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Combined COD & nutrients removal from slaughter-house wastewater in a sequencing batch reactor.


The industries like meat processing, slaughter-house, dairy, fermentation product, fertilizer, etc., discharge wastewater containing concentrated organic, nutrient--laden matter along with carbonaceous waste giving rise to high level of BOD and TKN. In aerobic stabilization of such wastewater organic / inorganic nitrogen is ultimately converted to nitrate as end product of metabolism. However, high level of nitrates in treated effluent to be discharged into the surface water is not desirable, since they are potentially toxic both for aquatic and human life. Formations of nitrosoamines, diseases like methanoglobinemia are the toxic effects to human being whereas eutrophication in lakes are caused by the discharge of these nutrients (nitrogen & phosphorus) which act as stimulants for algal bloom. Due to the carbonaceous and ammoniacal matters present in wastewater, D.O. level in the aquatic body where the wastewater is discharged, drastically depletes. Hence a treatment is very much necessary which would be combined in nature preferably by a cost-effective biological method.

The activated sludge process, which is especially suited for combined substrate (COD) and nutrient removal, is the sequencing batch reactor (SBR). Field conditions in SBR can be modified by controlling cycle length, D.O. and mixing. Reactor's contents can be mixed completely to ensure organism--substrate contact. Settling efficiency is also improved as organisms settle under completely quiescent condition. Several investigators have demonstrated the denitrification capabilities in SBR Ruya Tasli, D. Orhan and Nazik Artan (1999) 1 had successfully removed COD, nitrogen and phosphorus from domestic wastewater by adding acetate so as to keep COD/TKN ratio greater than 7.0 in influent wastewater. Artan et al (2002)2 carried out combined denitrifying and EBPR (enhanced biological phosphorus removal) experiments with various filling and aeration patterns. Keller et al (1996 and 1999)3'4 carried out experiments showing simultaneous nitrification and denitrification (SND) phenomena in a SBR by maintaining DO between 0.2--0.6mg/L within the reactor.

It was felt, that, the efficiency of SBR pertaining to nutrient removal should be explored in the laboratory so that further advancement can proceed for evaluation of kinetics and design parameters for its application for treatment of wastewater effluent from dairies, slaughterhouses, breweries, food-processing industries, tanneries, etc. It has been reported in earlier works by Ruya Tasli, Nazik Artan and D.Orhon [1,2] that both quality (i.e. percentage of soluble and readily biodegradable portion of COD in the wastewater concerned) and quantity of organic substrate as well anaerobic/anoxic/total cycle time lengths have an impact on denitrification and EBPR efficiency.

In this investigation, primary-treated wastewater was collected from a slaughterhouse-cum-meat processing industry in Howrah, West Bengal; and the effect of D.O., different substrates, filling strategies and cycle/phase times on denitrification and EBPR (enhanced biological phosphorus removal) efficiency of an SBR was investigated. The experiments were carried out in Jadavpur University Environmental Engineering Laboratory, Civil Engineering Department.

Materials and Methods Seed acclimatization

Aerobic and anaerobic/anoxic bacterial seeds were first cultured and acclimatized with the slaughter-house wastewater separately. During culture and acclimatization, a solution of Dextrose and micro-nutrients like [KH.sub.2]P[O.sub.4], [K.sub.2]HP[O.sub.4], MgS[O.sub.4],7H2O, Fe[Cl.sub.3], 6[H.sub.2]O, Ca[Cl.sub.2],2H2O, [(NH).sub.4][Mo.sub.7][O.sub.24], etc. (having a pre-determined composition) was added.

Experimental Setup

The experiment was carried out in a laboratory scale SBR of 18.0 L volume. The reactor was made up of plexiglass of 5 mm thickness. Photograph of the experimental set up is shown below. The feed is kept in a 25.0 L capacity carbuoy placed on an elevated wooden platform. A feed tube as inlet is connected from bottom of reservoir to inlet sprout of reactor. The air is supplied through a compressor (belt-driven) placed at floor level. The air from compressor is supplied to a diffuser system with small openings there in. The diffuser system is made up of glass tube with 2 mm opening size, which is a single assembly and kept 50mm above the bottom of the SBR. The compressed air is supplied to the reactor through the above openings. The treated water is taken from an outlet 100 mm above the bottom of the reactor. A stirrer of 0.3hp capacity is installed centrally for agitating and mixing the content of the reactor. The air supply is not given during anoxic mode of the react period. During this phase only stirrer was allowed to remain ON. A timer is also connected to compressor for controlling the aeration period. Two numbers of aquarium compressor pumps are also installed for additional supply of oxygen, if needed. A sludge withdrawal port is also provided near the bottom of the reactor.


