Microbial Population Database for Evaluating Biological Nutrient Removal Process in Kwa- Zulu Natal Sheena Kumari, Deepnarain N, Pillay K and Bux F Institute for Water and Wastewater Technology, Durban University of Technology, P O Box 1334, Durban, 4000 E-mail: sheenas@dut.ac.za
Activated sludge process was conceived and developed by Arden and Lockett in early 19 s Simple yet revolutionary idea of recycling the biomass Early Activated sludge process From then WWTP have dynamically progressed in design and operational structure and complexity Johannesburg Activated sludge process
Key objectives of current activated sludge processes? Oxidise Carbonaceous matter Nitrogen removal Phosphorus removal Reduction of pathogenic organisms
Objectives are achieved by a heterogeneous microbial community that is organized into flocs with most bacteria forming micro-colonies that are attached to the floc backbone. Heterotrophs Nitrifiers Denitrifiers Phosphate accumulating Organisms Filamentous bacteria An activated sludge floc
Operational parameters affecting process performance: Influent wastewater characteristics Temperature Dissolved oxygen concentration ph Sludge age General process operation etc...
To create a microbial population database by correlating plant operational parameters to functional microbial population dynamics
Primary objectives Identify and quantify functional microbial populations and their composition in two BNR plants. Correlate microbial population to the plant operating conditions and wastewater characteristics. Create an efficient microbial population database for South African BNR plants.
COD/N/P removal efficiency, aeration, Temp, ph Process design, volume of tank, addition of chemicals, mechanical problems.. Floc structure, Morphology, filament index, abundance etc Microbial Database
BNR1 Original design- UCT process configuration Currently working without internal recycle from aerobic to anoxic Treating primarily domestic sewage RAS passes to anoxic then to anaerobic
BNR2 Johannesburg process configuration (original design) Working without inernal recycle from aerobic to anoxic tank Treating primarily domestic sewage RAS passes to a pre-anoxic tank first then to anaerobic tank
Sampling on weekly basis Samples for microbiological analysis was obtained from the primary compartments of the BNR plant viz., aeration basins, anoxic and anaerobic zone and the secondary settling tanks. Chemical analysis was done for influent, effluent and aerobic tanks. Chemical data were obtained from the respective plant operators and an in-house testing also was done to compare the results.
Probe Target site (rrna) and position Specificity (target groups) ARCH917a,b EUB338a,b EUB338II EUB338III NONEUBb EUK516c ALF1bc BET42aa,b GAM42ac HGC69a LGC354A LGC354B LGC354C Nitrifiers Ntspa712b NIT3c NSO190a NSM156c PAO s &GAO spao846pao462 PAO651GAO Q431 GAO Q989GB Filamentous GLP1Gor 596Goam 192MPA60G123 Type CHL 1851G1BSNA 16S (917 934) 16S (338 355) 16S (338 355) 16S (338 355) 16S ( ) Control 18S (502 517) 16S (19 35) 23S (1027 1043) 23S (1027 1043) 23S (1901 1918) 16S (354-371) 16S (354-371) 16S (354-371) 16S (712 732) 16S (1035 1048) 16S (190 208) 16S (156 174) 16S (846 866)16S (462-485)16S(651 668)16S(431 448)16S(989 1006)16S (989 1006) 16S(174 193)16S(596 617)16S(192 209)16S (59-76)16S(697 714)16S(592 611)16S(1029 1046)16S(656 673) Archae Most bacteria Planctomycetales Verrucomicrobiales EUB338 Eucaryota a-proteobacteria b-proteobacteria c-proteobacteria Actinobacteria Firmicutes Firmicutes Firmicutes Nitrospira spp. Nitrobacter spp. AOB Nitrosomonas spp. Most members of the Candidatus Accumulibacter cluster, Candidatus Competibacter phosphatis, Candidatus "Competibacter phosphatis", novel gammaproteobacterial group Gordonia spp Gordonia spp G.amarae M. parvicella Thiothrix sp. filamentous Eikelboom Type 1851 Eikelboom type 021N group I, S. natans DenitrifiersDEN124DEN67 16S(124 143)16S (67-84) Acetate-denitrifying cluster Methanol-utilizing denitrifying cluster
Few general observations RESULTS AND DISCUSSION COD and Phosphorus removal efficiencies were consistent for both the plants Inconsistency in ammonia removal Bulking and foaming (Excessive growth of filamentous bacteria) Low DO (<1 mg/l) Low F/M ratio (<0.3 mg/l) High MLSS (>5000 mg/l) due to insufficient sludge wastage Mechanical failure of the aerators (loss of nitrification)
