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Hydrolyzed molasses as an external carbon source in biological nitrogen removal
Bioresource Technology 96 (2005) 1690–1695
Hydrolyzed molasses as an external carbon source in biological nitrogen removal Zhe-Xue Quan a, Yin-Shu Jin a, Cheng-Ri Yin b, Jay J. Lee c, Sung-Taik Lee
a,*
a
c
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea b Department of Chemistry, Yanbian University, Yanji 133-002, China Nakdong River Water Environment Laboratory, National Institute of Environmental Research, 239-3 Pyung-ri Dasan, Koryoung, Kyungpook, Republic of Korea Received in revised form 22 June 2004; accepted 17 December 2004 Available online 26 February 2005
Abstract Hydrolyzed molasses was evaluated as an alternative carbon source in a biological nitrogen removal process. To increase biodegradability, molasses was acidified before thermohydrolyzation. The denitrification rate was 2.9–3.6 mg N/g VSS h with hydrolyzed molasses, in which the percentage of readily biodegradable substrate was 47.5%. To consider the hydrolysate as a carbon source, a sequencing batch reactor (SBR) was chosen to treat artificial municipal wastewater. During the 14 days (28 cycles) of operation, the SBR using hydrolyzed molasses as a carbon source showed 91.6 ± 1.6% nitrogen removal, which was higher than that using methanol (85.3 ± 2.0%). The results show that hydrolyzed molasses can be an economical and effective external carbon source for the nitrogen removal process. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Carbon source; Denitrification; Hydrolyzed molasses; Sequencing batch reactor
1. Introduction In municipal wastewater treatment systems, nitrate removal is normally accomplished through denitrification, which relies on a carbon source as an electron donor. In many treatment plants, the readily degradable organic mater available in the wastewater may be a limiting factor for successful nitrogen removal (Æsøy et al., 1998; Mora et al., 2003). Many commercially available organic compounds, such as acetic acid (Her and Huang, 1995; Mohseni-Bandpi et al., 1999), glucose (Chou et al., 2003) and methanol (Louzeiro et al., 2002), can serve effectively as carbon sources for denitrification. Of these, methanol is the most commonly used *
Corresponding author. Tel.: +82 42 869 2617; fax: +82 42 863 5617. E-mail address: [email protected] (S.-T. Lee).
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.12.033
external carbon source due to its cost effectiveness (Louzeiro et al., 2003). However, there is certainly a need to find economical alternative carbon sources to those mentioned above to maintain the desired effluent quality. Many researchers have suggested various by-products or waste materials as alternative carbon sources. For example, wine distillery effluents (Bernet et al., 1996), the leachate of food waste (Lee et al., 2002), swine waste (Lee et al., 1997) and hydrolyzed sludge (Æsøy and Ødegaard, 1994; Aravinthan et al., 2001; Barlindhaug and Ødegaard, 1996). Another option that can be considered is the use of molasses. Molasses is a sugar production by-product with high sugar content (48–50%). This by-product is a cheap carbon source used for various industrial fermentations (Miranda et al., 1996; Najafpour and Shan, 2003). Some researchers have used molasses as an
Z.-X. Quan et al. / Bioresource Technology 96 (2005) 1690–1695
external carbon source for denitrification (Boaventura and Rodrigues, 1997; Ten Have et al., 1994). However, the major components in molasses, polysaccharides, have carbon chains that are too long to be used quickly by denitrifying bacteria and need to be hydrolyzed to reduced sugars such as sucrose, glucose and fructose (Najafpour and Shan, 2003). To increase the effect of molasses as a carbon source on denitrification, molasses were thermohydrolyzed under acidic conditions and the effect of hydrolyzed molasses on denitrification was determined. The effectiveness of hydrolyzed molasses for the treatment of artificial sewage in the SBR was compared with that of the commonly used carbon source, methanol.
2. Methods 2.1. Source of molasses and sludge The molasses (sugar cane), with sugar content of 45%, was supplied by M21 Environmental Technology Inc. (Korea) and the activated sludge was obtained from a sewage treatment plant in Daejeon, Korea. For the determination of the denitrification rate, the activated sludge was settled in an anoxic condition overnight for oxygen removal. 2.2. Molasses hydrolyzation For the hydrolysis, 1.0, 2.0, 3.0, and 4.0 ml of 3.1 mol/l sulfuric acid solution were added to 50 ml of 600 g/l molasses solution and heated to 90 °C for 2 h. The degree of hydrolysis was determined by measuring the content of reduced sugar in the hydrolysate.
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operated. At start-up, each reactor contained 2.7 l mixed liquor with 2000 mg/l MLVSS, prepared by mixing the synthetic wastewater with an appropriate volume of activated sludge. The MLVSS of each reactor was maintained at a constant value by checking the MLVSS value of the reactor and controlling the volume of sludge removed, and the sludge retention time was about 14 days. The synthetic wastewater was prepared so as to resemble common sewage in Korea, containing 140 mg glucose, 123 mg NH4Cl, 400 mg NaHCO3, 22 mg MgSO4 Æ 7H2O, 1 mg NaCl, 7 mg FeSO4 Æ 7H2O and 50 mg CaCl2 in 1 l. Both SBRs were operated on a 12 h cycle. The operational sequence of each cycle consisted of the following: 1 h FILL period with aeration, 10 h REACT period separated into 4 h aeration, 4 h anoxic and 2 h aeration stages, 0.5 h SETTLE, and 0.5 h DRAW and IDLE periods. The influent and effluent volumes were defined as 1.8 l. The activated sludge was kept suspended by mixing over the entire REACT period. Methanol and hydrolyzed molasses were added to individual reactors at the initial reactor COD value of 150 mg/l. To improve the efficiency of the carbon source utilization during denitrification, methanol and hydrolyzed molasses were slowly fed for 1 h at the beginning of the anoxic stage. 2.5. Analytical methods The COD, nitrate and ammonia concentrations were determined by Standard Methods (Clescerl et al., 1998). Content of reducing sugar was determined using the DNS (dinitrosalicylic acid) method (Chaplin and Kennedy, 1986).