Experimental Procedure

The primary treated slaughter-house wastewater was first characterised. Then this water, diluted as per our experimental needs, was used as feed to SBR. 2 sets of EBPR, 1 set of SND and 1 set of Nitrification / Denitrification experiments were performed during the next ten months using the primary treated water. Since the wastewater preserved for such a long period was bound to change its characteristics with time, therefore, characterization of the preserved wastewater was carried out before each run/set. The SBR experiments were carried out using different dilution factors, total cycle time, anoxic--aerobic sequence time, dissolved oxygen level and [V.sub.o]/[V.sub.F] (initial volume/ fill volume) ratios.

During each Set of experiment, 50mL of sample was collected from the effluent outlet of the reactor at every 1.0 hour interval, immediately after the fill period. The effluent samples were then filtered and the following parameters viz. MLSS, MLVSS, SCOD, [NH4.sup.+]-N, N[O.sub.2.sup.-]-N, N[O.sub.3.sup.-]-N, TP were measured as per Standard Methods.

Analytical methods for study of different parameters

To determine SCOD values, the wastewater was filtered before measurement of COD. In the laboratory DRB 200 Digital Reactor Block HR 50--150 mg/L (Hach, USA) was used for digestion of samples. Digestion was carried out for 2 hours at 150[degrees]C. Then COD was determined titrimetrically.

Concentration of [NH.sub.3] was determined by Orion 95-12 Ammonia Electrode.

Nitrite was determined by spectrophotometric method. The applicable range of the method is 10 to 1000 [micro]g N[O.sub.2.sup.-]-N/L at 543nm light wavelength (refer to page 4-129, Standard Methods, 17th Edition). Many of our samples (i.e. effluent taken every 1-hr from SBR) were diluted with a D.F = 50 while carrying out N[O.sub.2.sup.-] determination, so that the spectrophotometric method, valid within a range of 10 to 1000 pg N[O.sub.2.sup.-] - N/L, can be applied. Spectrophotometry was carried out using UV/Visible spectrophotometer (Varian).

Measurement of UV absorption at 220nm enables rapid determination of N[O.sub.3.sup.-]. Because dissolved organic matter also may absorb at 220nm, a second measurement was

done at 275nm to correct the N[O.sub.3.sup.-] value. Each sample was acidified with 1(N) HCL to prevent hydroxide or carbonate interference. For samples and standards, two times the absorbance reading at 275nm wavelength was subtracted from the reading at 220nm wavelength to obtain absorbance due to N[O.sub.3.sup.-]. Spectrophotometric measurements were done using Varian spectrophotometer.

Measurement of Total Phosphorus was carried out following the two steps:

(a) Conversion of the phosphorus form of interest to dissolved orthophosphate using Kjeldahl flasks.

(b) Colourimetric determination of dissolved orthophosphate using spectrophotometer.

Results and Discussion


The primary treated wastewater as collected from the slaughter-house was found to have the following characteristics

SCOD = 1620 mg/L

[SBOD.sub.5] = 300 mg/L

[NH.sub.3] = 468 ppm

N[O.sub.2] - N = 0.92 mg/L

N[O.sub.3.sup.-] - N = 24 mg /L

TP = 28.57 mg/L.

Experimental set-1 was carried out using [V.sub.o] = 10L and [V.sub.F] = 10L i.e. [V.sub.o]/[V.sub.F] = 1. Dilution factor (DF) of the feed wastewater was taken as 8.0. Due to dilution, the SCOD value fell to about 200 mg/l. To augment SCOD value, dextrose of about 20 gm was added to the feed solution, since it has been already reported by previous investigators in this field that for EBPR and denitrification to occur simultaneously, sufficient SCOD must be present in the wastewater. This experimental set-1, however, was conducted to investigate only the nitrification/denitrification aspect in SBR.

After dilution and subsequent addition of Dextrose, the feed to SBR had the following characteristics.

