Nitrate removal potential of different microbial consortia, feasible for wastewater treatment in RAS M.
1,2 Iordan ,
1Institute
A.C.
1 Dumitrache ,
I.I.
1 Ardelean ,
C.
1* Moisescu
of Biology Bucharest , Romanian Academy, Splaiul Independenei 296, Bucharest 060031 2RAJA Strada Răscoalei 1907, 5, Constanța 900178 *Corresponding author:
[email protected]
Abstract In Recirculating Aquaculture System (RAS) the water effluent from fish tanks is reused after cleansing with respect to organic substances, nitrate (NO3-), nitrite (NO2-), ammonia (NH4+) and phosphorus (PO4-) ions [1, 2, 3, 4]. One of the most used and promising cleansing techniques in modern RAS is the biological treatment of wastewaters performed by denitrifying bacteria which can convert the NO3- to N2. In this study we estimated the aerobic denitrifying performances of different microbial consortia isolated from various sources, feasible to be further used in RAS. The kinetics of NO3consumption in either batch or discontinuous conditions, on synthetic culture media with acetate or ethanol as sole carbon source and nitrate as the Results main final electron acceptor, has been characterized. The experimental data showed that the selected bacterial populations can clean water with efficiencies up to 90%, matching the requirements of international laws and making them suitable for employment in wastewater treatment in RAS.
Ratio C/N
mg NO3 /50ml mg NO3 /100ml activated Variant activated sludge/24 h sludge/24 h
250-1 250-1 250-1 250-1 250-1 250-1
a b a b a b
mg NO3 /1:3 Ratio C/N SAM/24 h
mg NO3 /50ml Ratio C/N Variant activated sludge/24 h
37,23
10-1 5-1 10-1 5-1
33,04 36,02 31,89 35,95
a b a b
250-1 250-1 10-1 10-1
63,63 63,5 51,38 50,82
13,75 35,47 66,75 41,79
36,13
Table. 1. NO3 consumption rates for different C/N ratios.
Results obtained on enriched microbial consortia 250
5.00 A1 A2 A3
4.00
A4
NO3 reduction rate (mg/ L/h)
NO3 concentration (µg/ml)
200
AF BOD 150
PBB PBS Raja3 S1
100
S2 S3
3.03 3.00
2.00
0
0.00 20
40
60
80
100
120
2.77
2.67
2.54 2.04
1.89
2.20
2.05
1.35 1.00
0
2.81 2.55
50
0.91
A1
140
A2
A3
A4
AF
Time (h)
BOD
PBb
PBs
Raja 3
S1
S2
S3
Microbial consortia
Fig. 2. NO3 removal rates of the denitrifying microbial populations.
Fig. 1. Denitrification capacity of the enriched denitrifying microbial consortia isolated from different sources.
48h 180
0.45
160
0.4
144h
140
0.35
120
0.3 NO3--N
100
0.25
OD660
80
0.2
60
0.15
40
0.1
20
0.05
NO3 removal efficiency (%)
100
Growth (OD660)
Bacteria, media and culture conditions. Different microbial consortia were isolated from different sources (microbiota from a fish farm biofilters and waters (A1, A2, A3, A4), activated sludge from a municipal waste water treatment plant (Raja 3), an aquaria biofilter (AF, BOD) and three soil samples (S1, S2, S3)) and enriched in (micro)aerophilic denitrifying bacteria using either acetate or ethanol as carbon source. Enrichment was performed according to literature [4] by using a series of transfers onto different selective media and maintained on denitrification medium (DM) slants at 4℃ in our laboratory (g/L): sodium acetate, 4.72; NaNO3, 1.62; MgSO4·7H2O, 1; KH2PO4, 1.50; Na2HPO4, 0.42; NH4Cl 0.6; casamino acids 5; trace element solution, 2.00 mL; pH 7.0-7.2. In experiments to test the effect of C/N ratio, ethanol served as the sole carbon source, and the concentration was varied to yield different C/N ratios (5-1, 10-1, 250-1). Reduction of nitrate. To obtain rapid population expansion for denitrification assay, cells were revived by aerobic cultivation for 24h, at 30℃ in DM medium. Cells were collected by centrifugation at 6500 r.p.m at 4℃, washed twice with water saline solution (0.9% NaCl) and 1% cell suspensions were inoculated into experimental flasks with nitrate denitrification medium (NDM, g/L): sodium acetate 5; NaNO3, 0.37; MgSO4·7H2O, 1; KH2PO4, 1; K2HPO4, 1; CaCl2 ·2H2O, 0.2; FeSO4 ·7H2O, 0.05; trace element solution, 2.00 mL; pH 7.0-7.2. For this assay, all of the cultivations were conducted at 30C without agitation. Time-dependent consumption of NO3 was measured using the Spectroquant® Nitrate test kit (1.09713.0001-Merck). All the NO3 concentrations were calculated using a standard curve prepared with Nitrate IC-STD solution 119811 (Merck).
Results obtained on enriched activated sludge
NO3 concentration (µg/ml)
Materials and methods
90 80 70 60 50 40 30 20 10 0
0
0 0
24
48
72
96
120
144
Time (h)
Fig. 3. Growth of BOD microbial consortia and its denitrification performance.
A1
A2
A3
A4
AF
BOD
PBb
PBs
Raja 3
S1
S2
S3
Microbial consortia
Fig. 4. NO3 removal efficiency of the isolated microbial consortia.
Conclusions Twelve consortia of microorganisms were isolated and enriched with respect to denitrification activity. All of them were able to reduce the NO3 concentration to at least 86% in 5 days cultivation time, one (A4) having a 100% efficiency in the first 48h. The conducted experiments gave us a clear idea of the effectiveness of each microbial consortia in depleting the nitrates in polluted wastewater and the stationary state solutions were found to match the requirements of international laws, making them feasible to be further used in RAS. The enriched populations together with expected purified strains could be further used to increase the cleaning/filtration capacities in fish farms (one task of ABAWARE Project) and (municipal) waste water plants, as well. 1. Dalsgaard, J., Lund, I., Thorarinsdottir, R., Drengstig, A., Arvonen, K., Pedersen, P.B., Farming different species in RAS in Nordic countries: Current status and future perspectives, Aqua. Engin. 2010 , doi:10.1016/j.aquaeng.2012.11.008 2. Chen R, Deng M, He X and Hou J Enhancing Nitrate Removal from Freshwater Pond by Regulating Carbon/Nitrogen Ratio. Front. Microbiol. 2017; 8:1712 doi: 10.3389/fmicb.2017.01712 3. Crab, R., Avnimelech, Y., Defoirdt, T., Bossier, P., Verstraete, W.,. Nitrogen removal techniques in aquaculture for a sustainable production. Aquaculture 2007 ; 270: 1–14 4. Takaya N, Catalan-Sakairi MAB, Sakaguchi Y, Kato I, Zhou Z, Shoun H. Aerobic denitrifying bacteria that produce low levels of nitrous oxide. Appl Environ Microbiol. 2003; 69:3152-3157
Acknowledgements: This work is funded by ABAWARE Project, financed under the ERA-NET Cofund WaterWorks2015 Call. This ERA-NET is an integral part of the 2016 Joint Activities developed by the Water Challenges for a Changing World Joint Programme Initiative (Water JPI). Thanks are also due to Prof. Dr. Crăciun Nicolae (AQUATERRA) , partner in ABAWARE project, for kindly providing microbiota from fish farm biofilter (Plutoniţa), PhD Corina Moga (DFR Systems) for kindly providing the SAM, and Vatca Ciprian (Merck) for analytical advices.
