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Protein recovery from excess sludge for its use as animal feed
Bioresource Technology 99 (2008) 8949–8954
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Protein recovery from excess sludge for its use as animal feed Jiyeon Hwang a, Lei Zhang a, Sunkeun Seo a, Yong-Woo Lee b, Deokjin Jahng a,* a b
Department of Environmental Engineering and Biotechnology, Myongji University, San 38-2, Namdong, Cheoin-Gu, Yongin, Gyeonggi-Do 449-728, South Korea R&D Center, Samsung Engineering Co. Ltd., 415-10, Woncheon-Dong, Youngtong-Gu, Suwon, Gyeonggi-Do 443-823, South Korea
a r t i c l e
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Article history: Received 15 January 2008 Received in revised form 1 May 2008 Accepted 1 May 2008 Available online 12 June 2008 Keywords: Protein recovery Animal feed Excess sludge
a b s t r a c t In this study, the possibility of using proteins recovered from excess sludge as animal feed was investigated. The proteins were recovered through the processes of sludge disintegration (alkali treatment followed by ultra-sonication), precipitation and drying of the soluble proteins. The compositions and the toxicants of the recovered proteins were analyzed, and the toxicity was assessed by Sprague-Dawley (SD) rat experiments. The results showed that the nutrient compositions were comparable with the commercial protein feeds. Heavy metals were found to be removed after the protein recovery process, and aflatoxin B1, ochratoxin A and Salmonella D groups were not detected. The rat toxicity tests showed that there were no effects on mortality, the incidence of clinical signs, body weight changes, and necropsy findings. The minimum lethal dose (MLD) was higher than 2000 mg/kg. Based on these results, the use of the crude protein recovered from excess sludge as animal feed appears to be technically feasible. Ó 2008 Published by Elsevier Ltd.
1. Introduction A large amount of excess sludge is generated from the activated sludge process due to consumption of organic pollutants in the wastewater and the concomitant microbial growth (Jung et al., 2001). As an example, the annual production of sewage sludge from municipal wastewater treatment facilities in Korea increased by approximately 159% from 1999 to 2005 and is expected to reach 10,071 tons/day in 2011 (MOE, 2006). To date, excess sludge has mainly been dealt with by soil application, landfill, combustion and ocean dumping. The capital and operating costs associated with excess sludge are known to be as high as 50% of the total cost of the wastewater treatment plant (Zhang et al., 2007). Furthermore, the costs of excess sludge treatment are increasing due to a lack of final disposal lands, strict regulations on air quality, and ocean environmental protection laws. Therefore, it is essential to develop new technologies for the efficient disposal and reuse of excess sludge (Jung et al., 2001). Reutilization of excess sludge as a resource is attracting more interest, which recovers useful biomaterials directly from excess sludge or transforms excess sludge into useful substances through physicochemical or biological processes. It is possible to realize this concept, as excess sludge typically consists of valuable organic substances such as nucleic acids, enzymes, proteins, and polysaccharides (Jung et al., 2002). Protein has been estimated to account for about 50% of the dry weight of bacterial cells (Shier and Purwono, 1994). On the other hand, protein is one of the most important * Corresponding author. Tel.: +82 31 330 6690; fax: +82 31 336 6336. E-mail address: [email protected] (D. Jahng). 0960-8524/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.biortech.2008.05.001
constituents in animal feed, furnishing energy and nitrogen. The so-called ‘green revolution’ has done much to eliminate calorie insufficiency by increasing the yields of cereal grains, but a shortage of grain proteins continues (Adebayo et al., 2004). Thus, various alternative sources of protein have been sought, and it has been noted that the only essentially unutilized source of protein large enough to mitigate the shortage is cellular proteins in the mixed microbial cultures of activated sludge from municipal sewage treatment plants (Shier and Purwono, 1994). For protein recovery, solubilization of intracellular materials in excess sludge must be achieved first. Many chemical, physical or biological treatments, such as alkali treatment (Navia et al., 2002) and ultra-sonication (Onyeche et al., 2002; Tiehm et al., 2001), have been reportedly used to disrupt the sludge floc structure and release the intracellular contents into the aqueous phase (Jung et al., 2001). After disintegration of the sludge, an increase in the concentrations of soluble chemical oxygen demand (SCOD) and soluble protein, as well as a decrease of the floc size and the amount of suspended solid (SS) were observed (Wang et al., 2006; Weemaes et al., 2000; Zhang et al., 2007). Therefore, protein recovery from excess sludge can also provide other important benefits. For example, the disintegration of microorganisms can result in enhanced dewaterability, and the sludge that has had the protein removed occupies less volume and contains fewer organic materials. Further treatment of this lower strength waste would be easier and less expensive compared to intact excess sludge. Methods of protein recovery from excess sludge have been investigated for years. Earlier attempts to utilize this protein source by directly feeding dried sludge to domestic animals has produced mixed results (Beszedits and Lugowski, 1981). The
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animals often showed reduced weight gain (Ray et al., 1982), presumably as a consequence of the toxic effects of heavy metals or other contaminants in the excess sludge (Shier and Purwono, 1994). In fact, it has been reported that toxic metals, organic pollutants and microbial toxins in sewage are concentrated in sewage sludge through the primary and secondary treatment processes. Hence, utilization of this protein source in the form of an animal feed supplement requires an efficient and economical process to extract the protein in a form free of toxic materials without producing significant amounts of residues or side products (Shier and Purwono, 1994). In this study, ultra-sonication followed by alkali treatment were used to release the intracellular proteins. Crude protein was then recovered from the cell-disrupted suspension. The compositions of the recovered protein (moisture, crude protein, crude fat, crude fiber, crude ash and nitrogen free extract), heavy metals, toxins (aflatoxin B1 and ochratoxin A) and harmful microorganisms (Salmonella D groups) were determined. Finally, toxicity of using the protein recovered from excess sludge was assessed through oral administration tests with Sprague-Dawley (SD) rats.
