jece-s-13-00441 (1)
TRANSCRIPT
Elsevier Editorial System(tm) for Journal of Environmental Chemical Engineering Manuscript Draft Manuscript Number: Title: Nitrogen and organic matter removal in an intermittently aerated fixed-bed reactor for post-treatment of anaerobic effluent from a slaughterhouse wastewater treatment plant Article Type: Original Article/Research Keywords: Nitrification; Denitrification; UASB effluent; Poultry slaughterhouse; Simultaneous Nitrification and Denitrification (SND); ANaerobic AMMonium OXidation (ANAMMOX) Corresponding Author: Dr. Ana Cláudia Barana, Ph.D. Corresponding Author's Institution: UEPG First Author: Ana Cláudia Barana, Ph.D. Order of Authors: Ana Cláudia Barana, Ph.D.; Deize D Lopes, Ph. D; Tiago H Martins, Ph.D; Eloisa Pozzi, Ph.D.; Márcia H Damianovic, Ph.D; Valeria D Nery, Ph.D; Eugenio Foresti, Ph.D Abstract: This study evaluated the performance of a lab scale, fixed bed reactor exposed to intermittent aeration for the removal of organic matter and nitrogen from anaerobic reactor effluent. The reactor was continuously fed with effluent from a UASB reactor used to treat wastewater from a poultry slaughterhouse. The hydraulic retention time (HRT) was maintained at 24 hours during the five operational phases that the reactor was subjected to. The phases differed only for the duration of periods with and without aeration. The best results regarding nitrogen removal efficiency were obtained in phase V (8 daily cycles of 1 hour of aeration and 2 hours without aeration). Under these conditions, for influent with total COD of 418 mg.L-1, 169 mg.L-1 of TKN and 112 mg.L-1 of NH4+-N, effluent with a total COD of 22 mg.L-1, 6.4 mg.L-1 of TKN, 6.4 mg.L-1 of NH4+-N, 58 mg.L-1 of NO3-N was obtained, and NO2-N was not detected. During this phase, the average nitrogen removal efficiency was 62%. Optical microscopy and molecular biology analyses associated with the study of microbial activity detected the activity of bacteria that perform Anammox, thereby contributing to the understanding of the processes involved. Suggested Reviewers: Cláudia ETCHEBEHERE Universidad de la República - Uruguai [email protected] Works with the theme Jules van Lier Delft University of Technology [email protected] He studies the theme for many years. Adalberto Robles Noyola UNAM [email protected] He works with the theme for many years.
Santiago Villaverde Gómez University of Valladolid [email protected] He works with the theme for many years. Reyes Sierra-Alvarez University of Arizona [email protected] He worsks with the theme for many years.
Universidade Estadual de Ponta Grossa – Departamento de Engenharia de Alimentos
Av.: Gal. Carlos Cavalcanti, 4748 – Bairro Uvaranas – Ponta Grossa/PR – 84030-900
Tel.: +55-42-99475836. Email: [email protected]
DDeeppaarrttmmeenntt ooff FFoooodd EEnnggiinneeeerriinngg Dr. Editor-in-Chief Journal of Environmental and Chemical Engineering
Ponta Grossa, April 04, 2013
Ref: Paper submission
Dear Editor-in-Chief
We are pleased to submit the manuscript entitled “Nitrogen and organic matter
removal in an intermittently aerated fixed-bed reactor for post-treatment of anaerobic
effluent from a slaughterhouse wastewater treatment plant” to Journal of Environmental
and Chemical Engineering. The manuscript is resulted from an original research work.
The authors Ana Cláudia Barana, Deize Dias Lopes, Tiago Henrique Martins,
Eloisa Pozzi, Marcia Helena Rissato Zamariolli Damianovic, Valéria Del Nery and Eugenio
Foresti agree to submit the article and attested that the work has not been published or
being submitted to another journal.
According with the description given in the journal home page, the authors believe
that Journal of Environmental and Chemical Engineering is an adequate journal for
publishing subjects on the application of biological process for wastewater treatment.
The manuscript word count is 3.906
Thank you for your attention.
Sincerely yours,
Ana Cláudia Barana
*Cover Letter
AUTHOR DECLARATION We wish to confirm that there are no known conflicts of interest associated with this publication and there
has been no significant financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all named authors and that there are no
other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order
of authors listed in the manuscript has been approved by all of us.
We confirm that we have given due consideration to the protection of intellectual property associated with
this work and that there are no impediments to publication, including the timing of publication, with
respect to intellectual property. In so doing we confirm that we have followed the regulations of our
institutions concerning intellectual property.