3. Results and discussion 2.3. Batch test for nitrate reduction using hydrolyzed molasses To determine the nitrate reduction rate with external carbon source, hydrolyzed molasses (final concentration of 100, 200, and 500 mg COD/l) was added to each bottle containing 100 mg/l nitrate and basal salts (1400 mg K2HPO4, 270 mg KH2PO4, 400 mg NaHCO3, 5 mg MgSO4 Æ 7H2O, 0.5 mg FeSO4 Æ 7H2O and 0.5 mg CaCl2 in 1 l) solution, and anoxic sludge was added to yield about 1800 mg/l of MLVSS (mixed liquor volatile suspended solids). To ensure anaerobic conditions, each bottle was flushed with nitrogen gas for 5 min at 500 ml/min. 2.4. SBR operation for nitrogen removal using hydrolyzed molasses as a carbon source To compare the effect of hydrolyzed molasses on nitrogen removal with that of methanol, two SBRs were
3.1. Molasses hydrolyzation Chemical, thermal, and thermo-chemical hydrolyses give concentrated and complex hydrolysates with respect to the distribution of organic compounds, containing low molecular weight and colloidal readily biodegradable matter and slowly hydrolyzable particulate materials (Æsøy et al., 1998). The readily biodegradable organic matter is preferred since this will give the highest denitrification rates. High molecular weight particulate materials must be hydrolyzed in order to be converted to low molecular weight or biodegradable colloidal matter. Hydrolyzation is the most common method used to prepare alternative carbon sources for use in denitrification. In this study, the acidic thermohydrolyzing method was chosen. Fig. 1 shows the change of pH and the content of reduced sugar after acidic thermohydrolyzation of molasses. After the addition of different volumes of sulfuric
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Fig. 1. pH and reduced sugar contents of thermohydrolyzed molasses with the addition of 3.1 mol/l sulfuric acid to 50 ml of 600 g/l molasses solution.
acid, the pH of the molasses solution changed from 4.6 to 4.3, 3.6, 2.7 and 1.9. When sulfuric acid was added to pH 3.6, the content of reduced sugar in hydrolyzed molasses increased from 170 to 520 g/kg-molasses. However, a further decrease in pH did not increase the content of reduced sugar. Therefore, the molasses hydrolyzed at pH 3.6 was used for further experiments. The COD value and reduced sugar contents of hydrolyzed molasses solution at pH 3.6 were 433 and 310 g/l, respectively. In other words, the content of reduced sugar in the COD of the hydrolyzed molasses was about 76%. 3.2. Batch test for nitrate reduction using hydrolyzed molasses as a carbon source To test the efficiency of nitrate reduction with hydrolyzed molasses, nitrate reduction and COD removal with three different concentrations of hydrolyzed molasses were monitored. When concentrations of hydrolyzed molasses were 100 and 200 mg COD/l, denitrification could be separated into two phases (Fig. 2). In the first rapid denitrification phase, a readily biodegradable substrate such as reduced sugar in hydrolyzed molasses would be used as a carbon source, and in the second slow denitrification phase, slowly biodegradable substrates such as unhydrolyzed polysaccharide in molasses would be used as the carbon source. Griffiths (1994) reported that during denitrification with wastewater as the electron donor and activated sludge originating from denitrifying–nitrifying systems, it is usual for three linear phases of nitrate reduction to occur simultaneously. The denitrification rate is highest when using soluble readily biodegradable substrate, followed by when using particulate slowly biodegradable substrate, and lowest in endogenous conditions.
Fig. 2. Denitrification with several concentrations of hydrolyzed molasses as carbon source. (A) 100 mg COD/l, (B) 200 mg COD/l, (C) 500 mg COD/l. The readily biodegradable fraction was calculated by determining the NO3 –N value, which is given by the difference between the initial NO3 –N concentration and the extrapolated value drawn from the second rate.
In denitrification processes, the fraction of readily biodegradable substrates in the alternative carbon source is a very important factor. Readily biodegradable organic matter is preferred since this will give the highest denitrification rate. The readily biodegradable fraction of the carbon source can be calculated by determining the DNO3 –N value, which is given by the difference between the initial NO3 –N concentration and the extrapolated value drawn from the second rate (Naidoo et al., 1998). In this experiment, the readily biodegradable substrate concentrations were 50 and 89 mg COD/l at
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Table 1 Denitrification with hydrolyzed molasses as an external carbon source Hydrolyzed molasses concentration (mg COD/l) Nitrate (mg/l) Denitrification rate (mg NO3 –N/g VSS h) Readily biodegradable substrate (mg COD/l) Substrate consumption (mg COD/mg NO3 –N)
1st phase 2nd phase
100
200
500
100 2.9 1.3 50
100 3.6 1.4 89
100 3.6 – –
4.3
5.0
5.8
different molasses concentrations (Table 1). Therefore, about 47.5 ± 2.5% of the organic compounds in hydrolyzed molasses were readily biodegradable, which was about two times higher than that in sewage, where the fraction of readily biodegradable substrate was about 10–20% of the total COD (Henze et al., 1994). The nitrate reduction rates in the first phase at the two different concentrations of molasses were 2.9 and 3.6 mg NO3 – N/g VSS h. The corresponding rates in the second phase were 1.3 and 1.4 mg NO3 –N/g VSS h (Table 1). This means that the readily biodegradable fraction of the hydrolyzed molasses stimulated nitrate reduction by more than two times. When the concentration of hydrolyzed molasses was increased to 500 mg COD/l, nitrate was linearly reduced (Fig. 2) with a nitrate reduction rate of 3.6 mg NO3 –N/g VSS h, because the readily biodegradable substrate in 500 mg COD/l hydrolyzed molasses was enough to completely remove initial nitrate. This shows that denitrification rate increased with increasing hydrolyzed molasses concentration. The substrate consumption rates were 4.3, 5.0, and 5.8 mg COD/mg NO3 –N with different molasses concentrations (Table 1). Compared to the hydrolyzate from sludge and household solid organic waste (8– 10 mg COD/mg NO3 –N, Æsøy et al., 1998), the amount of hydrolyzed molasses needed for nitrate reduction was only 50%, indicating that hydrolyzed molasses is a more effective carbon source than hydrolyzed sludge or solid organic waste. 3.3. Nitrogen removal in SBR using hydrolyzed molasses as a carbon source The SBR system, with the incorporation of alternating aerobic and anoxic/anaerobic stages in the REACT period, not only allows good removal of organic matter and suspended solids, but satisfactory removal of nitrogen by nitrification and denitrification is also achieved. Because of its simplicity and low capital and operational costs, the SBR is the most popular system for treating wastewater containing high nutrient concentrations in small and medium size cities with high population den-
Fig. 3. Effects of different external carbon sources on nitrogen removal in SBR over 14 days (28 cycles) operation.