2. Methods 2.1. Sludge disintegration The excess sludge containing 4500–5500 mg/L of suspended solid (SS) was obtained from a domestic wastewater treatment plant (Yongin, Korea). Three series of disintegration experiments were carried out, which included alkali treatment, ultra-sonication and alkali treatment followed by ultra-sonication. For the alkali treatment, the pH of the excess sludge was adjusted to pH 12 with 1.0 M NaOH and the sludge was stirred for 2 h. For the ultrasonication treatment, excess sludge was sonicated in a tube on ice using an ultrasonic processor emitting 20 kHz ultrasound through a tip (Sonicator VCX 600, Sonics & Materials, Newtown, USA). The input energy was varied from 0 to 3.84 1010 kJ/kg VSS (volatile suspended solid) to examine the effect of different amounts of energy on the disintegration of the sludge. After the sludge had disintegrated, the TCOD (total chemical oxygen demand), SCOD, SS, VSS and soluble protein levels were measured to evaluate the efficiencies of these sludge disintegration methods. SS and VSS were measured following the Standard Method (APHA, 2005). To measure the SCOD and soluble protein levels, the supernatant was obtained by centrifuging the disintegrated sludge at 7000 rpm for 30 min at a temperature of 4 °C. The COD concentration was measured by the HACH 8000 method using a HACH DC/2500 spectrophotometer. The protein concentration was measured by the Lowry method (Lowry et al., 1951) using BSA (bovine serum albumin) as a reference protein. The degree of disintegration in terms of COD solubilization rate was expressed as follows (Cui and Jahng, 2006):
COD solubilization rateð%Þ ¼
SCODa SCODi 100 TCODi SCODi
ð1Þ
where SCODi and SCODa are the soluble COD before and after the treatment, respectively, and TCODi is the initial TCOD.
2.2. Protein recovery Soluble protein in the supernatant of the disintegrated sludge obtained by centrifuging the disintegrated sludge at 7000 rpm and 4 °C for 30 min was precipitated by adjusting the pH of the supernatant to pH 1.0, 3.3 and 5.0 using 2.0 M H2SO4. After stirring for 15 min, precipitates were recovered by centrifugation at 7000 rpm for 30 min at 4 °C. The resulting pellet containing the precipitated protein was dried at 60 °C for 48 h. 2.3. Protein analysis Components of the recovered pellet including the moisture, crude protein, crude fat, crude fiber and ash were measured following the Korean Standard Method of Feed Analysis (MAF, 2007). To measure the concentrations of heavy metals, the recovered pellet was ground using a blender (HMF-347(E), Hanil Electric, Seoul, Korea), decomposed according to the aqua regia method, and then filtered using a filter paper (Quantitative Filter paper 5B, Advantec, Tokyo, Japan) (Ito et al., 2000). The concentrations of heavy metals in the filtrate were determined using an atomic absorption spectrometer (AAS) (220FS, Varian, Palo Alto, USA) and an inductively coupled plasma mass spectrometer (ICP-MS) (CCTX-7, Thermo elemental, Waltham, USA). Additionally, aflatoxin B1, ochratoxin A, and Salmonella D groups were measured following the guidelines of the Ministry of Agriculture and Forestry Republic of Korea (MAF, 2007). 2.4. Experiments on rats For acute toxicity analysis of the recovered protein, male and female Sprague-Dawley rats were divided into four groups, each of which consisted of five rats. The first group of rats as the control group was orally administered with 20 ml sterile water/kg. The other three groups were fed with 20 ml/kg for 500, 1000, and 2000 mg recovered protein/kg, respectively (Table 1). Mortality, incidence of clinical signs, and change of body weight within two weeks were monitored, and necropsy tests after the experiment period were performed. All experimental conditions followed the Korea Good Laboratory Practice (KGLP) of the Korea Food and Drug Administration (KFDA, 2005a,b), such that the temperature was 23 ± 3 °C, the relative humidity was 55 ± 15%, the number of awakenings was 10–20 times/h, and illumination was on for 12 h/day.
3. Results and discussion 3.1. Sludge disintegration After an alkali treatment of excess sludge at pH 12 for 2 h, the SSi, TCODi, SCODi and SCODa readings were 4580 mg/L, 5235 mg/L, 44 mg/L, 1285 mg/L, respectively, yielding COD solubilization rate of 23.9%. After the alkali treatment, the soluble protein concentration increased from 28.2 mg/L to 932.3 mg/L, and the protein concentration in the supernatant after the subsequent protein precipitation at pH 3.3 was 587.5 mg/L. Therefore, it was estimated
Table 1 Group organization of Sprague-Dawley rats Group
Sex
The number of rats
Number
Volumetric dose (ml/kg)
Protein dose (mg/kg)
G1 G2 G3 G4
Male/female Male/female Male/female Male/female
5/5 5/5 5/5 5/5
1–5/21–25 6–10/26–30 11–15/31–35 16–20/36–40
20 20 20 20
0 (sterile water) 500 1000 2000
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5000
2500
4000
2000 Soluble protein
3000
1500
SS
2000
1000
1000
Protein (mg/L)
SS and VSS (mg/L)
J. Hwang et al. / Bioresource Technology 99 (2008) 8949–8954
500
VSS
0
0
TCOD 70 COD solubilization rate 4000 60 SCOD 2000
COD solubilization rate (%)
TCOD and SCOD (mg/L)
80 6000
50
0 0.0
40 5.0e+9
1.0e+10 1.5e+10 2.0e+10 2.5e+10 3.0e+10 3.5e+10
Energy input (kJ/kg VSS) Fig. 1. Effects of ultra-sonication on the disintegration of excess sludge.
that 41.7% of the soluble protein released from the disintegrated sludge was precipitated at pH 3.3. Ultra-sonication of excess sludge was also carried out at energy levels ranging from 0 to 3.84 1010 kJ/kg VSS. Fig. 1 shows that the concentration of soluble protein increased as the input energy increased, particularly in the energy range from 5.49 109 kJ/kg VSS to 1.65 1010 kJ/kg VSS. At 1.65 1010 kJ/kg VSS, the percentages of COD solubilization and protein concentration were 68% and 1,818 mg/L, respectively. The combined treatment of an alkali treatment and a subsequent ultra-sonication led to a rapid increase in the protein concentration and COD solubilization to 5.49 1010 kJ/kg VSS. As shown in Fig. 2, VSS and SS decreased while the protein concentration increased as the ultra-sonication energy increased. The percentages of COD solubilization by ultra-sonication combined with the alkali treatment ranged from 32% to 84%. The concentration of soluble protein increased to 2672 mg/L, which was higher than the 2239 mg/L reading obtained by ultra-sonication alone. As the patterns of SS, VSS, COD solubilization rate and protein concentration were not linear with the energy input, ultra-sonication at 1.65 1010 kJ/kg VSS after an alkali treatment at pH 12 for 2 h was chosen for further experimentation. At these conditions, the percentages of COD solubilization rate and the protein concentration were 81% and 2626 mg/L, respectively, for the excess sludge with 4740 mg/L of the initial SS.