RESULTS AND DISCUSSION Influent Anaerobic Anoxic Aerobic Clarifier Removal Efficiency (%) Ammonia 27.34 20.58 25.57 0.56 0.09 99.67081 Nitrate 0.7 0.73 0.44 0.44 3.16 Nitrite 0 0 0 0 0 Phosphates 3.36 17.14 14.95 1.18 0 100 COD 710 38 94.64789 Influent Anaerobic Anoxic Aerobic Clarifier Removal efficiency (%) Ammonia 14.86 21.93 22.23 11.34 12.25 44.14045 Nitrate 1.3 1.19 0.68 0.49 0.8 Nitrite 0.06 0.01 0 0 0.05 Phosphates 1.88 9.74 7.89 0 0 100 COD 905 36 96.0221
RESULTS AND DISCUSSION Fig. 1 Average nutrient removal efficiency of BNR 1
RESULTS AND DISCUSSION Fig 2 Average nutrient removal efficiency of BNR 2
inf COD inf Amm Aerobic Nitrite aer Nitrate aer Amm effi DO mg Temp ph FM ratio RESULTS AND DISCUSSION Table SPSS analysis: Nitrifier population vs operational condtions BNR1 ntspa nit3c nso190 nsm total nitrifiers amx aear den 67 den 124 total den significant Insignificant Very strong positive relationship Strong positive relationship weak positive relationship Very strong negative relationship Strong negative relationship weak negative relationship
RESULTS AND DISCUSSION Microbial populations were relatively stable. Influent COD and TKN level showed strong correlations to the total bacterial populations. Influent ammonia concentration and temperature showed a strong positive correlation with nitrifying bacteria and nitrification efficiency of the plant. However, these results were not consistent over the two years study period and thus require further validation.
RESULTS AND DISCUSSION At low temperatures the activity of nitrifying bacteria seems to be inhibited and not the population size. It is further confirmed by correlating ammonia removal efficiency to the above operating parameters.
ammonia efficiency (%) ammonia efficiency (%) ammonia efficiency (%) RESULTS AND DISCUSSION 100 80 60 DO vs ammonia removal efficiency 100 90 80 70 temperature vs ammonia efficiency 40 60 20 50 0 0.5 1.0 1.5 DO (mg/l) 40 20 25 30 Temperature ( C) 100 influent COD vs ammonia efficiency 90 80 70 60 50 40 600 700 800 900 COD (mg/l) Fig. The scatter plots obtained for Nitrification efficiency vs operational parameters
Filamentous bulking RESULTS AND DISCUSSION High SVI (>150mg/L) and SRT(25-30 days) Thick chocolate brown filamentous foam BNR 1- Thiothrix sp, Type 1851, Type 021 and Type 0092- dominant filament observed (low F/M filaments) BNR 2- Thiothrix, Type 0092,Type 1851, Type 0675, Type 1701 and M. parvicella Seasonal shift in filamentous abundance and floc structure The relative composition of the different species were unique for each plant giving a characteristic fingerprint.
RESULTS AND DISCUSSION Fig Bulking sludge showing filamentous proliferation
Filament abundance Filament abundance RESULTS AND DISCUSSION BNR1 6.00 5.00 4.00 3.00 2.00 1.00 0.00 T 1851 Snatans Thio T 0041 T021N T 0092 Filaments 2011 2012 BNR 2 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 T 0092 Nocardia Mparvicella Thio T 0041 T 1701 filaments Fig. Relative abundance of filamentous bacteria 2011 2012
RESULTS AND DISCUSSION BNR 1 BNR 2 30 25 25 20 20 15 S.natans T021N 15 Mparvicella Gordonia spp. 10 T 1851 T 0092 10 T 1701 T 0092 5 T 0041 Thiothrix spp. 5 T 0041 Thiothrix spp. 0 0 Fig. Seasonal Variation in filamentous abundance
Since the data obtained for filamentous bacteria are categorical the ordinal logistic regression analyses were employed to correlate the data. Filamentous bacteria Influent COD RESULTS AND DISCUSSION Table 6: Cumulative logistic output, illustrating significant and non-significant relations with the organism and operational parameters (*significant relationship). Influent ammonia Significant factors (Pr>ChiSq) DO F/M Temp ph Aerated Phosphor Type 1851 *0.0059 *0.0077 *0.014 *0.0001 0.603 0.1939 *0.0163 T ype021n *0.0380 *0.0604 0.2277 0.3425 0.2131 0.6833 0.3668 Type 0092 *0.0041 *0.0043 *0.008 *0.0160 *0.0028 0.3440 0.7628 Thiothrix spp *0.0172 0.3970 0.1844 *0.0033 0.7691 0.1860 0.1158 Type 0041 0.7663 *0.0123 0.7880 0.3850 0.5441 *0.0175 0.5086 S.natans *0.0400 *0.0788 *0.0778 *0.0312 0.2280 0.5434 0.1732 us.