SCOD = 2202.5 mg/L

[NH.sub.3] = 58.5 ppm

N[O.sub.2.sup.-] -N = 0.115 mg /L

N[O.sub.3.sup.-] -N = 3 mg /L

TP = 3.57 mg/L.

Total cycle time selected was 9.5 hours with

a. 0.5h fill time

b. 4h React time (Aerobic)

c. 4h React time (Anaerobic)

d. 1h Settling period.



Nitritication/Denitrification aspect

Ammonia oxidation occurred during the Aerobic phase, due to the activity of species like Nitrosomonas and Nitrobacter. Theoretically, the reaction progresses as follows


During the Aerobic phase, N[O.sub.2.sup.-] -N first decreased, then increased. This may be due to their readily conversion to N[O.sub.3.sup.-] during initial stages which may occur because of dominance of nitrobacter sp.

Due to the presence of some residual DO, ammonia oxidation was extended even during anoxic period, but during the latter half of anoxic period ammonia oxidation was marginal because of dominating activity of facultative anaerobes and shortage of DO. During the later stage of anoxic period, there is a chance of further reduction of N[O.sub.3.sup.-] N to [NH.sub.4.sup.+]-N.


During Set 2, characterization of the primary-treated slaughter-house wastewater yielded the following results (before dilution):

SCOD = 1620 mg/L

SBOD = 300mg/L

[NH.sub.3] = 468 ppm as [NH.sub.3]

N[O.sub.2.sup.-] N = 923.5 [micro]g/L

N[O.sub.3.sup.-]-N = 24 mg/L

TP = 28.57 mg/L

TKN = 500 mg/L

Experimental set-2 was carried out using [V.sub.o] = 12L, [V.sub.F] = 8L i.e. [V.sub.o]/[V.sub.F] = 1.5

Dilution factor of feed waste water to SBR was taken as 6.0. About 15.52gm. of Sodium Acetate was added to the feed solution. This was done so as to augment the SCOD value of the wastewater. It has been reported in various journals that COD/TKN ratio is an important parameter while carrying out nutrient removal works in SBR. It has also been reported that salts of short-chain fatty acids like Sodium Acetate are the most suitable for the purpose. 0.6gm of commercially available superphosphate was then added to 8L of feed solution. This was done to increase Total Phosphorus concentration so that phosphorus removal (EBPR) effect can be highlighted, because after dilution TP concentration fell below 5 mg/1 (as shown below). This experiment was meant to investigate nitrification/denitrification as well as phosphorus removal in SBR.

After dilution and subsequent addition of NaAc and superphosphate, the feed to SBR had the following characteristics--

SCOD = 1078.33 mg/L

[NH.sub.3] =78 ppm as [NH.sub.3]

N[O.sub.2.sup.-] -N = 153.9 [micro]g/L

N[O.sub.3.sup.-]-N = 4mg/L

TP = 9.45mg/L as P

TKN = 83.3 mg/L

It is to be noted that we have consciously kept (SCOD/TKN) = (1078.33/83.3) = 12.9 (greater than 7) and (SCOD/TP) = (1078.33/9.45) = 114

Total cycle time selected was 10 hours with

(a) 2.5 h. of anaerobic/anoxic fill & mix phase including 2.0 h. of fill + mix and 0.5 h. of only mix.

(b) 2.5 h. of aerobic phase.

(c) 2 h. of anaerobic/anoxic fill & mix including 1.5 h. of fill+ mix and 0.5h. of only mix.

(d) 2 h. of aerobic phase.

(e) 1 h. settle period.



Carbonaceous matter oxidation

SCOD value during anaerobic/anoxic periods have initially increased since filling was in progress. SCOD values have however sharply decreased during aerobic periods.

Nitrification / denitrification aspect

In general, ammonia concentration has increased during anaerobic / anoxic period where as the same has decreased sharply during aerobic phase, as expected. However, in some cases, ammonia oxidation has extended till initial phases of anoxic period due to residual DO in the reactor.

N[O.sub.2] and N[O.sub.3.sup.-] concentration were found to have increased during aerobic while the same decreased during anaerobic / anoxic phase.