3.2. Protein recovery Excess sludge (SSi = 5330 mg/L) was disintegrated by an alkali treatment combined with ultra-sonication, and the protein concentration in the supernatant was found to be 3177.5 mg/L. Precipitation was performed by adjusting the pH value of the supernatant to pH 1.0, 3.3 and 5.0. As shown in Fig. 3, protein concentrations in the supernatant after precipitation at pH 1.0, 3.3 and 5.0 were 797.3 mg/L, 620 mg/L and 1572.3 mg/L, respectively. As the efficiency of the protein recovery method was the highest (80.5%) at pH 3.3, isoelectric precipitation at pH 3.3 was chosen to recover soluble proteins from the supernatant of disrupted excess sludge. 3.3. Nutritional analysis The nutritional components of the recovered pellet were analyzed to determine its nutritional value as animal feed. The largest part of the recovered pellet consisted of crude protein (50.1%). The remaining materials were NFE (21.8%), crude ash (15.4%), crude fat (9.0%), moisture (3.6%) and crude fiber (0.05%). The major components of the NFE were thought to be sugars, partial cellulose and hemi cellulose; lignin was also included in the NFE, which is expressed as follows (MAF, 2007):
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J. Hwang et al. / Bioresource Technology 99 (2008) 8949–8954
3000
5000
2500 Soluble protein 2000
3000 1500 2000
Protein (mg/L)
SS and VSS (mg/L)
4000
1000 SS VSS
1000
500
0 80
6000
TCOD 70 SCOD
4000
60
3000
50
2000
40
1000
30
0
COD solubilization rate (%)
TCOD and SCOD (mg/L)
COD solubilization rate 5000
20 0
5e+9
1e+10
2e+10
2e+10
3e+10
3e+10
Energy input (kJ/kg VSS) Fig. 2. Effects of ultra-sonication after an alkali treatment on the disintegration of excess sludge.
100 Protein conc. of supernatant after cell disruption Protein conc. of supernatant after precipitation Efficiency of protein recovery (%)
3000
80 2500 60
2000 1500
40 1000 20
500 0 Before pH adjustment
pH 1.0
pH 3.3
pH 5.0
Efficiency of protein recovery (%)
Protien concentration (mg/L)
3500
0
Fig. 3. Effect of pH on protein precipitation.
NFEð%Þ ¼ 100 ðmoisture þ crude protein þ crude fat þ crude ash þ crude fiberÞ
ð2Þ
In order to evaluate the nutrient potency of the recovered protein, the total digestible nutrients (TDN), digestible energy (DE), metabolizable energy (ME), net energy for maintenance (NEM), net energy for gain (NEG), and net energy for lactation (NEL) were calculated using the equations below (NRC, 2001).
TDN ð%Þ ¼ CP ð%Þ þ NFE ð%Þ þ Fiber ð%Þ þ CF ð%Þ 2:25
ð3Þ
DE ðMcal=kgÞ ¼ TDN ð%Þ 0:04409
ð4Þ
ME ðMcal=kgÞ ¼ 1:01 DE ðMcal=kgÞ 0:45
ð5Þ
2
3
ð6Þ
2
3
NEG ðMcal=kgÞ ¼ 1:42ME 0:174ðMEÞ þ 0:0122ðMEÞ 1:65
ð7Þ
NEL ðMcal=kgÞ ¼ 0:0929 CF ð%Þ þ 0:0547 CP ð%Þ þ 0:192
ð8Þ
NEM ðMcal=kgÞ ¼ 1:37ME 0:138ðMEÞ þ 0:0105ðMEÞ 1:12
The TDN, DE, ME, NEM, NEG and NEL levels in the recovered pellet were calculated according to nutritional component analytical results. These were compared with the values for major types of feeds. As shown in Table 2 the TDN, DE, ME, NEM, NEG and NEL levels of the recovered pellet were 92%, 4.07 Mcal/kg, 3.66 Mcal/kg, 2.56 Mcal/kg, 1.81 Mcal/kg, and 3.77 Mcal/kg, respectively. The content of crude protein in the recovered pellet was similar to that of dried Brewer’s yeast and bone meal, and the other items, TDN, NEM, NEG and NEL, were found at higher levels than in the major feeds. Therefore, the recovered protein from the excess sludge possesses nutritional values suitable for animal feed. 3.4. Hazardous materials analysis The measured concentrations of heavy metals in the original excess sludge and the recovered pellet are listed in Table 3 with regulatory standards. The original sludge contained a variety of
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J. Hwang et al. / Bioresource Technology 99 (2008) 8949–8954 Table 2 Nutritional values of major feeds (NRC, 1982) and the recovered pellet Feedstuff
Dried matter (%)
Ruminants TDN (%)
DE
ME
NEM
NEG
NEL
Crude protein (%)
Ash (%)
(Mcal/kg) Soybean mill Wheat grain Rice grain Barley grain Brewer’s yeast, dried Corn grain Bone meal Dry skimmed milk Oat grain
90 89 89 88 93 89 93 94 89
46 78 70 74 74 83 66 79 68
2.01 3.45 3.10 3.27 3.25 3.67 2.92 3.52 3.02
1.63 3.08 2.73 2.90 2.87 3.31 2.53 3.13 2.65
0.98 1.89 1.64 1.76 1.73 2.05 1.5 1.91 1.59
0.28 1.28 1.08 1.19 1.14 1.41 0.93 1.29 1.04
1.01 1.81 1.61 1.71 1.70 1.93 1.51 1.84 1.57
12.6 14.2 7.9 11.9 43.8 9.9 50.4 33.7 11.8
4.9 1.7 4.7 2.3 6.6 1.5 29.3 7.9 3.1
Recovered pellet
96
92
4.07
3.66
2.56
1.81
3.77
50.1
15.4
Table 3 Concentrations of heavy metals Element
Unit (mg/kg) Original excess sludge
Ag Al As Ca Cd Cr Cu Fe Hg Ni Pb Se Zn a b c
a
ND 7757.4 ND 1731.2 ND ND 278.5 5039.6 ND 53.22 23.6 ND 582.7
Recovered pellet
Legal standardc
ND 4642.8 ND 911.1 ND ND 246.9 2882.9 ND 15.12 6.93 ND 217.6
–b – 2 – 1 100 – – 0.4 – 10 2 –
Not detected. Not prescribed in the regulation. The lowest value in the legal standard of various feeds (MAF, 2004).