sities. An SBR system was therefore used in the present study to compare the nitrogen removal efficiencies of hydrolyzed molasses and methanol as external carbon sources. Fig. 3 shows the percentage of nitrogen removal in the two SBRs, where hydrolyzed molasses and methanol were used as the carbon sources over 14 days (28 cycles) of operation. Ammonia was completely oxidized and not detected in the effluent. The concentration of nitrate was 6–8 mg NO3 –N/l in the reactor with methanol (reMe) and 3–5 mg NO3 –N/l with hydrolyzed molasses (re-HM). This result shows that the reactors achieved stable nitrogen removal over 14 days of operation, and it is postulated that the denitrification activity would not have changed significantly for some time. When hydrolyzed molasses was the external carbon source, the percentage of nitrogen removed was 91.6 ± 1.6%; the corresponding percentage for methanol was 85.3 ± 2.0%. The t-test value of the results for both carbon sources was 11.2 and p < 0.001, which indicates that the two carbon sources had statistically different removal efficiencies. The unused carbon source accumulated in the wastewater and resulted in a high COD level in the effluent. The average COD values of the effluents from the two reactors were 51 and 53 mg/l, respectively. Some researchers (Her and Huang, 1995; Chou et al., 2003) reported that when C/N ratio was increased in denitrification, a great deal of methanol remained in the effluent while glucose was completely removed. It means that excess addition of methanol seriously inhibits the activity of denitrifying sludge and could result in residual methanol being discharged in treatment plant effluent. For the use of wastes or by-products as alternative external carbon sources, a high COD concentration in effluent is a frequent problem. Lee et al. (2002) reported that the use of anaerobically fermented leachate of food
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Fig. 4. Change of the COD and nitrate concentrations during the operation of SBR with different external carbon sources.
waste as a carbon source caused increased COD concentration in effluent because high molecular weight substrates were not used readily by the microorganisms. Fig. 4 shows the change of nitrate and COD during 1 cycle of SBR operation. In the aeration stage, nitrate concentration was continually increased by ammonia oxidation, and the final concentrations of nitrate were similar in both reactors. The organic carbon in re-Me was more quickly removed than in re-HM because the methanol left after the last stage of the cycle was readily biodegradable. On the other hand, in re-HM, organic matter left after the last stage would not be readily degradable. In re-Me, only 55% of nitrate was reduced in the 4 h anoxic stage, while nitrate was completely removed in the 2 h anoxic stage in re-HM. In re-HM, with the addition of hydrolyzed molasses, denitrifying bacteria would quickly get energy from the oxidation of the readily biodegradable fraction of hydrolyzed molasses and reduce nitrate. The COD in re-Me was increased to 130 mg/l in the first 1 h of the anoxic stage by the addition of an external carbon source, while in re-HM, COD was increased to 100 mg/l. The nitrate reduction rate in re-HM was 4.2 mg NO3 –N/g VSS h, which was higher than the nitrate reduction rate in re-Me (1.0 mg NO3 –N/g VSS h) and also higher than some values reported with methanol (1.3 mg NOx –N/g VSS h (Louzeiro et al., 2003); 2.2 mg NO3 –N/g VSS h (Min et al., 2002)). The nitrate reduction rate in re-HM was also higher than that with acidogenic fermented waste sludge (2.0 mg NO3 –N/g VSS h; Min et al., 2002). The nitrate reduction rate in re-Me was lower than that previously observed for methanol in a SBR (Tam et al., 1994). Louzeiro et al. (2003) reported that when methanol was used as an external carbon source a minimum of
55% increase in the denitrification rate was observed with acclimatized (five weeks) sludge compared with non-acclimatized sludge. The low denitrification rate with methanol in this experiment may have been partly caused by the short acclimation time for the methanol. The denitrification rate with glucose has been previously reported as much higher than that with methanol (Akunna et al., 1993). Since the main product of hydrolyzed molasses was reduced sugar similar to glucose, it would be the reason why the rate of denitrification in re-HM was higher than in re-Me. In the second aeration stage, the remaining nitrate concentration was 5.2–6.0 mg NO3 –N/l in re-Me, but in re-HM, the concentration of nitrate was 3.3 mg NO3 –N/l. The increase of nitrate concentration in reHM could be related to the oxidation of ammonia produced from the hydrolyzed molasses. The removal rate of COD in re-HM was lower than that in re-Me. This result coincides with the results obtained in the first aeration stage where the organic compounds remaining after denitrification in re-HM were slowly biodegradable. The COD value of the effluent in re-HM (51 mg/l) was higher than the COD regulation value for the sewage treatment plant in Korea (40 mg/l). The results for denitrification efficiency in re-HM showed that the carbon source used to remove the nitrate was a readily biodegradable fraction of the hydrolyzed molasses. If less hydrolyzed molasses were added, a fraction of slowly biodegradable substrates could also be used as a carbon source. The COD value of the effluent also decreased, though complete removal of the nitrate took longer because the denitrification rate with slowly biodegradable substrates was about 40% of the denitrification rate with readily biodegradable substrates (Table 1). Further study is needed to determine the optimum dosage of hydrolyzed molasses for efficient nitrate reduction and for prevention of a COD increase. Hydrolyzed molasses would be a more economical source of carbon than methanol. According to a report of the Korea Importers Association published in March 2003, the import of molasses costs US$60/ton whereas methanol costs US$300/ton. The cost of molasses is therefore only 20% of the cost of methanol. Only 17 l of sulfuric acid would be needed to hydrolyze 1 ton of molasses, and, based on figures quoted in the Chemical Market Reporter (February 3, 2003), the cost of this much sulfuric acid is only about 1 US dollar. Finally, a sewage treatment plant that treats 6000 tons of sewage a day needs only 1 ton of acidified molasses daily, and the acidic thermohydrolyzation could be processed in a 2 m3 heating tank. With a general boiler (LATTNER, USA), the heating energy required for the thermohydrolyzation of molasses is about 150 kWh/ton, which costs only about US$6/ton according to the price of industrial electricity in Korea.
