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Effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea
Bioresource Technology 98 (2007) 1028–1032
Effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea A.S.F. Arau´jo a
a,*
, R.T.R. Monteiro b, E.M.S. Carvalho
c
Universidade Estadual do Piauı´, Av. Nossa Senhora de Fa´tima, S/N, Parnaı´ba, PI 64202-220, Brazil b Centro de Energia Nuclear na Agricultura, P.O. Box 96, Piracicaba, SP 13400-970, Brazil c Universidade Federal do Piauı´ – Campus da Socopo, Teresina, PI 64049-550, Brazil Received 28 December 2005; received in revised form 13 April 2006; accepted 13 April 2006 Available online 19 June 2006
Abstract The effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea was evaluated in a greenhouse experiment. The compost was incorporated into soil at 0, 9.5, 19 and 38 t ha1 (bases upon the N requirement of the crops, i.e., 0, 50, 100 and 200 kg available N ha1). Growth, nodulation and shoot accumulation of nitrogen were evaluated 36 and 63 days after plant emergence. Nodule glutamine synthetase (GS) activity and leghemoglobin content were evaluated 63 days after emergence. Composted textile sludge did not show negative effects on nodule number and weight, nodule GS activity and leghemoglobin content. Nitrogen accumulation in shoot dry matter in soybean and cowpea was higher than other treatments with application of 19 t ha1 of compost. Composting can be an alternate technology for the management of solid textile mill sludge. This study verifies that the composted textile sludge was not harmful to growth, nodulation and nitrogen fixation of soybean and cowpea. 2006 Elsevier Ltd. All rights reserved. Keywords: Industrial wastes; Nitrogen fertilization; Symbiosis; Glutamine synthetase
1. Introduction Large volumes of organic waste is generated by the textile industry and released into the environment (Kaushik and Garg, 2003). Concerns about environmental quality have led to the introduction of alternative disposal methods such as the use as nutrient source for plants and as soil conditioners. Canellas et al. (2001) reported that the use of industrial organic matter in agricultural lands can be justified by the need of finding an appropriate destination for waste recycling. Textile sludge has a variable composition (Balan and Monteiro, 2001) and normally contains high organic matter, N, P, K and micronutrients contents (Martinelli et al., 2002). Additionally, dyes, heavy metals and pathogenic microorganisms may be present, and composting is
*
Corresponding author. Tel.: +55 86 321 1800; fax: +55 86 321 1825. E-mail address: [email protected] (A.S.F. Arau´jo).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.04.028
often required for utilization and transformation of these wastes into a soil amendment. During composting, the nutrients present in the wastes are converted by microbial action into soluble forms available to plants (Ndegwa and Thompson, 2001). Composting can also decrease or eliminate the toxicity of textile sludge (Arau´jo et al., 2001; Arau´jo and Monteiro, 2005). Kaushik and Garg (2003) evaluated the composting of textile sludge using worms, and concluded that the waste could be converted into a stabilized product for agricultural use. Little information about toxic effects of composted industrial wastes on soil microbiology and biochemical processes is available. Biological nitrogen fixation is a biochemical process that has been suggested as important measure of the effects of soil disturbances (Viser and Parkinson, 1992). Brookes (1995) recommended the measurement of biological nitrogen fixation as an indicator of soil stress resulting from pollutants, and Wetzel and Werner (1995) reported that nodulation is an important parameter related to toxic effects of pollution due to
A.S.F. Arau´jo et al. / Bioresource Technology 98 (2007) 1028–1032
compost application. Furthermore, reduction and assimilation of atmospheric nitrogen can be affected by contaminated compost. According to Gonnet and Diaz (2000), nodule glutamine synthetase (GS) (E.C.6.3.1.2) is crucial in the assimilatory process of atmospheric nitrogen, as it catalyzes the first step in the conversion of inorganic nitrogen (ammonium) into its organic form (glutamine). Therefore, reduction of GS activity, resulting from the application of toxic compounds to the soil, leads to a decrease in biological nitrogen fixation by legumes. The leghemoglobin content of the nodules also influences biological nitrogen fixation rates, since leghemoglobin provides a flux of O2 for rhizobial respiration and maintains O2 at a concentration that does not render the nitrogenase complex inactive (Appleby, 1984). Some research has been conducted on the effect of sewage sludge on biological nitrogen fixation in soybean (Angle et al., 1992; Vieira, 2001) and common bean (Vieira et al., 2004), but none on the effect of composted textile sludge on soil microbiology. The aim of this work was to evaluate the effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea. 2. Methods 2.1. Textile sludge compost The compost was produced with sludge obtained from the wastewater treatment plant of a textile mill located at Americana city, Sa˜o Paulo State, Brazil, and a structuring material (wood wastes) mixed in the ratio 1:1 (sludge to wood). The composting process was the Beltsville aerated-pile method (USDA, 1980) using 94,690 kg of mixture (60% humidity and C/N ratio 30:1) and was conducted for 25 days on a cemented pavement. The triangular pile was sieved and allowed to mature for another 65 days. Twenty single samples were collected at several sites of the stabilized compost to produce a composite sample. The chemical characteristics of the stabilized compost were determined by EPA 3051 method (USEPA, 1986).