hazardous heavy metals. It has been reported that heavy metals in wastewater are concentrated into sewage sludge through the primary and secondary treatment processes (Oliver and Cosgrove, 1974; Stephenson and Lester, 1987). For example, the heavy metals can be absorbed by biomass, precipitated by some anions such as sulfide, and accumulated in excess sludge (Ito et al., 2000). Through the recovery processes of protein, significant amounts of the heavy metals in the raw sludge were removed. Furthermore, the aflatoxin B1, ochratoxin A and Salmonella D groups, an indicator of pathogenic bacteria, were not detected (data not shown). In conclusion, heavy metals, harmful toxins and microorganisms, if any, included in the original excess sludge were found to be removed in significant amounts through the processes of cell disintegration and protein precipitation. 3.5. Toxicity test on rats To assess acute toxicity of the recovered pellet, male and female SD rats were fed by mouth with the recovered protein. During a two-week experimental period, mortality and specific incidences of clinical signs in rats were not detected. Table 4 showed that the body weights of the female group were higher 14 days after the administration of 500 and 2000 mg of recovered pellet/kg. In addition, statistic analyses showed that the increments of body weights were higher in the groups fed with 500, 1000 and 2000 mg of recovered pellet/kg compared to these increments in the control group. However, body weight increases were observed only in female groups, probably because the body weights of rats in
the female control group were accidentally low. Necropsy results in which red discoloration of the thymus was observed in a male group fed with 2000 mg of recovered pellet/kg. According to the necropsy estimation, red discoloration of thymus was thought to be an accidental change caused by the anesthesia and bloodletting processes (Greaves, 1990; Boorman, 1990). As mentioned above, at least three conditions should be fulfilled to use sludge protein as animal feed. First, the nutritional value of sludge protein must be equivalent to that of common feedstuffs. This requirement is thought to be satisfied according to the experimental data given in Table 2. Secondly, the sludge protein should be safe for domestic animals. It was shown in this study that the protein extracted from the excess sludge contained no heavy metals of which the concentrations were higher than the legal standards (Table 3). Additionally, toxins were not detected. The sludge protein was also free of Salmonella D groups, which served as an indicator of pathogenic bacteria, and did not show acute toxicity against rats. According to these results, it is possible to conclude tentatively that the protein extracted from the excess sludge is safe for animals, although additional analyses of the hazardous chemicals and microorganisms that may exist in the sludge are required. It is also necessary to determine through long-term studies to what type of domestic animals (e.g., ruminants, poultry, or pig) the sludge proteins are designated. Finally, the economic feasibility must be proven before the sludge protein can be utilized in agricultural industries. In fact, the extraction of proteins from excess sludge and its commercial use can provide economic benefits. Through the destruction of the microbial cells of the excess sludge and the recovery of cellular proteins, the mass for further treatment of the excess sludge residuals decreases, implying that the treatment expenses can be lowered. In this study, 5330 mg/L of the initial SS concentration was decreased to 1520 mg/L after the alkali and subsequent ultra-sonication treatments. This amount of sludge residuals represents only 28.5% of the original sludge. Furthermore, the dewaterability of the disintegrated sludge is expected to be significantly higher than intact excess sludge as the water inside cells is exposed when the cell walls are broken. Wastes with lower water contents are easier and less expensive to treat compared to wastes with higher water contents. Regarding the marketing of the product, sludge protein needs to compete with existing feedstuffs such as corn grain, fish mill, and dried yeast. The market price will be determined based on the manufacturing costs and other factors. To assess the manufacturing costs, it should be noted that the acquisition of excess sludge as a raw material creates earnings rather than expenditures, as wastewater treatment plants pay for the service of excess sludge disposal. In short, the extraction of cellular protein from excess sludge and its use as a feedstuff can provide many benefits regarding the management of the environment and food resources.
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Table 4 Body weight changes of SD rats fed with the sludge protein Dose (mg/kg)
Sex: male 0
500
1000
2000
Sex: female 0
500
1000
2000
Animal ID
Body weights (g) Day 0
Day 1
Day 3
Day 7
Day 14
Gains
Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N
224.70 9.07 5 221.58 7.14 5 222.00 8.05 5 221.08 6.24 5
251.30 10.64 5 247.94 7.17 5 248.84 8.78 5 250.20 7.94 5
266.85 9.62 5 264.58 7.39 5 264.14 9.61 5 265.86 7.59 5
292.63 10.54 5 292.75 8.18 5 292.48 12.00 5 293.01 6.76 5
336.42 10.46 5 329.41 10.27 5 331.16 14.53 5 339.42 9.04 5
111.72 2.92 5 107.83 9.53 5 109.16 7.56 5 118.33 7.05 5
Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N
160.55 5.21 5 161.94 5.07 5 160.57 7.01 5 161.29 7.48 5
178.60 6.90 5 178.68 5.50 5 178.88 6.21 5 174.82 6.07 5
187.54 6.40 5 187.83 7.86 5 185.75 6.73 5 185.85 5.94 5
199.95 6.08 5 199.40 10.35 5 191.03 8.72 5 198.50 7.26 5
201.79 5.62 5 216.35* 6.64 5 212.06 10.74 5 222.56** 9.22 5
41.24 2.28 5 54.40** 5.34 5 51.49** 4.39 5 61.28** 7.82 5
S.D.: standard deviation. Significantly different from control, p < 0.05. Significantly different from control, p < 0.01.
*
**
4. Conclusions Cellular proteins were prepared from excess sludge from a domestic wastewater treatment plant. Nutrient compositions of the recovered protein were similar to those of dried Brewer’s yeast and bone meal. The concentrations of heavy metals were lower than the legal standards. Aflatoxin B1, ochratoxin A and the Salmonella D groups were not detected. The acute toxicity tests using SD rats did not show significant effects on mortality, the incidence of clinical signs, and body weight changes. The minimum lethal dose was higher than 2000 mg/kg. From these results, it was concluded that the protein recovered from excess sludge appears to be feasible for use as animal feed. References Adebayo, O.T., Fagbenro, O.A., Jegede, T., 2004. Evaluation of Cassia fistula meal as a replacement for soybean meal in practical diets of Oreochromis niloticus fingerlings. Aquacult. Nutr. 10, 99–104. APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. American Public Health Association, Washington, DC. Beszedits, S., Lugowski, A., 1981. Utilizing Waste Activated Sludge for Animal Feeding. B&L Information Service, Toronto. Boorman, G.A., 1990. Pathology of the Fischer Rat: Reference and Atlas. Academic Press, San Diego, CA. Cui, R., Jahng, D., 2006. Enhanced methane production from anaerobic digestion of disintegrated and deproteinized excess sludge. Biotechnol. Lett. 28, 531–538. Greaves, P., 1990. Histopathology of Preclinical Toxicity Studies. Elsevier. Ito, A., Umita, T., Aizawa, J., Takachi, T., Morinaga, K., 2000. Removal of heavy metals from anaerobically digested sewage sludge by a new chemical method using ferric sulfate. Water Res. 344, 751–758. Jung, J., Xing, X.H., Matsumoto, K., 2001. Kinetic analysis of disruption of excess activated sludge by Dyno Mill and characteristics of protein release for recovery of useful materials. Biochem. Eng. J. 8, 1–7. Jung, J., Xing, X.H., Matsumoto, K., 2002. Recoverability of protease released from disrupted excess sludge and its potential application to enhanced hydrolysis of proteins in wastewater. Biochem. Eng. J. 10, 67–72.