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4. Conclusion The results show the effectiveness of the thermohydrolysate of molasses, prepared at pH 3.6 with sulfuric acid, as an alternative carbon source. It had a readily biodegradable substrate content of 47.5% and gave a denitrification rate of 2.9–3.6 mg N/g VSS h. In the SBR system, hydrolyzed molasses was more effective than methanol in removing nitrogen. Hydrolyzed molasses is therefore recommended as an economical and effective external carbon source for nitrogen removal. However, for full-scale replacement of methanol with hydrolyzed molasses, a long time is needed to stabilize the sludge. The optimum dosage of the carbon source also needs to be determined. Acknowledgements This work was supported by the Korean Ministry of Environments Eco-Technopia-21 program (Grant 121041-028). References Æsøy, A., Ødegaard, H., 1994. The nitrogen removal efficiency and capacity in biofilms with biologically hydrolysed sludge as carbon source. Water Science and Technology 30 (6), 63–71. Æsøy, A., Ødegaard, H., Bach, K., Pujol, R., Hamon, M., 1998. Denitrification in a packed bed biofilm reactor (BIOFOR)—experiments with different carbon sources. Water Research 32, 1463–1470. Akunna, J.C., Bizeau, C., Moletta, R., 1993. Nitrate and nitrite reductions with anaerobic sludge using various sources: glucose, glycerol, acetic acid, lactic acid and methanol. Water Research 27, 1303–1312. Aravinthan, V., Mino, T., Takizawa, S., Satoh, H., Matsuo, T., 2001. Sludge hydrolysate as a carbon source for denitrification. Water Science and Technology 43 (1), 191–199. Barlindhaug, J., Ødegaard, H., 1996. Thermal hydrolysis for the production of carbon source for denitrification. Water Science and Technology 34 (1–2), 371–378. Bernet, N., Habouzit, F., Moletta, R., 1996. Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater. Applied Microbiology and Biotechnology 46, 92–97. Boaventura, R.A.R., Rodrigues, A.E., 1997. Denitrification kinetics in a rotating disk biofilm reactor. Chemical Engineering Journal 65, 227–235. Chaplin, M.F., Kennedy, J.F., 1986. Carbohydrate Analysis: a Practical Approach. IRL Press, Oxford, UK, p .3. Chou, Y.J., Ouyang, C.F., Kuo, W.L., Huang, H.L., 2003. Denitrifying characteristics of the multiple stages enhanced biological
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nutrient removal process with external carbon sources. Journal of Environmental Science Health—Part A, Toxic/Hazardous Substances and Environmental Engineering 38, 339–352. Clescerl, L.S., Greenberg, A.E., Eaton, A.D., 1998. Standard Methods for the Examination of Water and Wastewater. APHA-AWWAWEF, Washington, USA. Griffiths, P., 1994. Modification of the IAWPRC task group general activated sludge model. Water Research 28, 657–664. Henze, M., Kristensen, G.H., Strube, R., 1994. Rate-capacity characterization of wastewater for nutrient removal processes. Water Science and Technology 29 (7), 101–107. Her, J.J., Huang, J.S., 1995. Influences of carbon source and C/N ratio on nitrate/nitrite denitrification and carbon breakthrough. Bioresource Technology 54, 45–51. Lee, C.Y., Shin, H.S., Chae, S.R., Nam, S.Y., Paik, B.C., 2002. Nutrient removal using anaerobically fermented leachate of food waste in the BNR process. Water Science and Technology 47 (1), 159–165. Lee, S.I., Park, J.H., Ko, K.B., Koopman, B., 1997. Effect of fermented swine wastes on biological nutrient removal in sequencing batch reactors. Water Research 31, 1807–1812. Louzeiro, N.R., Mavinic, D.S., Oldham, W.K., Meisen, A., Gardner, I.S., 2002. Methanol-induced biological nutrient removal kinetics in a full-scale sequencing batch reactor. Water Research 36, 2721– 2732. Louzeiro, N.R., Mavinic, D.S., Oldham, W.K., Meisen, A., Gardner, I.S., 2003. Process control and design considerations for methanolinduced denitrification in a sequencing batch reactor. Environmental Technology 24, 161–169. Min, K.S., Park, K.S., Jung, Y.J., Khan, A.R., Kim, Y.J., 2002. Acidogenic fermentation: utilization of wasted sludge as a carbon source in the denitrification process. Environmental Technology 23, 293–302. Miranda, M.P., Benito, G.G., Cristobal, N.S., Nieto, C.H., 1996. Color elimination from molasses wastewater by Aspergillus niger. Bioresource Technology 57, 229–235. Mohseni-Bandpi, A., Elliott, D.J., Momeny-Mazdeh, A., 1999. Denitrification of groundwater using acetate acid as a carbon source. Water Science and Technology 40 (2), 53–59. Mora, F.R., Ferrara de Giner, G., Andara, A.R., Esteban, J.L., 2003. Effect of organic carbon shock loading on endogenous denitrification in sequential batch reactors. Bioresource Technology 88, 215– 219. Naidoo, V., Urbain, V., Burckley, C.A., 1998. Characterization of wastewater and activated sludge from European municipal wastewater treatment plants using the NUR test. Water Science and Technology 38 (1), 303–310. Najafpour, G.D., Shan, C.P., 2003. Enzymatic hydrolysis of molasses. Bioresource Technology 86, 91–94. Tam, N.F.Y., Leung, G.L.W., Wong, Y.S., 1994. The effects of external carbon loading on nitrogen removal in sequencing batch reactors. Water Science and Technology 30 (6), 73–81. Ten Have, P.J.W., Willers, H.C., Derikx, P.J.L., 1994. Nitrification and denitrification in an activated-sludge system for supernatant from settled sow manure with molasses as an extra carbon source. Bioresource Technology 47, 135–141.