We understand that the Corresponding Author is the sole contact for the Editorial process (including
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Signed by all authors as follows:
- Ana Cláudia barana /UEPG – corresponding author
- Deize Dias lopes – UEL
- Márcia H.R.Z. Damianovic – USP
- Tiago Henrique Martins – USP
- Eloiza Pozzi – USP
- Valéria Del Nery – Céu Azul Alimentos
- Eugenio Foresti - USP
Conflict of Interest Form
1
Nitrogen and organic matter removal in an intermittently aerated fixed-bed reactor for 1
post-treatment of anaerobic effluent from a slaughterhouse wastewater treatment 2
plant 3
4
Barana, A.C.a, Lopes, D.D.b, Martins, T.H.c, Pozzi, E.c, Damianovic, M.H.R.Z.c, Del Nery, V.d, 5
Foresti, E.c 6
7
a Department of Food Engineering, State University of Ponta Grossa, Av.: Gal. Carlos 8
Cavalcanti, 4748, CEP 84030-900, Ponta Grossa, PR, Brazil 9
b Department of Civil Engineering, State University of Londrina, Rod. Celso Garcia Cid, km 10
380, CEP 86051-991, Londrina, PR, Brazil 11
c Department of Hydraulics and Sanitation, School of Engineering, University of São Paulo, 12
Av.: Trabalhador São-Carlense, 400, CEP 13566-590, São Carlos, SP, Brazil 13
d Céu Azul Alimentos Ltda., Rua Francisco Savaglia, 405, CEP 13569-590, São Carlos, SP, 14
Brazil 15
Abstract 16
This study evaluated the performance of a lab scale, fixed bed reactor exposed to 17
intermittent aeration for the removal of organic matter and nitrogen from anaerobic reactor 18
effluent. The reactor was continuously fed with effluent from a UASB reactor used to treat 19
wastewater from a poultry slaughterhouse. The hydraulic retention time (HRT) was 20
maintained at 24 hours during the five operational phases that the reactor was subjected to. 21
The phases differed only for the duration of periods with and without aeration. The best 22
results regarding nitrogen removal efficiency were obtained in phase V (8 daily cycles of 1 23
hour of aeration and 2 hours without aeration). Under these conditions, for influent with total 24
COD of 418 mg.L-1, 169 mg.L-1 of TKN and 112 mg.L-1 of NH4+-N, effluent with a total COD 25
of 22 mg.L-1, 6.4 mg.L-1 of TKN, 6.4 mg.L-1 of NH4+-N, 58 mg.L-1 of NO3-N was obtained, 26
and NO2-N was not detected. During this phase, the average nitrogen removal efficiency 27
was 62%. Optical microscopy and molecular biology analyses associated with the study of 28
*Manuscript
2
microbial activity detected the activity of bacteria that perform Anammox, thereby 29
contributing to the understanding of the processes involved. 30
31
Key-words - nitrification, denitrification, UASB effluent, poultry slaughterhouse, 32
simultaneous nitrification and denitrification (SND), ANaerobic AMMonium OXidation 33
(ANAMMOX) 34
35
1. INTRODUCTION 36
Poultry slaughterhouse wastewater is characterised by high concentrations of 37
organic matter, suspended solids, oil and grease, nitrogen and phosphorus. Blood, faeces 38
and fat are the main sources of organic matter and nutrients. Due to the high concentrations 39
of organic matter and nitrogen, biological treatment of these wastewaters usually occurs in 40
units sequentially arranged to remove organic matter prior to the removal of nitrogen. 41
Anaerobic processes normally removes significant fraction of organic matter but not 42
nitrogen. However, the anaerobic treatment prior to a nitrogen removal unit removes part of 43
the organic carbon leaving sufficient amount of COD for using in denitrification [1]. 44
In any case, nitrogen removal from wastewater usually involves sequential biological 45
processes of nitrification and denitrification. Autotrophic and heterotrophic bacteria 46
participate in these processes, under aerobic and anoxic conditions, respectively. The 47
denitrification stage can also occur through autotrophic processes. The ANAMMOX 48
(Anaerobic Ammonium Oxidation) process stands out as the most recent alternative for 49
nitrogen removal from wastewater with a low concentration of organic matter. 50
Conventional nitrogen removal systems involve the installation of several units of 51
sequential operations, requiring large areas for the deployment of full-scale systems. One 52
way to reduce deployment costs is by using systems that integrate nitrification and 53
denitrification within a single unit [2, 3]. 54
Several authors reported on the use of sequencing batch reactors (SBR) as a 55
complementary treatment of anaerobic processes of slaughterhouse wastewater. Keller et 56
3