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Protein recovery from excess sludge for its use as animal feed Jiyeon Hwang a, Lei Zhang a, Sunkeun Seo a, Yong-Woo Lee b, Deokjin Jahng a,* a b
Department of Environmental Engineering and Biotechnology, Myongji University, San 38-2, Namdong, Cheoin-Gu, Yongin, Gyeonggi-Do 449-728, South Korea R&D Center, Samsung Engineering Co. Ltd., 415-10, Woncheon-Dong, Youngtong-Gu, Suwon, Gyeonggi-Do 443-823, South Korea
a r t i c l e
i n f o
Article history: Received 15 January 2008 Received in revised form 1 May 2008 Accepted 1 May 2008 Available online 12 June 2008 Keywords: Protein recovery Animal feed Excess sludge
a b s t r a c t In this study, the possibility of using proteins recovered from excess sludge as animal feed was investigated. The proteins were recovered through the processes of sludge disintegration (alkali treatment followed by ultra-sonication), precipitation and drying of the soluble proteins. The compositions and the toxicants of the recovered proteins were analyzed, and the toxicity was assessed by Sprague-Dawley (SD) rat experiments. The results showed that the nutrient compositions were comparable with the commercial protein feeds. Heavy metals were found to be removed after the protein recovery process, and aflatoxin B1, ochratoxin A and Salmonella D groups were not detected. The rat toxicity tests showed that there were no effects on mortality, the incidence of clinical signs, body weight changes, and necropsy findings. The minimum lethal dose (MLD) was higher than 2000 mg/kg. Based on these results, the use of the crude protein recovered from excess sludge as animal feed appears to be technically feasible. Ó 2008 Published by Elsevier Ltd.
1. Introduction A large amount of excess sludge is generated from the activated sludge process due to consumption of organic pollutants in the wastewater and the concomitant microbial growth (Jung et al., 2001). As an example, the annual production of sewage sludge from municipal wastewater treatment facilities in Korea increased by approximately 159% from 1999 to 2005 and is expected to reach 10,071 tons/day in 2011 (MOE, 2006). To date, excess sludge has mainly been dealt with by soil application, landfill, combustion and ocean dumping. The capital and operating costs associated with excess sludge are known to be as high as 50% of the total cost of the wastewater treatment plant (Zhang et al., 2007). Furthermore, the costs of excess sludge treatment are increasing due to a lack of final disposal lands, strict regulations on air quality, and ocean environmental protection laws. Therefore, it is essential to develop new technologies for the efficient disposal and reuse of excess sludge (Jung et al., 2001). Reutilization of excess sludge as a resource is attracting more interest, which recovers useful biomaterials directly from excess sludge or transforms excess sludge into useful substances through physicochemical or biological processes. It is possible to realize this concept, as excess sludge typically consists of valuable organic substances such as nucleic acids, enzymes, proteins, and polysaccharides (Jung et al., 2002). Protein has been estimated to account for about 50% of the dry weight of bacterial cells (Shier and Purwono, 1994). On the other hand, protein is one of the most important * Corresponding author. Tel.: +82 31 330 6690; fax: +82 31 336 6336. E-mail address: [email protected] (D. Jahng). 0960-8524/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.biortech.2008.05.001
constituents in animal feed, furnishing energy and nitrogen. The so-called ‘green revolution’ has done much to eliminate calorie insufficiency by increasing the yields of cereal grains, but a shortage of grain proteins continues (Adebayo et al., 2004). Thus, various alternative sources of protein have been sought, and it has been noted that the only essentially unutilized source of protein large enough to mitigate the shortage is cellular proteins in the mixed microbial cultures of activated sludge from municipal sewage treatment plants (Shier and Purwono, 1994). For protein recovery, solubilization of intracellular materials in excess sludge must be achieved first. Many chemical, physical or biological treatments, such as alkali treatment (Navia et al., 2002) and ultra-sonication (Onyeche et al., 2002; Tiehm et al., 2001), have been reportedly used to disrupt the sludge floc structure and release the intracellular contents into the aqueous phase (Jung et al., 2001). After disintegration of the sludge, an increase in the concentrations of soluble chemical oxygen demand (SCOD) and soluble protein, as well as a decrease of the floc size and the amount of suspended solid (SS) were observed (Wang et al., 2006; Weemaes et al., 2000; Zhang et al., 2007). Therefore, protein recovery from excess sludge can also provide other important benefits. For example, the disintegration of microorganisms can result in enhanced dewaterability, and the sludge that has had the protein removed occupies less volume and contains fewer organic materials. Further treatment of this lower strength waste would be easier and less expensive compared to intact excess sludge. Methods of protein recovery from excess sludge have been investigated for years. Earlier attempts to utilize this protein source by directly feeding dried sludge to domestic animals has produced mixed results (Beszedits and Lugowski, 1981). The
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animals often showed reduced weight gain (Ray et al., 1982), presumably as a consequence of the toxic effects of heavy metals or other contaminants in the excess sludge (Shier and Purwono, 1994). In fact, it has been reported that toxic metals, organic pollutants and microbial toxins in sewage are concentrated in sewage sludge through the primary and secondary treatment processes. Hence, utilization of this protein source in the form of an animal feed supplement requires an efficient and economical process to extract the protein in a form free of toxic materials without producing significant amounts of residues or side products (Shier and Purwono, 1994). In this study, ultra-sonication followed by alkali treatment were used to release the intracellular proteins. Crude protein was then recovered from the cell-disrupted suspension. The compositions of the recovered protein (moisture, crude protein, crude fat, crude fiber, crude ash and nitrogen free extract), heavy metals, toxins (aflatoxin B1 and ochratoxin A) and harmful microorganisms (Salmonella D groups) were determined. Finally, toxicity of using the protein recovered from excess sludge was assessed through oral administration tests with Sprague-Dawley (SD) rats.