RESULTS AND DISCUSSION Table Maximum likelihood with std error and Odds Ratio Factors Type 1851 Type 021N Type 0092 Inflluent COD Max likelihood Std error Point Estimate Max likelihoo d Std error Point Estimate Max likelihoo d Std error Point Estimate 1.3884 0.5043 4.009 1.0205 0.4919 2.775-1.588 0.5532 0.204 Temp - - - - - - 1.8823 0.6286 6.569 F/M 3.4963 0.7333 32.992 - - - -1.4602 0.6061 0.232 DO 1.7498 0.7125 5.754 - - - -1.9217 0.7256 0.146 Influent 1.4931 0.5600 4.451-1.0493 0.5588 0.350 1.7875 0.6286 0.167 NH 4 Phos 1.8526 0.7710 6.377 - - - - - - Factors Thiothrix spp Type 0041 S.natans Inflluent COD Max likelihood Std error Point Estimate Max likelihoo d Std error Point Estimate Max likelihoo d Std error Point Estimate 1.2614 0.5043 3.530 - - - 1.0236 0.4984 2.783 Temp - - - - - - - - - F/M 1.9805 0.6742 7.246 - - - 1.3050 0.6058 3.688 DO - - - - - - 1.1529 0.6538 3.167 Influent - - - 1.5334 0.6122 0.216 1.0324 0.4873 2.808 NH 4 Phos 1.1415 0.7257 3.131 - - - 0.9017 0.6620 -
RESULTS AND DISCUSSION Fig. The probability graph for Type 1851 at various operational conditions (F/M, phosphate, DO, Inlfunet COD and NH3)
RESULTS AND DISCUSSION Fig graphs display the probability of abundance of Type 0092 within each of the low (0) and high (1) factors (i.e. influent COD, F/M, Influent ammonia, DO and Temperature respectively).
RESULTS AND DISCUSSION High levels of COD, F/M, influent ammonia, DO, and phosphorus shows a greater probability that Type 1851 receives lower rankings of 2 or 3 Type 1851, Thiothrix and type 0047 are reported to be low F/M filaments (Jenkins et al., 2004). Can thrive both in aerobic and anoxic conditions and are capable of using both oxygen and Nitrite as electron acceptors High levels of COD, F/M, influent ammonia, DO and low temperature shows a greater probability that Type 0092 receives higher rankings Type 0092 are strictly aerobic (Jenkins et al., 2004)
Fig. Poly bar graphs of Type 1851.The graphs represents the probability of Type 1851being in abundance when all factors grouped together (influent COD, F/M, DO and influent ammonia)
Fig. Poly bar graphs of Type 021N Fig. Poly bar graphs of Type 0092
CONCLUSIONS The first intensive study on full scale BNR plants in South Africa Nitrification efficiency of the plants were affected by low DO, higher COD and low temperature No significant variation in microbial population (Heterotrophs, Nitrifiers, Denitrifiers and PAOs) except during the periods of mechanical failures of aerators Insufficient sludge wastage - Sludge blanket rising in the clarifier
CONCLUSIONS Filamentous bulking and foaming High COD, low F/M (<0.3 mg/l), low DO (<0.5 mg/l) contributed for filamentous proliferation Seasonal variation in the dominance of filamentous bacterial population More than one filamentous bacteria are involved in bulking and foaming incidents in these plants The relative composition of the different species were unique for each of these plants giving a characteristic fingerprint
Future Recommendations Evaluation of the efficiency of developed filamentous population model to other wastewater treatment plants across South Africa. Lab scale experiments to confirm the uniqueness of Type 0092 over other filamentous bacteria. The role filamentous bacteria on nutrient removal process also require a further verification at laboratory scale Microbial analysis using high-throughput sequencing to explore the microbial diversity Lab-scale experiments to explore nitrifying bacteria and its interaction within the system.
Acknowledgements Kingsburgh WWTW Umgeni WWTW Water Research Commission of South Africa Durban University of Technology
Thank You For Your Attention Tel: +27 31 373 2346 Fax: +27 31 373 2777 Email: sheenas@dut.ac.za Web: http://www.dut.ac.za/iwwt