al. [1] obtained final effluent with a concentration of 20 mgN/L and 5 mgP/L. Cassidy and 57
Belia [4] found over 97% removal of nitrogen. De Nardi et al. [5] obtained 64% removal of 58
COD and 100% removal of NH3-N in a lab scale SBR using anaerobic reactor effluent. 59
There are criticisms regarding the use of SBR for nitrification and denitrification in the 60
same batch: the intermittently exposition of autotrophic and heterotrophic bacteria to 61
unfavorable environmental conditions; the increase of operating costs if supplementary 62
alkalinity and organic carbon are required for nitrification and denitrification, respectively. 63
Some authors studied SBR for the treatment of raw effluent from slaughterhouses. 64
Shengquan et al. [6] studied a membrane reactor (SBR), in which COD was reduced by 98% 65
and NH3-N by 95%. Li et al. [7] obtained 96% reduction of COD, 96% reduction of total 66
nitrogen, and 99% reduction of total phosphorous. 67
Other system configurations used to remove of organic matter and nitrogen from 68
slaughterhouse wastewater are successful. Systems composed of anaerobic and aerobic 69
reactors fed continuously and with recirculation of the nitrified effluent to the anaerobic 70
reactor achieve high efficiencies. Reginatto et al. [8] obtained 95% removal of nitrogen and 71
verified the presence of Anammox-like microorganisms in the biomass. Nuñes and Martinez 72
[9] obtained 85% removal of carbon and 75% nitrogen removal, without the addition of an 73
external carbon source. 74
Bench-scale fixed-bed reactors such as the SBBR - sequencing batch immobilized 75
biomass reactor, were also able to remove organic matter and nitrogen [3, 10, 11]. Recently, 76
a continuous flow reactor containing a structured fixed-bed reactor [12] succeeded in 77
removing organic matter from synthetic effluent. Further on, this reactor was subjected to 78
intermittent aeration and removed organic carbon and nitrogen efficiently [13]. These 79
reactors use fixed-bed of polyurethane (PU) foam considered the best biomass support in a 80
previous study [14]. 81
Based on Moura et al. [13], this study aimed to identify the biological processes 82
involved in the removal of residual organic matter and nitrogen from the effluent of a full-83
scale UASB reactor treating poultry slaughterhouse wastewater. 84
4
85
2. MATERIAL AND METHODS 86
87
2.1. Installation for the experiment 88
89
2.1.1. Reactor 90
The cylindrical shape reactor constructed of acrylic was 80 cm in height with an 91
internal diameter of 14.5 cm. The reactor had two inlets near the base and two outlets near 92
the top. The inlets controlled the feed of the influent, and the entry of the recirculation flow, 93
respectively. The outlets from the top corresponded to the outlet of the treated effluent, 94
located 65 cm from the base of the reactor, and to the outlet of the recirculation pump. The 95
recirculation ratio relative to input flow (QR:Q) was 6:1. The feeding and recirculation were 96
carried out with the aid of ProMinent Beta Series, Concept Plus model, diaphragm metering 97
pumps, controlled by timers. The system was aerated with a Regent, Model 8500, aquarium 98
aerator. The system was kept in a climatised chamber at 30±1ºC (Fig. 1). 99
The high value of QR:Q caused the reactor to operate in a regime of complete 100
mixture. 101
102
2.1.2. Support media 103
The reactor was filled with 13 cylindrical tubes of polyurethane foam (PU) with a 104
diameter of 2 cm and 70 cm length, which were used as support media for the biomass. The 105
foam used was 22 g.L-1 in density and had a porosity of 92%. 106
107
2.2. Reactor operation 108
109
2.2.1. Inoculum 110
5
The reactor was inoculated with biomass from an activated sludge reactor, with 111
nitrifying activity, from a wastewater treatment plant at the Volkswagen, in São Carlos, São 112
Paulo, and it was immobilised following the method proposed by Zaiat et al. [15]. 113
114
Fig. 1. Outline of the reactor bed (A: influent entrance, B: recirculation entrance, C: effluent 115
outlet; D: recirculation exit; E: air entrance). Source: Moura et al. [13] 116
117
2.2.2. Adaptation phase 118
After inoculation, the reactor was fed with effluent from an UASB reactor used for the 119
treatment of a poultry slaughterhouse and it was operated in batch under continuous 120
aeration for 7 days to allow the development of nitrifying biomass. 121
122
2.2.3. Aerobic/anoxic periods 123
After the adaptation phase, the reactor was supplied continuously with HRT for 24 124