2. Methods 2.1. Sludge disintegration The excess sludge containing 4500–5500 mg/L of suspended solid (SS) was obtained from a domestic wastewater treatment plant (Yongin, Korea). Three series of disintegration experiments were carried out, which included alkali treatment, ultra-sonication and alkali treatment followed by ultra-sonication. For the alkali treatment, the pH of the excess sludge was adjusted to pH 12 with 1.0 M NaOH and the sludge was stirred for 2 h. For the ultrasonication treatment, excess sludge was sonicated in a tube on ice using an ultrasonic processor emitting 20 kHz ultrasound through a tip (Sonicator VCX 600, Sonics & Materials, Newtown, USA). The input energy was varied from 0 to 3.84 1010 kJ/kg VSS (volatile suspended solid) to examine the effect of different amounts of energy on the disintegration of the sludge. After the sludge had disintegrated, the TCOD (total chemical oxygen demand), SCOD, SS, VSS and soluble protein levels were measured to evaluate the efficiencies of these sludge disintegration methods. SS and VSS were measured following the Standard Method (APHA, 2005). To measure the SCOD and soluble protein levels, the supernatant was obtained by centrifuging the disintegrated sludge at 7000 rpm for 30 min at a temperature of 4 °C. The COD concentration was measured by the HACH 8000 method using a HACH DC/2500 spectrophotometer. The protein concentration was measured by the Lowry method (Lowry et al., 1951) using BSA (bovine serum albumin) as a reference protein. The degree of disintegration in terms of COD solubilization rate was expressed as follows (Cui and Jahng, 2006):
COD solubilization rateð%Þ ¼
SCODa SCODi 100 TCODi SCODi
ð1Þ
where SCODi and SCODa are the soluble COD before and after the treatment, respectively, and TCODi is the initial TCOD.
2.2. Protein recovery Soluble protein in the supernatant of the disintegrated sludge obtained by centrifuging the disintegrated sludge at 7000 rpm and 4 °C for 30 min was precipitated by adjusting the pH of the supernatant to pH 1.0, 3.3 and 5.0 using 2.0 M H2SO4. After stirring for 15 min, precipitates were recovered by centrifugation at 7000 rpm for 30 min at 4 °C. The resulting pellet containing the precipitated protein was dried at 60 °C for 48 h. 2.3. Protein analysis Components of the recovered pellet including the moisture, crude protein, crude fat, crude fiber and ash were measured following the Korean Standard Method of Feed Analysis (MAF, 2007). To measure the concentrations of heavy metals, the recovered pellet was ground using a blender (HMF-347(E), Hanil Electric, Seoul, Korea), decomposed according to the aqua regia method, and then filtered using a filter paper (Quantitative Filter paper 5B, Advantec, Tokyo, Japan) (Ito et al., 2000). The concentrations of heavy metals in the filtrate were determined using an atomic absorption spectrometer (AAS) (220FS, Varian, Palo Alto, USA) and an inductively coupled plasma mass spectrometer (ICP-MS) (CCTX-7, Thermo elemental, Waltham, USA). Additionally, aflatoxin B1, ochratoxin A, and Salmonella D groups were measured following the guidelines of the Ministry of Agriculture and Forestry Republic of Korea (MAF, 2007). 2.4. Experiments on rats For acute toxicity analysis of the recovered protein, male and female Sprague-Dawley rats were divided into four groups, each of which consisted of five rats. The first group of rats as the control group was orally administered with 20 ml sterile water/kg. The other three groups were fed with 20 ml/kg for 500, 1000, and 2000 mg recovered protein/kg, respectively (Table 1). Mortality, incidence of clinical signs, and change of body weight within two weeks were monitored, and necropsy tests after the experiment period were performed. All experimental conditions followed the Korea Good Laboratory Practice (KGLP) of the Korea Food and Drug Administration (KFDA, 2005a,b), such that the temperature was 23 ± 3 °C, the relative humidity was 55 ± 15%, the number of awakenings was 10–20 times/h, and illumination was on for 12 h/day.
3. Results and discussion 3.1. Sludge disintegration After an alkali treatment of excess sludge at pH 12 for 2 h, the SSi, TCODi, SCODi and SCODa readings were 4580 mg/L, 5235 mg/L, 44 mg/L, 1285 mg/L, respectively, yielding COD solubilization rate of 23.9%. After the alkali treatment, the soluble protein concentration increased from 28.2 mg/L to 932.3 mg/L, and the protein concentration in the supernatant after the subsequent protein precipitation at pH 3.3 was 587.5 mg/L. Therefore, it was estimated
Table 1 Group organization of Sprague-Dawley rats Group
Sex
The number of rats
Number
Volumetric dose (ml/kg)
Protein dose (mg/kg)
G1 G2 G3 G4
Male/female Male/female Male/female Male/female
5/5 5/5 5/5 5/5
1–5/21–25 6–10/26–30 11–15/31–35 16–20/36–40
20 20 20 20
0 (sterile water) 500 1000 2000
8951
5000
2500
4000
2000 Soluble protein
3000
1500
SS
2000
1000
1000
Protein (mg/L)
SS and VSS (mg/L)
J. Hwang et al. / Bioresource Technology 99 (2008) 8949–8954
500
VSS
0
0
TCOD 70 COD solubilization rate 4000 60 SCOD 2000
COD solubilization rate (%)
TCOD and SCOD (mg/L)
80 6000
50
0 0.0
40 5.0e+9
1.0e+10 1.5e+10 2.0e+10 2.5e+10 3.0e+10 3.5e+10
Energy input (kJ/kg VSS) Fig. 1. Effects of ultra-sonication on the disintegration of excess sludge.
that 41.7% of the soluble protein released from the disintegrated sludge was precipitated at pH 3.3. Ultra-sonication of excess sludge was also carried out at energy levels ranging from 0 to 3.84 1010 kJ/kg VSS. Fig. 1 shows that the concentration of soluble protein increased as the input energy increased, particularly in the energy range from 5.49 109 kJ/kg VSS to 1.65 1010 kJ/kg VSS. At 1.65 1010 kJ/kg VSS, the percentages of COD solubilization and protein concentration were 68% and 1,818 mg/L, respectively. The combined treatment of an alkali treatment and a subsequent ultra-sonication led to a rapid increase in the protein concentration and COD solubilization to 5.49 1010 kJ/kg VSS. As shown in Fig. 2, VSS and SS decreased while the protein concentration increased as the ultra-sonication energy increased. The percentages of COD solubilization by ultra-sonication combined with the alkali treatment ranged from 32% to 84%. The concentration of soluble protein increased to 2672 mg/L, which was higher than the 2239 mg/L reading obtained by ultra-sonication alone. As the patterns of SS, VSS, COD solubilization rate and protein concentration were not linear with the energy input, ultra-sonication at 1.65 1010 kJ/kg VSS after an alkali treatment at pH 12 for 2 h was chosen for further experimentation. At these conditions, the percentages of COD solubilization rate and the protein concentration were 81% and 2626 mg/L, respectively, for the excess sludge with 4740 mg/L of the initial SS.