Hydrolyzed molasses as an external carbon source in biological nitrogen removal Zhe-Xue Quan a, Yin-Shu Jin a, Cheng-Ri Yin b, Jay J. Lee c, Sung-Taik Lee
a,*
a
c
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea b Department of Chemistry, Yanbian University, Yanji 133-002, China Nakdong River Water Environment Laboratory, National Institute of Environmental Research, 239-3 Pyung-ri Dasan, Koryoung, Kyungpook, Republic of Korea Received in revised form 22 June 2004; accepted 17 December 2004 Available online 26 February 2005
Abstract Hydrolyzed molasses was evaluated as an alternative carbon source in a biological nitrogen removal process. To increase biodegradability, molasses was acidified before thermohydrolyzation. The denitrification rate was 2.9–3.6 mg N/g VSS h with hydrolyzed molasses, in which the percentage of readily biodegradable substrate was 47.5%. To consider the hydrolysate as a carbon source, a sequencing batch reactor (SBR) was chosen to treat artificial municipal wastewater. During the 14 days (28 cycles) of operation, the SBR using hydrolyzed molasses as a carbon source showed 91.6 ± 1.6% nitrogen removal, which was higher than that using methanol (85.3 ± 2.0%). The results show that hydrolyzed molasses can be an economical and effective external carbon source for the nitrogen removal process. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Carbon source; Denitrification; Hydrolyzed molasses; Sequencing batch reactor
1. Introduction In municipal wastewater treatment systems, nitrate removal is normally accomplished through denitrification, which relies on a carbon source as an electron donor. In many treatment plants, the readily degradable organic mater available in the wastewater may be a limiting factor for successful nitrogen removal (Æsøy et al., 1998; Mora et al., 2003). Many commercially available organic compounds, such as acetic acid (Her and Huang, 1995; Mohseni-Bandpi et al., 1999), glucose (Chou et al., 2003) and methanol (Louzeiro et al., 2002), can serve effectively as carbon sources for denitrification. Of these, methanol is the most commonly used *
Corresponding author. Tel.: +82 42 869 2617; fax: +82 42 863 5617. E-mail address: [email protected] (S.-T. Lee).
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.12.033
external carbon source due to its cost effectiveness (Louzeiro et al., 2003). However, there is certainly a need to find economical alternative carbon sources to those mentioned above to maintain the desired effluent quality. Many researchers have suggested various by-products or waste materials as alternative carbon sources. For example, wine distillery effluents (Bernet et al., 1996), the leachate of food waste (Lee et al., 2002), swine waste (Lee et al., 1997) and hydrolyzed sludge (Æsøy and Ødegaard, 1994; Aravinthan et al., 2001; Barlindhaug and Ødegaard, 1996). Another option that can be considered is the use of molasses. Molasses is a sugar production by-product with high sugar content (48–50%). This by-product is a cheap carbon source used for various industrial fermentations (Miranda et al., 1996; Najafpour and Shan, 2003). Some researchers have used molasses as an
Z.-X. Quan et al. / Bioresource Technology 96 (2005) 1690–1695
external carbon source for denitrification (Boaventura and Rodrigues, 1997; Ten Have et al., 1994). However, the major components in molasses, polysaccharides, have carbon chains that are too long to be used quickly by denitrifying bacteria and need to be hydrolyzed to reduced sugars such as sucrose, glucose and fructose (Najafpour and Shan, 2003). To increase the effect of molasses as a carbon source on denitrification, molasses were thermohydrolyzed under acidic conditions and the effect of hydrolyzed molasses on denitrification was determined. The effectiveness of hydrolyzed molasses for the treatment of artificial sewage in the SBR was compared with that of the commonly used carbon source, methanol.
2. Methods 2.1. Source of molasses and sludge The molasses (sugar cane), with sugar content of 45%, was supplied by M21 Environmental Technology Inc. (Korea) and the activated sludge was obtained from a sewage treatment plant in Daejeon, Korea. For the determination of the denitrification rate, the activated sludge was settled in an anoxic condition overnight for oxygen removal. 2.2. Molasses hydrolyzation For the hydrolysis, 1.0, 2.0, 3.0, and 4.0 ml of 3.1 mol/l sulfuric acid solution were added to 50 ml of 600 g/l molasses solution and heated to 90 °C for 2 h. The degree of hydrolysis was determined by measuring the content of reduced sugar in the hydrolysate.
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operated. At start-up, each reactor contained 2.7 l mixed liquor with 2000 mg/l MLVSS, prepared by mixing the synthetic wastewater with an appropriate volume of activated sludge. The MLVSS of each reactor was maintained at a constant value by checking the MLVSS value of the reactor and controlling the volume of sludge removed, and the sludge retention time was about 14 days. The synthetic wastewater was prepared so as to resemble common sewage in Korea, containing 140 mg glucose, 123 mg NH4Cl, 400 mg NaHCO3, 22 mg MgSO4 Æ 7H2O, 1 mg NaCl, 7 mg FeSO4 Æ 7H2O and 50 mg CaCl2 in 1 l. Both SBRs were operated on a 12 h cycle. The operational sequence of each cycle consisted of the following: 1 h FILL period with aeration, 10 h REACT period separated into 4 h aeration, 4 h anoxic and 2 h aeration stages, 0.5 h SETTLE, and 0.5 h DRAW and IDLE periods. The influent and effluent volumes were defined as 1.8 l. The activated sludge was kept suspended by mixing over the entire REACT period. Methanol and hydrolyzed molasses were added to individual reactors at the initial reactor COD value of 150 mg/l. To improve the efficiency of the carbon source utilization during denitrification, methanol and hydrolyzed molasses were slowly fed for 1 h at the beginning of the anoxic stage. 2.5. Analytical methods The COD, nitrate and ammonia concentrations were determined by Standard Methods (Clescerl et al., 1998). Content of reducing sugar was determined using the DNS (dinitrosalicylic acid) method (Chaplin and Kennedy, 1986).