hours. The duration of the aerobic periods was reduced gradually, starting with continuous 125
aeration and ending after one hour. This approach aimed to assess the relationship between 126
the aerated and non-aerated periods which resulted in the most efficient removal of nitrogen. 127
The operation of the system was divided into five phases (Table 1), depending on the 128
duration of the aerobic/anoxic phases. 129
130
Table 1 131
Duration of aerobic and anoxic periods of each experimental phase. 132
133
2.3. Substrate and operating conditions 134
135
2.3.1. Wastewater 136
The wastewater used throughout the experiments was effluent from a UASB reactor 137
at Céu Azul poultry slaughterhouse, located in Sorocaba, São Paulo (Table 2). 138
6
139
Table 2 140
Mean values and standard deviation of parameters analysed in the influent of each phase of 141
the experiment. 142
n.a.: not analysed; SD: standard deviation 143
144
The CODT/BOD relationship of the substrate showed values between 1.6 and 3.6, 145
indicating that this substrate is biodegradable. The analysis of organic acids by high 146
performance liquid chromatography did not reveal the presence of organic acids in the 147
wastewater used. The presence of nitrite and nitrate was also not detected. These results 148
were to be expected, since it was UASB effluent. 149
150
2.4. Monitoring of the reactor 151
152
2.4.1. Variables analysed 153
During the experiment, the following variables were analysed: pH, alkalinity, TKN 154
(Total Kjeldahl Nitrogen), NH4+-N (ammonium nitrogen), NO2
--N (nitrite nitrogen), NO3--N 155
(nitrate nitrogen), TSS (Total Suspended Solids), VSS (Volatile Suspended Solids), DO 156
(Dissolved Oxygen) and COD (Chemical Oxygen Demand). The analyses of nitrate and 157
nitrite were made using the Dionex Ion Chromatograph ICS 5000 (USA). The conditions 158
used for chromatography were: eluent of 4.5mM of Na2CO3/0,8mM of NaHCO3 at a flow rate 159
of 1.0 mL.min-1, IonPac AS23 column (4 x 250 mm) and IonPac AG23 Guard pre-column (4 160
x 50 mm) at a temperature of 30°C, electrochemichal conductivity detector, with gradient 161
pump and an anion self-regenerating suppressor (ASRS 300). The alkaline analysis was 162
carried out using the method described by Ripley [16]. All further analyses were made by 163
methods described in APHA [17]. 164
Influent and effluent alkalinity and pH were measured daily for the correction of 165
alkalinity necessary for nitrification. The average alkalinity by ammonia concentration in the 166
7
influent was 4.8:1. It was necessary a supplementation of alkalinity in the influent of 8.64 mg 167
of HCO3- per mg of ammonia removed from the system, which meant the addition of 50-680 168
mg.L-1 of NaHCO3. 169
170
2.4.2. Efficiency of the reactor 171
The efficiency of the reactor in removing total nitrogen was calculated for each 172
phase, using Equation I. The presence of nitrite was not observed during all the experiment. 173
The efficiency of nitrification was calculated for each phase, using Equation II. 174
Tota nitrogen removal (%) = (TKNA – TKNE - NO3--NE)/TKNA x 100 (Equation I)
Nitrification (%) = (TKNA - TKNE)/TKNA x 100 (Equation II)
Denitrification (%) = (TKNA - TKNE - NO3--NE)/ (TKNA - TKNE) x 100 (Equation III)
Where: 175
- TKNA: Total Kjeldahl Nitrogen Influent 176
- TKNE: Total Kjeldahl Nitrogen Effluent 177
- NO3--NE: Nitrate Nitrogen Effluent 178
The nitrogen removal processes evaluated were heterotrophic denitrification, using 179
the available carbon source in the wastewater, and the Anammox process due to the low 180
ratio of COD/N of the substrate used (Table 2). 181
182
2.5. Microscopic analyses 183
184
The microscopic analyses of the sludge in the reactor at the end of the experiment 185
were performed using a normal optical microscope, with phase and epifluorescence 186
contrast, using an Olympus BX60 model coupled with a camera with image capture and 187
Image-Pro Plus software. 188
189
2.6. Study of Anammox activity 190
191
8
To corroborate the Anammox activity, three reactors with 250 mL volume each, 192
were fitted and operated in batch. Culture medium was added to each reactor that would 193
provide the growth of Anammox bacteria consisting of N-NO2 e N-NH4, adapted from de Van 194
de Graaf et al. [18]: NH4Cl (126mg.L-1), NaNO2 (121mg.L-1), KHCO3 (1000mg.L-1), KH2PO4 195
(27.2mg.L-1), MgSO4.7H2O (300mg.L-1), CaCl2.2H2O (180mg.L-1).To this medium was added 196
1 mL of trace solution I, adapted from Van de Graaf et al. [18]: EDTA (5g.L-1) and 197