3.2. Protein recovery Excess sludge (SSi = 5330 mg/L) was disintegrated by an alkali treatment combined with ultra-sonication, and the protein concentration in the supernatant was found to be 3177.5 mg/L. Precipitation was performed by adjusting the pH value of the supernatant to pH 1.0, 3.3 and 5.0. As shown in Fig. 3, protein concentrations in the supernatant after precipitation at pH 1.0, 3.3 and 5.0 were 797.3 mg/L, 620 mg/L and 1572.3 mg/L, respectively. As the efficiency of the protein recovery method was the highest (80.5%) at pH 3.3, isoelectric precipitation at pH 3.3 was chosen to recover soluble proteins from the supernatant of disrupted excess sludge. 3.3. Nutritional analysis The nutritional components of the recovered pellet were analyzed to determine its nutritional value as animal feed. The largest part of the recovered pellet consisted of crude protein (50.1%). The remaining materials were NFE (21.8%), crude ash (15.4%), crude fat (9.0%), moisture (3.6%) and crude fiber (0.05%). The major components of the NFE were thought to be sugars, partial cellulose and hemi cellulose; lignin was also included in the NFE, which is expressed as follows (MAF, 2007):
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3000
5000
2500 Soluble protein 2000
3000 1500 2000
Protein (mg/L)
SS and VSS (mg/L)
4000
1000 SS VSS
1000
500
0 80
6000
TCOD 70 SCOD
4000
60
3000
50
2000
40
1000
30
0
COD solubilization rate (%)
TCOD and SCOD (mg/L)
COD solubilization rate 5000
20 0
5e+9
1e+10
2e+10
2e+10
3e+10
3e+10
Energy input (kJ/kg VSS) Fig. 2. Effects of ultra-sonication after an alkali treatment on the disintegration of excess sludge.
100 Protein conc. of supernatant after cell disruption Protein conc. of supernatant after precipitation Efficiency of protein recovery (%)
3000
80 2500 60
2000 1500
40 1000 20
500 0 Before pH adjustment
pH 1.0
pH 3.3
pH 5.0
Efficiency of protein recovery (%)
Protien concentration (mg/L)
3500
0
Fig. 3. Effect of pH on protein precipitation.
NFEð%Þ ¼ 100 ðmoisture þ crude protein þ crude fat þ crude ash þ crude fiberÞ
ð2Þ
In order to evaluate the nutrient potency of the recovered protein, the total digestible nutrients (TDN), digestible energy (DE), metabolizable energy (ME), net energy for maintenance (NEM), net energy for gain (NEG), and net energy for lactation (NEL) were calculated using the equations below (NRC, 2001).
TDN ð%Þ ¼ CP ð%Þ þ NFE ð%Þ þ Fiber ð%Þ þ CF ð%Þ 2:25
ð3Þ
DE ðMcal=kgÞ ¼ TDN ð%Þ 0:04409
ð4Þ
ME ðMcal=kgÞ ¼ 1:01 DE ðMcal=kgÞ 0:45
ð5Þ
2
3
ð6Þ
2
3
NEG ðMcal=kgÞ ¼ 1:42ME 0:174ðMEÞ þ 0:0122ðMEÞ 1:65
ð7Þ
NEL ðMcal=kgÞ ¼ 0:0929 CF ð%Þ þ 0:0547 CP ð%Þ þ 0:192
ð8Þ
NEM ðMcal=kgÞ ¼ 1:37ME 0:138ðMEÞ þ 0:0105ðMEÞ 1:12
The TDN, DE, ME, NEM, NEG and NEL levels in the recovered pellet were calculated according to nutritional component analytical results. These were compared with the values for major types of feeds. As shown in Table 2 the TDN, DE, ME, NEM, NEG and NEL levels of the recovered pellet were 92%, 4.07 Mcal/kg, 3.66 Mcal/kg, 2.56 Mcal/kg, 1.81 Mcal/kg, and 3.77 Mcal/kg, respectively. The content of crude protein in the recovered pellet was similar to that of dried Brewer’s yeast and bone meal, and the other items, TDN, NEM, NEG and NEL, were found at higher levels than in the major feeds. Therefore, the recovered protein from the excess sludge possesses nutritional values suitable for animal feed. 3.4. Hazardous materials analysis The measured concentrations of heavy metals in the original excess sludge and the recovered pellet are listed in Table 3 with regulatory standards. The original sludge contained a variety of
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J. Hwang et al. / Bioresource Technology 99 (2008) 8949–8954 Table 2 Nutritional values of major feeds (NRC, 1982) and the recovered pellet Feedstuff
Dried matter (%)
Ruminants TDN (%)
DE
ME
NEM
NEG
NEL
Crude protein (%)
Ash (%)
(Mcal/kg) Soybean mill Wheat grain Rice grain Barley grain Brewer’s yeast, dried Corn grain Bone meal Dry skimmed milk Oat grain
90 89 89 88 93 89 93 94 89
46 78 70 74 74 83 66 79 68
2.01 3.45 3.10 3.27 3.25 3.67 2.92 3.52 3.02
1.63 3.08 2.73 2.90 2.87 3.31 2.53 3.13 2.65
0.98 1.89 1.64 1.76 1.73 2.05 1.5 1.91 1.59
0.28 1.28 1.08 1.19 1.14 1.41 0.93 1.29 1.04
1.01 1.81 1.61 1.71 1.70 1.93 1.51 1.84 1.57
12.6 14.2 7.9 11.9 43.8 9.9 50.4 33.7 11.8
4.9 1.7 4.7 2.3 6.6 1.5 29.3 7.9 3.1
Recovered pellet
96
92
4.07
3.66
2.56
1.81
3.77
50.1
15.4
Table 3 Concentrations of heavy metals Element
Unit (mg/kg) Original excess sludge
Ag Al As Ca Cd Cr Cu Fe Hg Ni Pb Se Zn a b c
a
ND 7757.4 ND 1731.2 ND ND 278.5 5039.6 ND 53.22 23.6 ND 582.7
Recovered pellet
Legal standardc
ND 4642.8 ND 911.1 ND ND 246.9 2882.9 ND 15.12 6.93 ND 217.6
–b – 2 – 1 100 – – 0.4 – 10 2 –
Not detected. Not prescribed in the regulation. The lowest value in the legal standard of various feeds (MAF, 2004).