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respectively. The available N of compost was calculated according to the formula: Nc ¼ 0:1ðNK NAm Þ þ NAm þ NNit ðCETESB; 1999Þ Nc = available N in the compost (kg kg1); NK = total N in the compost (Kjeldahl method); NAm = NH4–N; NNit = NO3–N. The rate of applied compost (dry weight) was calculated: Rate of compost (kg ha1) = Recommended N (kg ha1)/Available N in the compost (kg kg1). Pots of 5-kg capacity were filled with the amended soils, wetted with distilled water to 40% of water holding capacity of the mixture and incubated for one week before sowing. Soybean and cowpea seeds were inoculated with a commercial inoculum at a level of 1 kg per 50 kg of seeds. Six seeds per pot were sowed at 0.5 cm depth. After emergence, seedlings were thinned to two plants per pot. Plants were harvested at 36 and 63 days after emergence to measure growth, nodule number, nodule fresh matter and shoot nitrogen accumulation. Glutamine synthetase (GS) activity and leghemoglobin content were determined at 63 days. The shoots and roots were dried at 60 C for 72 h for dry weigh determination. A randomized complete block design with six replications of all treatments was used and data were analyzed by analysis of variance at the 5% level of probability by Duncan’s multiple range test. 2.3. Preparation of nodule crude extracts A composite sample of five representative nodules from each treatment was used for the preparation of nodule extracts. Nodule samples (0.3 g) were ground with liquid N2 in a chilled mortar with a pestle and extracted with 2 ml of 25 mM Tris–HCl buffer, pH 7.6, containing 5 mM EDTA, 10 mM 2-mercaptoethanol, polyvinylpyrrolidone (PVP) 5% (w/v), and 1 mM MgCl2 Æ 6H2O, for 5 min. The suspension was centrifuged at 15,000g at 4 C for 15 min, and the supernatant was used to determine GS activity.
2.2. Greenhouse study
2.4. Glutamine synthetase (GS) activity and leghemoglobin content
A yellow Podzol soil sample was selected to be amended with the composted textile sludge. Yellow Podzol soils are very permeable, have a low carbon content, poor water retention and low fertility and therefore allow for the differentiation of the effect of the organic compost. The soil sample presented the following chemical characteristic (Tedesco et al., 1995): pH, 5.7; organic matter, 19 g dm3; P, 12 mg dm3; K, 9; Ca, 7; Mg, 5 and CEC, 47 mmolc dm3. Yellow Podzol soil were collected at a depth of 0–20 cm and amended into soil at four rates: 0· (0 t ha1), 0.5· (9.5 t ha1), 1· (19 t ha1) and 2· (38 t ha1). The 1· (recommended rate) rate was applied to supply 100 kg available N ha1. The 0.5· and 2· application rates supplied 50 and 200 kg available N ha1,
GS activity (EC 6.3.1.2) was determined by the hydroxamate biosynthesis method (Farnden and Robertson, 1980). The reaction mixture consisted of 600 ll of 250 mM Tris– HCl buffer, pH 7.0, 200 ll of 30 mM glutamate, 200 ll of 30 nM ATP, 200 ll of 500 mM MgSO4 Æ 7H2O, 500 ll of enzyme extract and 200 ll of 1 M hydroxylamine. The mixture was incubated at 30 C and measured spectrophotometrically at 540 nm. Hydroxylamine was omitted from the control. The GS activity was expressed as lmoles of glutamyl-hydroxamate h1 g1 fresh nodules. Nodule leghemoglobin was extracted with Drabkin reagent and its concentration determined by the cyanmethemoglobin method (Wilson and Reisenauer, 1963) using human hemoglobin as standard.