FeSO4.7H2O (9.17 g.L-1), and 1 mL of trace solution II, also adapted from Van de Graaf et al. 198
[18]: EDTA (15 g.L-1), ZnSO4.7H2O (0.43 g.L-1), CoCl2.6H2O (0.24 g.L-1), MnCl2.4H2O (0.99 199
g.L-1), CuSO4.5H2O (0.25 g.L-1), NaMoO.2H2O (0.22 g.L-1), NiCl2.6H2O (0.19 g.L-1), Na2SeO3 200
(0.09 g.L-1) and H3BO3 (0.014 g.L-1). The reactors were inoculated with 1.6 g of biomass 201
(VSS) extracted from the fixed bed reactor and stored at 30°C without stirring. To maintain 202
anaerobic conditions, prior to beginning the tests, the reactors were bubbled with argon for 203
15 minutes. Samples were taken regularly over a period of 39 hours for analysis of NH4-N, 204
NO2-N and NO3--N. 205
206
2.7. Molecular biology analysis 207
208
After 131 days of operation a sample of the sludge immobilised in the support 209
material of the reactor was withdrawn in order to extract the nucleic acid according to the NG 210
et al. [19] protocol. From the DNA extracted from the samples, PCR product of the RNAr16S 211
gene was obtained using specific PLA 46Frc/AMX 820R primers for Anammox with 212
approximately 780 base pairs [20, 21]. A master cycle (Eppendorf) thermal cycler was used 213
for the amplification of DNA fragments. 214
Agarose gel electrophoresis was used to evaluate the product resulting from 215
extraction of the nucleic acid and amplification by PCR. To evaluate the product of PCR 216
amplification, 1% agarose was utilised and Low was used as a marker of low molecular 217
weight. 218
219
9
2.8. Statistical analysis 220
221
The mean values of total nitrogen removal of each phase were statistically analysed 222
by ANOVA and the means were compared by the Tukey test (p = 0.05). 223
224
3. RESULTS AND DISCUSSION 225
226
3.1. Efficiency of the reactor 227
Nitrogen removal occurred during all the experimental phases. Nitrogen removal 228
efficiencies increased with the decrease of the aerated periods and increase of the non 229
aerated periods. On the other hand, COD removal efficiencies were higher than 88% along 230
all the experimental period. 231
Data of the reactor effluent in each experimental phase and removal of nitrogen and 232
organic material are shown in Table 3. Nitrite was not detected in effluent samples. 233
234
Table 3 235
Mean values and standard deviation of the results of the characterisation of the reactor 236
effluent during the different evaluated phases. 237
n.a.: not analysed; CODTC/: Total COD Consumed; NO3-NR: NO3-N Removed 238
239
At the start of phase I (Table 1), when the concentration of ammonium nitrogen in 240
the effluent began to decrease (Fig. 2) with formation of nitrate (Fig. 4), all the alkalinity of 241
the medium was consumed, with a consequent decrease in pH (Fig. 3) due to release of H+ 242
in the medium. To keep the proportion of 8.64 mg of HCO3- per mg of nitrogen removed [22] 243
the influent alkalinity was corrected with NaHCO3-, depending on the values of alkalinity 244
obtained in the effluent and NH4+-N removed. The effluent alkalinity resulted from its 245
consumption during nitrification and production during denitrification. Values of pH below 7.0 246
cause an immediate reduction in nitrification rates [23]. During this phase, it was possible to 247
10
remove about 88% of total COD, indicating the intense action of heterotrophic bacteria in the 248
removal of carbonaceous organic matter. During this phase, there were no anoxic periods 249
and the conversion of reduced nitrogen compounds to nitrate was 76%. At high DO 250
concentrations, facultative denitrifying bacteria use free oxygen instead of nitrate, as an 251
electron acceptor, thereby stopping denitrification [24]. Liu et al. [25] observed a decrease in 252
denitrification from 60% to less than 30% when the concentration of DO in the reactor 253
increased from 1.0 to 1.5 mg.L-1. Denitrification process was not significantly affected with 254
the DO concentration up to 0.6 mg.L-1 [26] .The removal of total nitrogen from the system 255
can be explained by the existence of an anoxic region inside the foam cylinders used for the 256
immobilisation of biomass. In these anoxic zones, heterotrophic denitrification due to the 257
absence of free oxygen may have occurred. Another additional possibility would be the 258
synthesis of biomass, which is generated at a rate of 0.15 g of biomass per g of oxidised 259
NH4+-N [23]. 260
In phases II to V, nitrification, calculated according to Equation II, was greater than 261
94%, despite the introduction of anoxic periods. The denitrification in phase II, calculated 262