Total phosphorus removal aspect

During the anaerobic stage, phosphorus release is associated with storage of organic substrate within biomass, thus increasing TP concentration. In the aerobic stage, phosphorus uptake takes place at the expense of stored organics which also serve as carbon source for the growth of Acinetobacter sp. SCOD/TKN value was kept greater than 7.0 and SCOD/TP ratio was kept above 20 so that amount of substrate will be sufficient both for denitrification and PHB (poly R hydroxyl butyrate) storage for EBPR.


During Set 3, characterization of the primary treated slaughter-house yielded the following results (before dilution)

SCOD = 750 mg/L

[NH.sub.3] = 223 ppm

TKN = 250 mg/L

N[O.sub.2.sup.-]-N = 118 mg /L

N[O.sub.3.sup.-]-N= 27 mg /L

Experiment Set 3 was carried out using [V.sub.o] = 10L, [V.sub.F] = 10L i.e. [V.sub.o]/[V.sub.F] = 1.0. D.F of feed wastewater to SBR was chosen as 6.0. DO level during the entire experiment was kept low at 0.2-0.6 mg/L with DO value higher at initial stages of the react period but gradually approaching 0.2 mg/L during the later phase. This experiment was meant to investigate Simultaneous Nitrification & Denitrification (SND) phenomena in SBR.

After dilution using DF = 6, the feed to SBR had the following characteristics:

SCOD = 125 mg/L

[NH.sub.3 =] 37.17 ppm as [NH.sub.3]

TKN = 41.67 mg/L

N[O.sub.2.sup.-]-N = 19.67 mg/L

N[O.sub.3.sup.-]-N = 4.5 mg/L

Total cycle time selected was 7 hours with

a. 0.5 h anoxic filling,

b. 5.5 h react period with DO ranging from 0.2 to 0.6 mg/L

c. 1 h settle period.



Carbonaceous matter oxidation

SCOD values have readily diminished during the initial stages and then slowly as DO becomes deficient.

Nitrification / Denitrification profile

Ammonia oxidation had progressed steadily during initial stages. With time, during later phases of the react period, N[O.sub.3.sup.-] concentration declines while N[O.sub.2] concentration increases. It implies inhibition of the second step of nitrification (oxidation of nitrite to nitrate) with time. Removal of nitrogen via nitrite may thus be achieved by reducing the activity of Nitrobacter and giving Nitrosomonas sp. growth advantages. Microbiologists have long reported existence of aerobic denitrifiers. Moreover nitrification under fully anaerobic conditions has been shown possible in many previous literatures.

It is to be noted that NOZ concentration in the reactor declines only after almost all ammonia has been oxidized.


During Set 4, characterization of the primary treated slaughter-house wastewater yielded the following results (before dilution).

SCOD = 750 mg/L

[NH.sub.3] = 223 ppm as [NH.sub.3]

TKN = 250 mg/L

N[O.sub.2.sup.-]-N = 118 mg/L

N[O.sub.3.sup.-]-N = 27 mg/L

TP = 35 mg/L

Experimental Set-4 was carried out using [V.sub.o] = 12L, [V.sub.F] = 8L i.e. [V.sub.o]/[V.sub.F] = (12L/8L) = 1.5. D.F. of the feed wastewater was taken as 4. About 4.8 gms of NaAc was added to the feed solution, [V.sub.F.] This experiment was meant to investigate nitrification / denitrification as well as TP removal in SBR.

After dilution with DF = 4 and addition of NaAc, the feed to SBR had the following characteristics

SCOD = 437.5 mg/L

[NH.sub.3]= 55.75 ppm

N[O.sub.2.sup.-] = 29.5 mg /L

N[O.sub.3.sup.-]-N = 6.75 mg/L

TKN = 62.5 mg/L

TP = 8.75 mg/L

Thus (SCOD/TKN) ratio is thus kept (437.5/62.5) = 7 & (SCOD/TP) is kept (437.5/8.75) = 50 in the feed.

Total cycle time selected was 10 hours with

(a) 2.5 h of anaerobic / anoxic fill & mix phase including 2.0 h of fill + mix and 0.5 h of only mix.

(b) 2.5 h of aerobic phase.

(c) 2.0 h of anaerobic / anoxic fill & mix including 1.5 hrs of fill + mix and 0.5 h of only mix.

(d) 2.0 h of aerobic phase

(e) 1 h of settle period.