3. Results and discussion 2.3. Batch test for nitrate reduction using hydrolyzed molasses To determine the nitrate reduction rate with external carbon source, hydrolyzed molasses (final concentration of 100, 200, and 500 mg COD/l) was added to each bottle containing 100 mg/l nitrate and basal salts (1400 mg K2HPO4, 270 mg KH2PO4, 400 mg NaHCO3, 5 mg MgSO4 Æ 7H2O, 0.5 mg FeSO4 Æ 7H2O and 0.5 mg CaCl2 in 1 l) solution, and anoxic sludge was added to yield about 1800 mg/l of MLVSS (mixed liquor volatile suspended solids). To ensure anaerobic conditions, each bottle was flushed with nitrogen gas for 5 min at 500 ml/min. 2.4. SBR operation for nitrogen removal using hydrolyzed molasses as a carbon source To compare the effect of hydrolyzed molasses on nitrogen removal with that of methanol, two SBRs were
3.1. Molasses hydrolyzation Chemical, thermal, and thermo-chemical hydrolyses give concentrated and complex hydrolysates with respect to the distribution of organic compounds, containing low molecular weight and colloidal readily biodegradable matter and slowly hydrolyzable particulate materials (Æsøy et al., 1998). The readily biodegradable organic matter is preferred since this will give the highest denitrification rates. High molecular weight particulate materials must be hydrolyzed in order to be converted to low molecular weight or biodegradable colloidal matter. Hydrolyzation is the most common method used to prepare alternative carbon sources for use in denitrification. In this study, the acidic thermohydrolyzing method was chosen. Fig. 1 shows the change of pH and the content of reduced sugar after acidic thermohydrolyzation of molasses. After the addition of different volumes of sulfuric
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Fig. 1. pH and reduced sugar contents of thermohydrolyzed molasses with the addition of 3.1 mol/l sulfuric acid to 50 ml of 600 g/l molasses solution.
acid, the pH of the molasses solution changed from 4.6 to 4.3, 3.6, 2.7 and 1.9. When sulfuric acid was added to pH 3.6, the content of reduced sugar in hydrolyzed molasses increased from 170 to 520 g/kg-molasses. However, a further decrease in pH did not increase the content of reduced sugar. Therefore, the molasses hydrolyzed at pH 3.6 was used for further experiments. The COD value and reduced sugar contents of hydrolyzed molasses solution at pH 3.6 were 433 and 310 g/l, respectively. In other words, the content of reduced sugar in the COD of the hydrolyzed molasses was about 76%. 3.2. Batch test for nitrate reduction using hydrolyzed molasses as a carbon source To test the efficiency of nitrate reduction with hydrolyzed molasses, nitrate reduction and COD removal with three different concentrations of hydrolyzed molasses were monitored. When concentrations of hydrolyzed molasses were 100 and 200 mg COD/l, denitrification could be separated into two phases (Fig. 2). In the first rapid denitrification phase, a readily biodegradable substrate such as reduced sugar in hydrolyzed molasses would be used as a carbon source, and in the second slow denitrification phase, slowly biodegradable substrates such as unhydrolyzed polysaccharide in molasses would be used as the carbon source. Griffiths (1994) reported that during denitrification with wastewater as the electron donor and activated sludge originating from denitrifying–nitrifying systems, it is usual for three linear phases of nitrate reduction to occur simultaneously. The denitrification rate is highest when using soluble readily biodegradable substrate, followed by when using particulate slowly biodegradable substrate, and lowest in endogenous conditions.
Fig. 2. Denitrification with several concentrations of hydrolyzed molasses as carbon source. (A) 100 mg COD/l, (B) 200 mg COD/l, (C) 500 mg COD/l. The readily biodegradable fraction was calculated by determining the NO3 –N value, which is given by the difference between the initial NO3 –N concentration and the extrapolated value drawn from the second rate.
In denitrification processes, the fraction of readily biodegradable substrates in the alternative carbon source is a very important factor. Readily biodegradable organic matter is preferred since this will give the highest denitrification rate. The readily biodegradable fraction of the carbon source can be calculated by determining the DNO3 –N value, which is given by the difference between the initial NO3 –N concentration and the extrapolated value drawn from the second rate (Naidoo et al., 1998). In this experiment, the readily biodegradable substrate concentrations were 50 and 89 mg COD/l at
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Table 1 Denitrification with hydrolyzed molasses as an external carbon source Hydrolyzed molasses concentration (mg COD/l) Nitrate (mg/l) Denitrification rate (mg NO3 –N/g VSS h) Readily biodegradable substrate (mg COD/l) Substrate consumption (mg COD/mg NO3 –N)
1st phase 2nd phase
100
200
500
100 2.9 1.3 50
100 3.6 1.4 89
100 3.6 – –
4.3
5.0
5.8
different molasses concentrations (Table 1). Therefore, about 47.5 ± 2.5% of the organic compounds in hydrolyzed molasses were readily biodegradable, which was about two times higher than that in sewage, where the fraction of readily biodegradable substrate was about 10–20% of the total COD (Henze et al., 1994). The nitrate reduction rates in the first phase at the two different concentrations of molasses were 2.9 and 3.6 mg NO3 – N/g VSS h. The corresponding rates in the second phase were 1.3 and 1.4 mg NO3 –N/g VSS h (Table 1). This means that the readily biodegradable fraction of the hydrolyzed molasses stimulated nitrate reduction by more than two times. When the concentration of hydrolyzed molasses was increased to 500 mg COD/l, nitrate was linearly reduced (Fig. 2) with a nitrate reduction rate of 3.6 mg NO3 –N/g VSS h, because the readily biodegradable substrate in 500 mg COD/l hydrolyzed molasses was enough to completely remove initial nitrate. This shows that denitrification rate increased with increasing hydrolyzed molasses concentration. The substrate consumption rates were 4.3, 5.0, and 5.8 mg COD/mg NO3 –N with different molasses concentrations (Table 1). Compared to the hydrolyzate from sludge and household solid organic waste (8– 10 mg COD/mg NO3 –N, Æsøy et al., 1998), the amount of hydrolyzed molasses needed for nitrate reduction was only 50%, indicating that hydrolyzed molasses is a more effective carbon source than hydrolyzed sludge or solid organic waste. 3.3. Nitrogen removal in SBR using hydrolyzed molasses as a carbon source The SBR system, with the incorporation of alternating aerobic and anoxic/anaerobic stages in the REACT period, not only allows good removal of organic matter and suspended solids, but satisfactory removal of nitrogen by nitrification and denitrification is also achieved. Because of its simplicity and low capital and operational costs, the SBR is the most popular system for treating wastewater containing high nutrient concentrations in small and medium size cities with high population den-
Fig. 3. Effects of different external carbon sources on nitrogen removal in SBR over 14 days (28 cycles) operation.