according to Equation III, was approximately 30% and in phase III it was 25%. An increase 263
in the average efficiency of removal of total nitrogen, from 42% (phase IV) to 64% (phase V) 264
followed the reduction of aerated periods and increase of anoxic periods. This result 265
reinforced the hypothesis of the need for the removal of dissolved oxygen from the bulk 266
liquid to favour the denitrification process in this type of reactor. 267
Throughout the experimental period the concentration of dissolved oxygen in the 268
aerobic steps was around 2.6 mg.L-1. 269
270
Table 4 271
Mean values of total nitrogen applied and removed during the experiment. 272
Means followed by the same letters in the line of Total N Removed (%) do not differ (Tukey’s 273
test, p = 0.05) 274
275
11
The results of ANOVA and Tukey's test performed at a significance level of p≤0.05, 276
indicate no significant differences between nitrogen removal in phases II and III, represented 277
by the means with same letter in the line of Total N Removed (Table 4). However, phases I, 278
IV and V are significantly different from each other and different from phases II and III, 279
represented by different letters in the line of Total N Removed (Table 4). Therefore, changes 280
in aeration times interfere with denitrification, because in phases II and III the reactor 281
remained under anoxic conditions for 8 hours per day. In phases I, IV and V the reactor 282
remained under anoxic conditions for 0, 12 and 16 hours per day. 283
The CODTC/ NO3--NR ratio was around 5.5 for phase IV and 3.8 for phase V. The 284
CODTC/N-NO3-R ratio necessary for heterotrophic denitrification varies depending on the 285
organic material used. Fernández-Nava et al. [27] studied heterotrophic denitrification using 286
different carbon sources and observed a CODTC/N-NO3-R ratio between 5.6 and 7.8 when 287
using effluent from a candy manufacturer as a carbon source; a ratio of 3. 6 to 4.2 when the 288
carbon source was from effluent from a soft drink industry; and about 3.0 to 3.5 when dairy 289
effluent was the carbon source. Carrera et al. [28] found a CODTC/ NO3--NR of 7.1, when the 290
source of carbon was ethanol. Phillips et al. [24] calculated the theoretical ratio of CODTC/ 291
NO3--NR for denitrification using glucose as a carbon source at 2.7. In practice, however, 292
they observed that a ratio greater than 4.0 was necessary for complete conversion to N2. 293
294
Fig. 2 – Values of TKN and N-ammonia of influent and effluent throughout the period 295
of study (n≥ 5) 296
297
298
Fig. 3 – Evolution of pH and alkalinity values measured in the influent and effluent during all 299
phases of the experiment (n ≥ 5) 300
301
302
12
Fig. 4 – Values of nitrate effluent and filtered COD, and total influent and effluent 303
304
3.2. Study of Anammox activity 305
According to Henze et al. [29] in a denitrification system in which no part of the 306
carbon source is lost through oxidation by oxygen, the CODTC/ NO3--NR ratio is in the range 307
of 3.5 to 4.5. The CODTC/ NO3--NR ratio in phase V was 3.8 and part of the COD was 308
removed by aerobic heterotrophic bacteria during the aerobic periods. As heterotrophic 309
denitrification was limited by the amount of COD available, other processes might have 310
occurred. It is well known that Anammox bacteria can use nitrite as an electron acceptor and 311
NH4+-N producing N2 and nitrate. 312
To verify the existence of bacteria in the sludge capable of performing the Anammox 313
process tests were performed with biomass taken from the reactor and synthetic medium 314
prepared with nitrite and ammonia. The tests were positive regarding the presence of 315
microorganisms that consume NO2--N and NH4
+-N and produce NO3-N (Fig. 5). The results 316
confirmed the hypothesis of the occurrence of Anammox as an additional denitrification 317
process. 318
The abiotic reactor did not exhibit any removal of NH4+-N and NO2
--N, indicating that 319
there was no other cause for the removal of nitrogen than biological. 320
The agarose gel image of the PCR reaction (Fig. 6) confirmed the presence of 321
Anammox bacteria in the sludge, since the reaction was performed with specific primers for 322
that microbial group. Channel 1 shows the bands of the low molecular weight marker; 323
Channel 2 shows the band of the PCR product of the sludge sample from the reactor; and 324
Channel 3 shows the band of the PCR product of the positive control culture. The Chanel 3 325