Carbonaceous matter oxidation

SCOD values had increased during the initial phase of anaerobic / anoxic phase due to filling. However, during the aerobic period it has decreased sharply.

Nitrification / Denitrification aspect

In general, N[O.sub.2] and N[O.sub.3.sup.-] values have increased during aerobic period and decreased during anoxic phase. However in certain cases, NOZ has increased during anoxic/anaerobic period due to filling of feed solution. Also during the initial periods of aerobic phase, NOZ values have gone down implying predominance of denitrifying bacterial species till early aerobic period.

Total phosphorus removal aspect

Phosphorus values have increased during anaerobic phase while it has decreased during aerobic phase as expected.


(1) From literature reviews, we know that in SBR process for N and P removal, an anoxic phase is inherently established before truly anaerobic conditions leading to PHB storage and P release. It has already been reported by several researchers in this field that for successful simultaneous removal of Nitrogen and Phosphorus--COD value, or more appropriately, SCOD value must be high. Researchers like Ruya Tasli, Nazik Artan [1,2] in their various papers have shown that COD/TKN ratio should be a minimum of 7.0 for simultaneous removal of N & P. Otherwise, only Nitrogen will be removed with little or no removal of Phosphorus due to shortage of substrate-COD. Denitrification preferentially consumes available organic carbon thus blocking biological processes related to EBPR. Therefore, SBR is not likely to perform efficiently with respect to EBPR, for domestic sewage with low RBCOD (readily biodegradable fraction of COD) content. In case of Phosphorus removal, one should be very careful with regard to availability of substrate-COD as well as proper development of anaerobic phase. We have thus added Dextrose (in the first experiment) and Sodium Acetate (in the second experiment) so that substrate-COD does not become a limiting condition. Researchers like Nazik Artan, Ruya Tasli [1,2] have already shown that addition of salts of short-chain fatty acids (like NaAc) so as to augment substrate-COD, produces good results as far as EBPR is concerned.

(2) Any biological process like SBR needs efficient / active bacterial cells. Proper development of anoxic, anaerobic, aerobic and poly-P bacterial seeds is needed before venturing wastewater treatment by SBR. Thus, in our experiments, we have devoted some time to develop bacterial culture and seed acclimatization. References

[1] Tasli, R., Orhon, D. and Artan, N., July 1999, "The effect of substrate composition on the nutrient removal potential of sequencing batch reactors", Water SA, 25 (3), 337-343

[2] Artan, N., Wilderer, P., Orhon, D., Tasli, R., E. Morgenroth, E., October 2002, "Model evaluation and optimization of nutrient removal potential for sequencing batch reactors". Water SA, 28 (4), 423-432

[3] Munch, E.V., Lant, P., and Kellar, J., 1996, "Simultaneous nitrification and denitrification in bench scale Sequencing Batch Reactors", Water Research, Vol. 30(2), 277-284.

[4] Pochana, K. and Kellar, J., 1999, "Study of factors affecting simultaneous nitrification and denitrification". Water Science Technology, 39(6), 61-68.

[5] Metcalfe and Eddy., Wastewater Engineering, Treatment, Disposal and Reuse. McGraw Hill.

[6] 6 Coelho, M.A.Z., Russo, C. and Araujo, O.Q.F., 2000, 'Optimisation of a Sequencing Batch Reactor for biological nitrogen removal', Water Research, 34(10), 2809-2817

[7] Gupta S. K. and Sharma, R., 1993, "Biological oxidation of high strength nitrogenous wastewater", Water Research, 30(3)

[8] Norcros, K.L.,1992 "Sequencing Batch Reactors-An Overview", Water Science Technology.

[9] Nawghare, P., Srivastabha, A., Kartick, M. and Kaul, S.N., 2004, "Sequencing Batch Reactor-An emerging technology for wastewater treatment", Journal IAEM 31,13-33

[10] Collado, N.H. and Forresti, E., 2001, "Removal of organic carbon, nitrogen and phosphorus in sequencing batch reactors integrating the aerobic/anaerobic processes", Water Science and Technology, 44(4)

[11] Masse, D.L. and Masse, L., 2000, "Treatment of slaughter house wastewater in anaerobic sequencing batch reactors", Canadian Agricultural Engineering, 42(3)

Koushik Paul

Lecturer, BIT Mesra, Ranchi, Civil Engineering Department

(former student of Jadavpur University, Kolkata)

Table 1: Results for Experiment Set-1.

