Tunisian – Japan Symposium (TJS Water 2018) Innovation discovery For Sustainable Water Resource Management November 2018 Comparative study on pilots of Nitrogen removal performance in Constructed Wetlands between a partially saturated Vertical Flow and a Hybrid systems for rural wastewater treatment K. Kraiem 1, 2, H.Kallali 1 and N. Jedidi 1 1 Effluent Treatment and Valorization Laboratory (ETV), Water Research and Technology Center (WATREC) Tunisia, Technopark Touristic Road of Soliman, Nabeul PO-box N° Soliman, Tunisia 2 Faculty of Sciences of Tunis, University of Tunis El Manar, B.P. no ROMMANA, 1068 Tunis, Tunisia.

1. Introduction 2.Material and Methodes 3.Results 4.Conclusion and perspectives

Nitrogen is essential to the production of plant and animal tissue. It is used primarily by plants and animals to synthesize protein. But an overdose supply of nitrogen can be harmful to all of them. For that reason, the excessive discharge of wastewater without proper treatment directly into Soil degradation quality and quantity of plants Death of microorganisms P + N: Eutrophication Hypoxia Death of microorganisms

 Constructed wetlands represent an alternative to conventional technologies for nitrogen remove. It had an increasing attention due to : Simplicity and Low cost operation Have a great capacity to degrate pollutant from many different origins Environemental friendly  Several biological treatment technologies ( membrane bioreactor and activated sludge ) have been widely used to treat nitrogen polluted wastewaters Demand high operation costs they are still very limited in rural areas

What is a Constructed Wetland? Constructed wetlands are man-made systems, constructed to treat wastewater using the natural processes typical of natural wetlands. These natural processes are an interaction and combination of wetland plants, soil and microbial life.

Constructed wetlands Surface flow constructed wetland, where the surface of the water is exposed to the atmosphere Subsurface flow constructed wetland, where the surface of the water is below ground level. Vertical subsurface flow (VSSF), the water flows vertically through the substrate Horizontal subsurface flow (HSSF), the water flows horizontally through the substrate

Constructed Wetlands – Nitrogen removal in constructed wetlands

Constructed Wetlands – Limits of nitrogen removal in constructed wetland

Constructed Wetlands – Hybrid systems  The use of a single vertical flow constructed wetland with a saturated layer where an alternative microbial pathway ANAMMOX for nitrogen removal was discovered  This process can remove large amounts of ammonium and nitrite without the need high amount of oxygen or organic matter.  This alternative route decreases 40 % of greenhouse gas production compared to nitrification-denitrification.

The objective of this research was to evaluate the nitrogen removal in a subsurface vertical flow constructed wetland (SSVF) where the ANAMMOX process is favored by imposing a saturated zone at the down of the basin compared to a classic nitrification-denitrification process performed in a hybrid constructed wetland

Material and methods

25-40 mm:5 cm mm 2-4 mm 2-4mm : 60 cm mm Vertical flow constructed wetlands Horizontal constructed wetlands The four pilots were fed with rural wastewater taken from Khanguet Al Hojjej. ( withHydraulic Loading Rate (HLR) was 20 l/d given in five sequences with operation cycle 2 days feeding period followed by a 5 days resting period

Material and methods Chemical parameters Chemical water quality parameters were determined as described in the Standard Methods for the examination of Water and Wastewater [APHA, 2005.].  Total Kjeldahl Nitrogen (TKN): using Kjeldahl Method.  Ammonia (NH 4 + ), and nitrate (NO 3 - : using Spectrophotometer Bacteria identification  After four months of operation, biomass was taken from the media at - 25cm depth and biomass was taken from saturated layer at -40 cm depth for all pilot units.  All the biomass samples were prepared according to standard method of Fluorescence In Situ Hybridization (FISH) analysis (Zwirglmaier 2005).

Results  The results indicated that the pH at the outlet was greater than pH inlet for the four pilots units; this can be suppose there is anammox reaction pH inlet pH outlet SSVF 2 pH outlet SSVF 3 pH outlet SSVF 4 pH outlet (SSVF 1 + SSHF 1 ) 27 April6,967,437,237,437,78 25 May7.127,338,127,127,87 29 June6.457,767,517,677,56 27 July7.557,397,457,547,87 25 August7.387,547,437,647,67 means7.097,567,617,357,72

Results The statistical analysis showed that:  the design of pilots and plant species have a significant effect (P < 0.05) on the ammonium remove Average removal efficiency (%) of NH4+ for (SSVF1+SSHF1),(SSVF2),(SSVF3) and (SSVF4) 60% : Hybrid system 49%: subsurface flow constructed, 40% planted with Phragmites (SSVF2) 53%: subsurface flow constructed, 40% planted with Typha (SSVF3) 57%: subsurface flow constructed, 20% planted with phragmites (SSVF4)

Results  No significant difference between hybrid system and SSVF3  there is significant difference between SSVF3 and SSVF2 due to the effect of plant species  there is significant difference between SSVF3 and SSVF4 due to the effect of saturation height Average removal efficiency (%) of NTK of (SSVF1+SSHF1), (SSVF2),(SSVF3) and (SSVF4) 54% : Hybrid system 48%: subsurface flow constructed, 40% planted with Phragmites (SSVF2) 51%: subsurface flow constructed, 40% planted with Typha (SSVF3) 43%: subsurface flow constructed, 20% planted with Typha(SSVF4)

Results There is increase the concentration of nitrate in SSVF2, SSVF3 and SSVF4, this results may be explained by not all nitrate produced in unsaturated media were reduced in saturated media by conventional denitrification or by existence of another mechanism of nitrogen removal that produces nitrate, combined with denitrifiaction reaction concentration of nitrate of raw water,SSVF1+SSHF1),(SSVF2),(SSVF3), and SSVF4)

Results  FISH analysis showed that ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were detected in the four pilot units at -20cm depth.  SSVF1and SSVF4 had the high proportion of nitrifying bacteria, Relative abundance of AOB and NOB bacteria in SSVF1+ SSHF1, SSVF2 and SSVF3 and SSVF4

Results  Five genus of the most dominant phylum Proteobacteria reported in denitrification was identified in –40cm depth of each pilot.  The configuration and the plant species significantly affected the abundance and the composition of these denitrifying bacteria SSVF1+SSFH1SSVF2 SSVF4 SSVF3

Results SSVF4 SSVF1+ SSHF1 SSV3 SSVF2 Detection of anammox bacteria by FISH DAPI technique (all DNA (blue)), EUB338mix probe (all bacteria (fluos, green)), Amx368 probe (all anammox (Cy3, red) for the four pilots

Conclusions and perspectives  This research highlights the one stage with saturated layer cannot only get performance as good as two stage system but FISH analysis indicated that ANAMMOX process can be realized in constructed wetlands, which provide a potential new alternate process for enhancing the nitrogen removal with low operating costs and low required area  This study highlights also the role played by the plant species in favoring the ANAMMOX bacteria development  Optimizing ANAMMOX process in constructed wetlands needs more investigations such as the optimal depth of saturation and the overall depth of the VSSF, the speciation of ANAMMOX bacteria present in the biofilm populations, and effect of certain inhibitors.