band originated from a reactor operated under conditions conducive to the development of 326
Anammox bacteria [30]. It can be seen that the DNA fragments amplified by PCR align with 327
the fragment of 800pb, relative to the size of the 16S rRNA gene fragments selected by the 328
specific primers. 329
330
13
331
Fig. 5 – Graph with the average results of Anammox activity tests in three reactors 332
333
334
Fig. 6 – Agarose gel image of the PCR reaction, where Channel 1: "low mass" marker; 335
Channel 2: sample reactor (Anammox) Channel 3: positive control (specific primers- 336
Anammox = PLA 46Frc/AMX 820R) 337
338
The biomass was also analysed by optical microscopy. We observed the presence 339
of cocci clusters similar to the bacteria responsible for the Anammox process reported by 340
literature [31, 32, 33, 34]. 341
342
Probably, the anammox in the reactor occurred inside of the bioparticule 343
(polyurethane foam plus biomass) where the dissolved oxygen was zero, in order words, 344
anoxic niches. Ono [35], utilizing a micro sensor to determine the oxygen profile in a biofilm 345
of polyurethane foam-supported for nitrifying biomass adhesion, observed that in depths 346
greater than 400 μm the biofilm is in anoxic conditions. So, in that conditions nitrification and 347
denitrification can occur. 348
349
The influence of oxygen in the anammox process was investigated by Strous et al. 350
[36] in a fluidized bed reactor and batch. The first reactor was monitored for 20 days, with 351
alternating cycles of 2 h with aerobic (O2) and anaerobic (argon) conditions. The anammox 352
process was observed only in microaerophilic conditions, when oxygen concentrations were 353
equal to 2, 1 and 0.5% of saturation. However, the case was reinstated under anaerobic 354
conditions, demonstrating that the inhibition by oxygen was reversible. 355
356
4. CONCLUSIONS 357
14
The structured fixed-bed reactor operated under continuous flow and intermittent 358
aeration proved suitable for the post-treatment of slaughterhouse wastewater and for the 359
removal of residual COD and ammonia. 360
In all of the experimental phases studied, nitrification efficiency was above 90%. In 361
phase V, which operated in cycles of 3 hours – 1 hour aerobic followed by 2 hours anoxic - 362
the reactor achieved nitrogen and COD removal of 62% and 95%, respectively. The effluent 363
generated at this stage had TKN concentrations of 6.0 mg.L-1, 58.5 mg.L-1 of N-NO3, and 19 364
mg.L-1of COD. 365
After 131 days of operation (phase V) the presence of anammox bacteria was 366
verified. These microorganisms should have been part of the pool of microorganisms that 367
were active in nitrogen removal. Molecular biology analyses detected the presence of 368
anammox bacteria, contributing to a better understanding of the processes involved. 369
The use of structured fixed-bed reactors subjected to intermittent aeration was 370
successful for the post-treatment of effluents from anaerobic reactors treating poultry 371
slaughterhouse effluents. Considering the results obtained with this particular wastewater 372
the reactor assayed is recommended for the removal of residual organic matter and nitrogen 373
present in effluents of anaerobic reactors. 374
375
Acknowledgments 376
The authors acknowledge Fundação Araucária – Fundação de Amparo à Pesquisa 377
do Estado do Paraná, CNPq - Conselho Nacional de Pesquisa Científica e Tecnológica, and 378
FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support, 379
and Céu Azul Alimentos for technical support. 380
381
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TABLES
Table 1. Duration of aerobic and anoxic periods of each experimental phase.
Phase Aerobic
(hours)
Anoxic
(hours)
HRT
(hours)
Duration
(days)
I 6 0 24 24
II 4 2 24 11
III 2 1 24 8
IV 1.5 1.5 24 25
V 1 2 24 56
Table 2. Mean values of the parameters analysed in the influent of each phase of the
experiment with their standard deviations.
Parameters Phase I
(SD)
Phase II
(SD)
Phase III
(SD)
Phase IV
(SD)
Phase V
(SD)
pH 7.4 to 8.1 7.7 to 8.2 7.3 to 7.9 7.6 to 8.0 7.3 to 8.1
Alkalinity (mg CaCO3.L-1) 817 (129) 739 (140) 673 (103) 711 (103) 736 (156)
TKN (mg.L-1) 147 (13) 127 (3) 142 (24) 152 (11) 169 (9)
NH4-N (mg.L-1) 110 (14) 112 (18) 115 (11) 115 (3) 112 (15)
COD filtered (mg.L-1) 126 (79) 243 (23) 204 (65) 130 (49) 139 (16)
COD total (mg.L-1) 383 (197) 370 (18) 319 (26) 376 (78) 418 (150)
BOD filtered (mg.L-1) 113 (n.e.) n.e. 107 (n.e.) 122 (n.e.) 69 (n.e.)