Time                                  Aerobic Phase

Parameter          0.5h       1.5h       2.5h       3.5h       4.5h

SCOD               826        426        373        350        320

N[H.sub.3]         29.9      15.80       12.2       8.22       6.87

N[O.sub.2.          57         36       24.48       24.2       42.3

N[O.sub.3.         10.0      10.88      11.73      11.34       13.8

MLSS               800        960        1600       1700       1060

MLVSS              540        640        1240       1320       740

Time                          Anaerobic Phase                 Settle

Parameter          5.5h       6.5h       7.5h       8.5h       9.5h

SCOD               270        267        254        214        160

N[H.sub.3]         6.43       5.74       5.68       6.09       6.87

N[O.sub.2.         43.7      39.54       39.2       37.5      36.26

N[O.sub.3.         12.9       12.9       12.2       10.0       9.4

MLSS               1580       1420       1200       600        240

MLVSS              1200       1180       960        400         80

Table 2: Results for Experiment Set-2.

Time                Anaerobic/ Anoxic              Aerobic

Parameters      1 h       2h     2.5 h      3h       4h       5h

SCOD            650      750      600      600      500      150

N[H.sub.3]      47.3    57.05     62.1     9.0      8.5      7.5

N[O.sub.2.      88.2     167      97.0     176      422      482

N[O.sub.3.       0        0        0       2.6      5.9      6.1

TP              13.0     19.6      20       14      11.7     10.7
(mg/L as P)

MLSS            4000     4460     2920     2380     3020     3000

MLVSS           3000     3060     2080     1560     1920     2140

Time              Anaerobic          Aerobic       Settle

Parameters       6h       7h       8h       9h      10h

SCOD            350      400      350      250      150

N[H.sub.3]      6.5     13.85     7.0      2.5      2.64

N[O.sub.2.      490       78      264      318      350

N[O.sub.3.      2.2      1.9      1.33     3.8      3.0

TP              9.28    18.57     15.7     7.4      8.1
(mg/L as P)

MLSS            2420     3140     2840     2640     160

MLVSS           1680     2400     1980     1780     104

Table 3: Results for Experiment Set-3.

Time              Anoxic              React period
Parameters         0.5h         1h          2h          3h

SCOD                450         300         250         210

N[H.sub.3]          37          22          12          8.2

N[O.sub.2.          588          0           0          341

N[O.sub.3.           2         5.48         10         13.4

MLSS               1480        2320        3000        3360

MLVSS              1040        1860        2280        2740

Time                      React period                Settle

Parameters          4h          5h          6h          7h

SCOD                179         127         90          8.8

N[H.sub.3]          5.5         4.0         3.4         2.9

N[O.sub.2.         2882        5882        9705        4117

N[O.sub.3.         15.94       05.0          0          0.7

MLSS               2512        2700        2800         240

MLVSS              1920        2160        2212         90

Table 4: Results for Experiment Set 4.

Time                 Anaerobic / Anoxic              Aerobic

Parameter        1h       2h       2.5h       3h       4h        5h

SCOD             90       350       280      200       150      130

N[H.sub.3]       38      42.2      24.3      3.57     2.44      2.33

N[O.sub.2.      3.59    11.058    15.294     7.65    12.941     15.3

N[O.sub.3.      6.29     6.12      5.92      3.95     6.15      8.27

TP              7.14     7.86      12.85     9.28     7.14      5.71

MLSS            4420     4120      4510      2280     4760      4100

MLVSS           4348     3800      4000      1560     4044      3720

Time               Anaerobic/            Aerobic       Settle
Parameter        6h        7h        8h        9h        10h

SCOD             200       200       150       130       100

N[H.sub.3]      32.2      29.6      24.0      17.2      15.4

N[O.sub.2.      12.6      10.29     12.26     18.4      12.6

N[O.sub.3.      8.02      6.08       7.1       7.4      5.51

TP              15.0      18.56     10.71      8.0       8.1

MLSS            3860      4120      3720      3620       700

MLVSS           3000      3260      2900      2814       280
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Title Annotation:chemical oxygen demand
Author:Paul, Koushik
Publication:International Journal of Applied Environmental Sciences
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2009
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