sities. An SBR system was therefore used in the present study to compare the nitrogen removal efficiencies of hydrolyzed molasses and methanol as external carbon sources. Fig. 3 shows the percentage of nitrogen removal in the two SBRs, where hydrolyzed molasses and methanol were used as the carbon sources over 14 days (28 cycles) of operation. Ammonia was completely oxidized and not detected in the effluent. The concentration of nitrate was 6–8 mg NO3 –N/l in the reactor with methanol (reMe) and 3–5 mg NO3 –N/l with hydrolyzed molasses (re-HM). This result shows that the reactors achieved stable nitrogen removal over 14 days of operation, and it is postulated that the denitrification activity would not have changed significantly for some time. When hydrolyzed molasses was the external carbon source, the percentage of nitrogen removed was 91.6 ± 1.6%; the corresponding percentage for methanol was 85.3 ± 2.0%. The t-test value of the results for both carbon sources was 11.2 and p < 0.001, which indicates that the two carbon sources had statistically different removal efficiencies. The unused carbon source accumulated in the wastewater and resulted in a high COD level in the effluent. The average COD values of the effluents from the two reactors were 51 and 53 mg/l, respectively. Some researchers (Her and Huang, 1995; Chou et al., 2003) reported that when C/N ratio was increased in denitrification, a great deal of methanol remained in the effluent while glucose was completely removed. It means that excess addition of methanol seriously inhibits the activity of denitrifying sludge and could result in residual methanol being discharged in treatment plant effluent. For the use of wastes or by-products as alternative external carbon sources, a high COD concentration in effluent is a frequent problem. Lee et al. (2002) reported that the use of anaerobically fermented leachate of food
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Fig. 4. Change of the COD and nitrate concentrations during the operation of SBR with different external carbon sources.
waste as a carbon source caused increased COD concentration in effluent because high molecular weight substrates were not used readily by the microorganisms. Fig. 4 shows the change of nitrate and COD during 1 cycle of SBR operation. In the aeration stage, nitrate concentration was continually increased by ammonia oxidation, and the final concentrations of nitrate were similar in both reactors. The organic carbon in re-Me was more quickly removed than in re-HM because the methanol left after the last stage of the cycle was readily biodegradable. On the other hand, in re-HM, organic matter left after the last stage would not be readily degradable. In re-Me, only 55% of nitrate was reduced in the 4 h anoxic stage, while nitrate was completely removed in the 2 h anoxic stage in re-HM. In re-HM, with the addition of hydrolyzed molasses, denitrifying bacteria would quickly get energy from the oxidation of the readily biodegradable fraction of hydrolyzed molasses and reduce nitrate. The COD in re-Me was increased to 130 mg/l in the first 1 h of the anoxic stage by the addition of an external carbon source, while in re-HM, COD was increased to 100 mg/l. The nitrate reduction rate in re-HM was 4.2 mg NO3 –N/g VSS h, which was higher than the nitrate reduction rate in re-Me (1.0 mg NO3 –N/g VSS h) and also higher than some values reported with methanol (1.3 mg NOx –N/g VSS h (Louzeiro et al., 2003); 2.2 mg NO3 –N/g VSS h (Min et al., 2002)). The nitrate reduction rate in re-HM was also higher than that with acidogenic fermented waste sludge (2.0 mg NO3 –N/g VSS h; Min et al., 2002). The nitrate reduction rate in re-Me was lower than that previously observed for methanol in a SBR (Tam et al., 1994). Louzeiro et al. (2003) reported that when methanol was used as an external carbon source a minimum of
55% increase in the denitrification rate was observed with acclimatized (five weeks) sludge compared with non-acclimatized sludge. The low denitrification rate with methanol in this experiment may have been partly caused by the short acclimation time for the methanol. The denitrification rate with glucose has been previously reported as much higher than that with methanol (Akunna et al., 1993). Since the main product of hydrolyzed molasses was reduced sugar similar to glucose, it would be the reason why the rate of denitrification in re-HM was higher than in re-Me. In the second aeration stage, the remaining nitrate concentration was 5.2–6.0 mg NO3 –N/l in re-Me, but in re-HM, the concentration of nitrate was 3.3 mg NO3 –N/l. The increase of nitrate concentration in reHM could be related to the oxidation of ammonia produced from the hydrolyzed molasses. The removal rate of COD in re-HM was lower than that in re-Me. This result coincides with the results obtained in the first aeration stage where the organic compounds remaining after denitrification in re-HM were slowly biodegradable. The COD value of the effluent in re-HM (51 mg/l) was higher than the COD regulation value for the sewage treatment plant in Korea (40 mg/l). The results for denitrification efficiency in re-HM showed that the carbon source used to remove the nitrate was a readily biodegradable fraction of the hydrolyzed molasses. If less hydrolyzed molasses were added, a fraction of slowly biodegradable substrates could also be used as a carbon source. The COD value of the effluent also decreased, though complete removal of the nitrate took longer because the denitrification rate with slowly biodegradable substrates was about 40% of the denitrification rate with readily biodegradable substrates (Table 1). Further study is needed to determine the optimum dosage of hydrolyzed molasses for efficient nitrate reduction and for prevention of a COD increase. Hydrolyzed molasses would be a more economical source of carbon than methanol. According to a report of the Korea Importers Association published in March 2003, the import of molasses costs US$60/ton whereas methanol costs US$300/ton. The cost of molasses is therefore only 20% of the cost of methanol. Only 17 l of sulfuric acid would be needed to hydrolyze 1 ton of molasses, and, based on figures quoted in the Chemical Market Reporter (February 3, 2003), the cost of this much sulfuric acid is only about 1 US dollar. Finally, a sewage treatment plant that treats 6000 tons of sewage a day needs only 1 ton of acidified molasses daily, and the acidic thermohydrolyzation could be processed in a 2 m3 heating tank. With a general boiler (LATTNER, USA), the heating energy required for the thermohydrolyzation of molasses is about 150 kWh/ton, which costs only about US$6/ton according to the price of industrial electricity in Korea.