hazardous heavy metals. It has been reported that heavy metals in wastewater are concentrated into sewage sludge through the primary and secondary treatment processes (Oliver and Cosgrove, 1974; Stephenson and Lester, 1987). For example, the heavy metals can be absorbed by biomass, precipitated by some anions such as sulfide, and accumulated in excess sludge (Ito et al., 2000). Through the recovery processes of protein, significant amounts of the heavy metals in the raw sludge were removed. Furthermore, the aflatoxin B1, ochratoxin A and Salmonella D groups, an indicator of pathogenic bacteria, were not detected (data not shown). In conclusion, heavy metals, harmful toxins and microorganisms, if any, included in the original excess sludge were found to be removed in significant amounts through the processes of cell disintegration and protein precipitation. 3.5. Toxicity test on rats To assess acute toxicity of the recovered pellet, male and female SD rats were fed by mouth with the recovered protein. During a two-week experimental period, mortality and specific incidences of clinical signs in rats were not detected. Table 4 showed that the body weights of the female group were higher 14 days after the administration of 500 and 2000 mg of recovered pellet/kg. In addition, statistic analyses showed that the increments of body weights were higher in the groups fed with 500, 1000 and 2000 mg of recovered pellet/kg compared to these increments in the control group. However, body weight increases were observed only in female groups, probably because the body weights of rats in
the female control group were accidentally low. Necropsy results in which red discoloration of the thymus was observed in a male group fed with 2000 mg of recovered pellet/kg. According to the necropsy estimation, red discoloration of thymus was thought to be an accidental change caused by the anesthesia and bloodletting processes (Greaves, 1990; Boorman, 1990). As mentioned above, at least three conditions should be fulfilled to use sludge protein as animal feed. First, the nutritional value of sludge protein must be equivalent to that of common feedstuffs. This requirement is thought to be satisfied according to the experimental data given in Table 2. Secondly, the sludge protein should be safe for domestic animals. It was shown in this study that the protein extracted from the excess sludge contained no heavy metals of which the concentrations were higher than the legal standards (Table 3). Additionally, toxins were not detected. The sludge protein was also free of Salmonella D groups, which served as an indicator of pathogenic bacteria, and did not show acute toxicity against rats. According to these results, it is possible to conclude tentatively that the protein extracted from the excess sludge is safe for animals, although additional analyses of the hazardous chemicals and microorganisms that may exist in the sludge are required. It is also necessary to determine through long-term studies to what type of domestic animals (e.g., ruminants, poultry, or pig) the sludge proteins are designated. Finally, the economic feasibility must be proven before the sludge protein can be utilized in agricultural industries. In fact, the extraction of proteins from excess sludge and its commercial use can provide economic benefits. Through the destruction of the microbial cells of the excess sludge and the recovery of cellular proteins, the mass for further treatment of the excess sludge residuals decreases, implying that the treatment expenses can be lowered. In this study, 5330 mg/L of the initial SS concentration was decreased to 1520 mg/L after the alkali and subsequent ultra-sonication treatments. This amount of sludge residuals represents only 28.5% of the original sludge. Furthermore, the dewaterability of the disintegrated sludge is expected to be significantly higher than intact excess sludge as the water inside cells is exposed when the cell walls are broken. Wastes with lower water contents are easier and less expensive to treat compared to wastes with higher water contents. Regarding the marketing of the product, sludge protein needs to compete with existing feedstuffs such as corn grain, fish mill, and dried yeast. The market price will be determined based on the manufacturing costs and other factors. To assess the manufacturing costs, it should be noted that the acquisition of excess sludge as a raw material creates earnings rather than expenditures, as wastewater treatment plants pay for the service of excess sludge disposal. In short, the extraction of cellular protein from excess sludge and its use as a feedstuff can provide many benefits regarding the management of the environment and food resources.
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Table 4 Body weight changes of SD rats fed with the sludge protein Dose (mg/kg)
Sex: male 0
500
1000
2000
Sex: female 0
500
1000
2000
Animal ID
Body weights (g) Day 0
Day 1
Day 3
Day 7
Day 14
Gains
Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N
224.70 9.07 5 221.58 7.14 5 222.00 8.05 5 221.08 6.24 5
251.30 10.64 5 247.94 7.17 5 248.84 8.78 5 250.20 7.94 5
266.85 9.62 5 264.58 7.39 5 264.14 9.61 5 265.86 7.59 5
292.63 10.54 5 292.75 8.18 5 292.48 12.00 5 293.01 6.76 5
336.42 10.46 5 329.41 10.27 5 331.16 14.53 5 339.42 9.04 5
111.72 2.92 5 107.83 9.53 5 109.16 7.56 5 118.33 7.05 5
Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N
160.55 5.21 5 161.94 5.07 5 160.57 7.01 5 161.29 7.48 5
178.60 6.90 5 178.68 5.50 5 178.88 6.21 5 174.82 6.07 5
187.54 6.40 5 187.83 7.86 5 185.75 6.73 5 185.85 5.94 5
199.95 6.08 5 199.40 10.35 5 191.03 8.72 5 198.50 7.26 5
201.79 5.62 5 216.35* 6.64 5 212.06 10.74 5 222.56** 9.22 5
41.24 2.28 5 54.40** 5.34 5 51.49** 4.39 5 61.28** 7.82 5
S.D.: standard deviation. Significantly different from control, p < 0.05. Significantly different from control, p < 0.01.
*
**
4. Conclusions Cellular proteins were prepared from excess sludge from a domestic wastewater treatment plant. Nutrient compositions of the recovered protein were similar to those of dried Brewer’s yeast and bone meal. The concentrations of heavy metals were lower than the legal standards. Aflatoxin B1, ochratoxin A and the Salmonella D groups were not detected. The acute toxicity tests using SD rats did not show significant effects on mortality, the incidence of clinical signs, and body weight changes. The minimum lethal dose was higher than 2000 mg/kg. From these results, it was concluded that the protein recovered from excess sludge appears to be feasible for use as animal feed. References Adebayo, O.T., Fagbenro, O.A., Jegede, T., 2004. Evaluation of Cassia fistula meal as a replacement for soybean meal in practical diets of Oreochromis niloticus fingerlings. Aquacult. Nutr. 10, 99–104. APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. American Public Health Association, Washington, DC. Beszedits, S., Lugowski, A., 1981. Utilizing Waste Activated Sludge for Animal Feeding. B&L Information Service, Toronto. Boorman, G.A., 1990. Pathology of the Fischer Rat: Reference and Atlas. Academic Press, San Diego, CA. Cui, R., Jahng, D., 2006. Enhanced methane production from anaerobic digestion of disintegrated and deproteinized excess sludge. Biotechnol. Lett. 28, 531–538. Greaves, P., 1990. Histopathology of Preclinical Toxicity Studies. Elsevier. Ito, A., Umita, T., Aizawa, J., Takachi, T., Morinaga, K., 2000. Removal of heavy metals from anaerobically digested sewage sludge by a new chemical method using ferric sulfate. Water Res. 344, 751–758. Jung, J., Xing, X.H., Matsumoto, K., 2001. Kinetic analysis of disruption of excess activated sludge by Dyno Mill and characteristics of protein release for recovery of useful materials. Biochem. Eng. J. 8, 1–7. Jung, J., Xing, X.H., Matsumoto, K., 2002. Recoverability of protease released from disrupted excess sludge and its potential application to enhanced hydrolysis of proteins in wastewater. Biochem. Eng. J. 10, 67–72.
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