treat raw wastewater of small communities in the Rhone-Alpes region of France (Table
VFCWs characteristics.
Units | Glandieu | Queige | Allèves | Evieu | Colomieu | |
---|---|---|---|---|---|---|
People equivalent | Pe | 250 | 500 | 250 | 100* | 200 |
First stage area | m2 | 233 | 850 | 360 | — | 300 |
Second stage area | m2 | 164 | 750 | 230 | — | 225 |
Initial organic load | g BOD5 m-2 d-1 | 64 | 32 | — | — | 48 |
Starting date | year | 1999 | 1998 | 1999 | 2003 | 1993 |
* Evieu was only connected to 30 Pe for almost 3 months during the sampling campaign (November 2003).
Each stage was made of two to four beds fed according to 5–7 days rotation. Beds were 0.7 m. deep and planted with
Average performances (mass removal in %) observed in the first stage of the different VFCWs (adapted from Chazarenc and Merlin, [
Name | Spring-Summer (average) | Autumn-winter (average) | ||||
TSS | COD | N-TKN | TSS | COD | N-TKN | |
Allèves | 95.2 | 92.3 | 84.2 | 95* | 83.9* | 85.6* |
Colomieu | 96.6 | 93,4 | 98.4 | 96.1 | 92.3 | 76.7 |
Glandieu | 99.6* | 98.7* | 93.3* | 98.5* | 95.7* | — |
Queige | 92.5 | 91.0 | 77.7 | 90.1 | 83.0 | 38.3 |
* only one measurement available.
For the first campaign, samples were taken at Colomieu, Glandieu, Allèves, and Queige at four different dates, 2002 (March, April, June, October). For the second campaign, samples were taken at Colomieu and Evieu, in November 2003. A 60 cm long core, divided in three 20 cm sections, was taken at 50 cm of an inlet pipe. Between 2 and 15 grams of cores were first washed with a phosphate buffer solution (K2HPO4, 9.3 g L-1; KH2PO4 1.8 g L-1; pH = 7.2) to separate gravel and obtain a final solution concentration having 2% w/w of extracted matter and was screened on a 5 mm filter. This method was effective in normalising the cell number density in the final inoculum for the Biolog EcoPlate. Carbon sources utilization pattern was realized using Biolog EcoPlate (Hayward, CA, USA). Each plate contains 31 of the most useful carbon sources for soil community analysis [
The extraction solution was diluted 20 times using phosphate buffer and vortexed for 10 minutes. Finally 150
Microbial communities sampled in CWs were generally able to use all of the 31 carbon sources. The most important growths were observed on L-asparagine, L-serine, lactose, mannitol, acetyl glucosamine, glycogen, and cellobiose sources. As expected, elementary sugars or polymers such as glycogen were greatly metabolised, most probably in Kreb’s cycle by oxidative phosphorylation. Complex carbon source and carbohydrates provided the best growth, most probably because they are the more energetic compounds. The smallest microbial growths were reported on carbon sources containing aromatic cycle.
Principal component 1 accounted for 61% of total variability and was mainly associated to carbon source B1, D2, and E2 (Figure
PCA of Biolog data of the first sampling campaign. A code identifies each sample: the first two letters refer to the CW name, followed by I for first stage and II for the second; we referred to the depth as 20 for 0–20 cm, 40 for 20–40, cm and 60 for 40–60; the feeding mode was expressed as F for bed fed for 3 days, R for bed in rest for 3 days, and RR for bed in rest for 8 to10 days. For example, COIF20RR refers to Colomieu, stage I, 0–20 cm layer, in rest for 10 days.
Seasonal metabolic response of samples taken in surface of the fed bed, first stage of Colomieu.
Seasonal variations of bacterial community response in the upper layer of the fed bed in Colomieu illustrate well the seasonal changes observed in all samples (Figure
Figure
Ordination produced from PCA analysis of Biolog profile obtained during the second campaign. Samples connected with lines represent sampling from the same bed at different depths; full line: Colomieu stage 1; indented line: Colomieu stage 2; dotted line: Evieu. See legend in Figure
Carbon sources in Biolog EcoPlate wells. Codification matrix and groups of carbon source isolated (real configuration is in triplicate).
Profiles distribution along principal component 1 was mostly related to community diversity and activity. Slight differences were observed when comparing profiles for bed fed and in rest for 3 days in the first stage of Colomieu (1–3 compared to 4–6) (Figure
Because the second stages were shown to be mostly efficient for TKN removal [
One can suppose that wastewater greatly contribute to microbial establishment in CW; it could have been very interesting to compare profiles of influent versus CW effluent to see and study the wetland contribution as showed by Hench et al. [
Season was the most important parameter generating variability between bacterial community profiles. Small differences between same samples taken at different depth were observed except for the one of Colomieu 40–60 cm. Profile differences between fed beds and bed in rest for almost 10 days suggest that other degradation mechanisms such as endogenous respiration appeared over time (this could explain low sludge production in French VFCWs). Biolog Ecoplates are a quantitative tool for a simple comparison of microbial metabolic pattern in VFCWs. However care must be taken in interpreting results as the statistical analysis highlights mostly relative differences from a sample profile to another. Furthermore a wide range of bacteria such as strict anaerobic ones are not detected using microplates. Finally, this tool enabled us to see clearly differences between samples taken in the same CW and could be useful to optimize batch loading, to identify dead volumes or bypass, or to estimate dysfunction. In the future, wastewater contribution to bacteria communities should be studied.