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4. Conclusion The results show the effectiveness of the thermohydrolysate of molasses, prepared at pH 3.6 with sulfuric acid, as an alternative carbon source. It had a readily biodegradable substrate content of 47.5% and gave a denitrification rate of 2.9–3.6 mg N/g VSS h. In the SBR system, hydrolyzed molasses was more effective than methanol in removing nitrogen. Hydrolyzed molasses is therefore recommended as an economical and effective external carbon source for nitrogen removal. However, for full-scale replacement of methanol with hydrolyzed molasses, a long time is needed to stabilize the sludge. The optimum dosage of the carbon source also needs to be determined. Acknowledgements This work was supported by the Korean Ministry of Environments Eco-Technopia-21 program (Grant 121041-028). References Æsøy, A., Ødegaard, H., 1994. The nitrogen removal efficiency and capacity in biofilms with biologically hydrolysed sludge as carbon source. Water Science and Technology 30 (6), 63–71. Æsøy, A., Ødegaard, H., Bach, K., Pujol, R., Hamon, M., 1998. Denitrification in a packed bed biofilm reactor (BIOFOR)—experiments with different carbon sources. Water Research 32, 1463–1470. Akunna, J.C., Bizeau, C., Moletta, R., 1993. Nitrate and nitrite reductions with anaerobic sludge using various sources: glucose, glycerol, acetic acid, lactic acid and methanol. Water Research 27, 1303–1312. Aravinthan, V., Mino, T., Takizawa, S., Satoh, H., Matsuo, T., 2001. Sludge hydrolysate as a carbon source for denitrification. Water Science and Technology 43 (1), 191–199. Barlindhaug, J., Ødegaard, H., 1996. Thermal hydrolysis for the production of carbon source for denitrification. Water Science and Technology 34 (1–2), 371–378. Bernet, N., Habouzit, F., Moletta, R., 1996. Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater. Applied Microbiology and Biotechnology 46, 92–97. Boaventura, R.A.R., Rodrigues, A.E., 1997. Denitrification kinetics in a rotating disk biofilm reactor. Chemical Engineering Journal 65, 227–235. Chaplin, M.F., Kennedy, J.F., 1986. Carbohydrate Analysis: a Practical Approach. IRL Press, Oxford, UK, p .3. Chou, Y.J., Ouyang, C.F., Kuo, W.L., Huang, H.L., 2003. Denitrifying characteristics of the multiple stages enhanced biological
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nutrient removal process with external carbon sources. Journal of Environmental Science Health—Part A, Toxic/Hazardous Substances and Environmental Engineering 38, 339–352. Clescerl, L.S., Greenberg, A.E., Eaton, A.D., 1998. Standard Methods for the Examination of Water and Wastewater. APHA-AWWAWEF, Washington, USA. Griffiths, P., 1994. Modification of the IAWPRC task group general activated sludge model. Water Research 28, 657–664. Henze, M., Kristensen, G.H., Strube, R., 1994. Rate-capacity characterization of wastewater for nutrient removal processes. Water Science and Technology 29 (7), 101–107. Her, J.J., Huang, J.S., 1995. Influences of carbon source and C/N ratio on nitrate/nitrite denitrification and carbon breakthrough. Bioresource Technology 54, 45–51. Lee, C.Y., Shin, H.S., Chae, S.R., Nam, S.Y., Paik, B.C., 2002. Nutrient removal using anaerobically fermented leachate of food waste in the BNR process. Water Science and Technology 47 (1), 159–165. Lee, S.I., Park, J.H., Ko, K.B., Koopman, B., 1997. Effect of fermented swine wastes on biological nutrient removal in sequencing batch reactors. Water Research 31, 1807–1812. Louzeiro, N.R., Mavinic, D.S., Oldham, W.K., Meisen, A., Gardner, I.S., 2002. Methanol-induced biological nutrient removal kinetics in a full-scale sequencing batch reactor. Water Research 36, 2721– 2732. Louzeiro, N.R., Mavinic, D.S., Oldham, W.K., Meisen, A., Gardner, I.S., 2003. Process control and design considerations for methanolinduced denitrification in a sequencing batch reactor. Environmental Technology 24, 161–169. Min, K.S., Park, K.S., Jung, Y.J., Khan, A.R., Kim, Y.J., 2002. Acidogenic fermentation: utilization of wasted sludge as a carbon source in the denitrification process. Environmental Technology 23, 293–302. Miranda, M.P., Benito, G.G., Cristobal, N.S., Nieto, C.H., 1996. Color elimination from molasses wastewater by Aspergillus niger. Bioresource Technology 57, 229–235. Mohseni-Bandpi, A., Elliott, D.J., Momeny-Mazdeh, A., 1999. Denitrification of groundwater using acetate acid as a carbon source. Water Science and Technology 40 (2), 53–59. Mora, F.R., Ferrara de Giner, G., Andara, A.R., Esteban, J.L., 2003. Effect of organic carbon shock loading on endogenous denitrification in sequential batch reactors. Bioresource Technology 88, 215– 219. Naidoo, V., Urbain, V., Burckley, C.A., 1998. Characterization of wastewater and activated sludge from European municipal wastewater treatment plants using the NUR test. Water Science and Technology 38 (1), 303–310. Najafpour, G.D., Shan, C.P., 2003. Enzymatic hydrolysis of molasses. Bioresource Technology 86, 91–94. Tam, N.F.Y., Leung, G.L.W., Wong, Y.S., 1994. The effects of external carbon loading on nitrogen removal in sequencing batch reactors. Water Science and Technology 30 (6), 73–81. Ten Have, P.J.W., Willers, H.C., Derikx, P.J.L., 1994. Nitrification and denitrification in an activated-sludge system for supernatant from settled sow manure with molasses as an extra carbon source. Bioresource Technology 47, 135–141.