BOD total (mg.L-1) 169 (n.e.) n.e. 194 (n.e.) 157 (n.e.) 117 (n.e.)
TSS (mg.L-1) 214 (180) 384 (81) 129 (12) 525 (276) 834 (11)
VSS (mg.L-1) 162 (121) 268 (50) 104 (1) 299 (84) 543 (1)
Oils and greases (mg.L-1) 38 (n.e.) n.e. 28 (n.e.) 36 (n.e.) 21 (n.e.)
TKN load (kg/m3.day) 0.147 0.127 0.142 0.153 0.169
CODT/BOD 2.2 n.e. 1.6 2.4 3.6
CODT Influent/TKNInfluent 2.6 2.4 2.2 2.4 2.5
n.e.: not evaluated; SD: standard deviation
Table 3. Mean values of the results of the characterisation of the reactor effluent during the
different evaluated phases.
Parameters Phase I
(SD)
Phase II
(SD)
Phase III
(SD)
Phase IV
(SD)
Phase V
(SD)
pH 6.4 to 8.0 6.1 to 8.2 5.6 to 7.3 6.4 to 8.1 6.9 to 8.2
jhazmat tables (3).doc
Alkalinity (mg CaCO3.L-1) 114 (65) 136 (30) 15 (18) 75 (2) 160 (76)
TKN (mg.L-1) 35.5 (32) 9.6 (0) 9.0 (1) 7.0 (3) 6.4 (5)
NH4+-N (mg.L-1) 19.7 (20) 8.0 (4) 7.5 (4) 5.3 (2) 6.4 (6)
NO3--N (mg.L-1) 103 (16) 92 (11) 100 (8) 84 (12) 58 (11)
CODF (mg.L-1) 18 (2) 4 (0) 49 (11) 31 (17) 5 (2)
CODT (mg.L-1) 45 (9) 24 (2) 69 (6) 31 (25) 22 (2)
TSS (mg.L-1) 30 (17) 8 (2) 8,5 (4) 17 (3) 1,5 (1)
VSS (mg.L-1) 28 (13) 8 (2) 8,5 (4) 15 (5) 0 (0)
CODTC/NO3-NR 14.2 14.3 7.18 5.5 3.8
NT removed load (kg
N/m3.day)
0.024 0.027 0.035 0.063 0.104
SD: standard deviation; CODTC: Total COD Consumed; NO3-NR: NO3-N Removed
Table 4. Mean values of total nitrogen applied and removed during the experiment.
Parameters Phase I Phase II Phase III Phase IV Phase V
Duration of phase (days) 24 13 11 27 56
TKN (kg/m3.day) 0.147 0.127 0.142 0.153 0.169
Total N (kg/m3.d) removed 0.024 0.027 0.035 0.063 0.104
NH4+-N (kg/m3.d) removed 0.112 0.117 0.133 0.146 0.163
Total N removed (%) 8 a 30 b 25 b 42 c 62 d
Means followed by the same letters in the line of Total N Removed (%) do not differ (Tukey’s
test, p = 0.05)
FIGURES
Figure1
0
50
100
150
200
am
moniu
m (
mg N
H4-N
.L-1)
Phases
I II III IV V
influent
0
20
40
60
effluent
0
100
200
300
400
TK
N (
mg
NH
4-N
.L-1)
Phases
I II III IV V
influent
0
20
40
60
80
effluent
Figure 2
jhazmat figures.doc
6
7
8
9
pH
Phases
I II III IV V
influent
5
6
7
8
effluent
400
600
800
1000
1200
A
alk
alin
ity
(mg
Ca
CO
3.L
-1)
Phases
I II III IV V
influent
0
100
200
300
400
effluent
Figure 3
40 60 80 100 120 140 160 180
0
100
200
300
400
500
600
0
25
50
75
100
125
150 I II III IV V
CO
D (
mg O
2.L
-1)
Time (days)
CODF influent COD
F effluent COD
T influent
CODT effluent Nitrate
Nitra
te (m
g N
O3-N
.L-1)
FIGURE 4
0 5 10 15 20 25 30 35 40 450
5
10
15
20
25
30
35
Nitro
gen (
mg.L
-1)
Time (h)
NH4-N
NO2-N
NO3-N
FIGURE 5
FIGURE 6