A.S.F. Arau´jo et al. / Bioresource Technology 98 (2007) 1028–1032
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3. Results and discussion The chemical properties of the stabilized compost are given in Table 1. From the data it is clearly evident that the composted sludge presents a neutral pH and contained appreciable quantities of N, P and K. Additionally, the total metal content was below the limits permitted by CETESB (1999). The total dry matter production by soybean and cowpea at 36 and 63 days after emergence (DAE) are given in Table 2. Toxic effects on soybean plants were observed at 36 and to cowpea at 63 DAE for the 2· rate of composted textile sludge application, compared to soil control, while no difference was observed among any of the other treatments. The decrease in shoot and root dry weight at the first harvest of soybean supplied with 2· compost suggests a possible toxic effect to soybean plants. These results are in agreement with the findings of Eivazi (1990) who observed a decrease in the dry weight of soybean after application of sewage sludge at high rates to the soil. Except for the roots of soybean which showed a lower nodule weight at the 2· rate of compost application at 36 Table 1 Chemical composition of stabilized compost
pH (CaCl2) Organic matter (%) Total N (g kg1) Ammonium N (g kg1) Nitrate N (g kg1) Total P (g kg1) Total K (g kg1) Sodium (mg kg1) Iron (%) Mn (mg kg1) Mo (mg kg1) Cu (mg kg1) Zn (mg kg1) Cr (mg kg1) Pb (mg kg1) Cd (mg kg1) Ni (mg kg1) Co (mg kg1) a
Stabilized compost
Limits of heavy metal permitteda
6.8 29.27 1.31 1.61 51.51 8.70 3.40 0.29 5.61 700.0 <4.0 110.8 397.0 73.9 33.4 <0.3 30.1 14.6
– – – – – – – – – – – 4300 7500 3000 75 85 420 840
CETESB (1999).
DAE, there were no differences in either the number or weight of root nodules of soybean and cowpea among the treatments (Table 3). The composted textile sludge caused no negative effects on the formation of nodules, contrary to the common view that nodulation is suppressed in soil high in N (Angle et al., 1992). However, it is possible that nodule formation on the compost-amended soil occurred only after much of the readily available N had been depleted. These results are similar to those of Angle et al. (1992) and Vieira (2001) for sewage sludge applied to the soil. Nodule glutamine synthetase (GS) activity and leghemoglobin content of the soybean and cowpea were not affected by the application of compost (Table 4). Moreover, there was a higher nodule leghemoglobin content of plants grown in soil amended with 0.5· compost, compared plants grown in control soil. These results confirm that there were no toxic effects of the compost on nitrogen fixation by soybean and cowpea. Additionally, the initial stage of Bradyrhizobium-legumes symbiosis was favored by the application of compost at a low rate. All nodules presented a pink-colored interior due to leghemoglobin pigment. Leghemoglobin is a symbiotic product whose globin part is synthesized by the plant in response to the bacterial infection (Verma and Long, 1983), and leghemoglobin formation occurs before the beginning of nitrogen fixation (Freire, 1992). At 36 DAE no significant differences were observed in the accumulation of N by soybean shoots, while cowpea accumulated more N when the compost was applied at the 1· rate (Table 5). At 63 DAE shoots of soybean and cowpea presented higher accumulated N than other treatments when the compost was applied at the 1· rate. The highest amount of N in shoots, compared to control soil, may be related to the uptake by the plants of nitrogen derived from compost. In contrast, the decrease in the shoot-accumulated N at the 2· rate of composted sludge may be due the excess of N and may also be partially responsible for reducing the plant dry weight. There are conflicting reports on the effects of sludge on soil population of rhizobia, N fixation and yield of legumes (Angle et al., 1992). Some papers reported that sludgeborne heavy metals significantly reduced soil population of Bradyrhizobium japonicum (Reddy et al., 1983) and
Table 2 Effect of different rates of composted textile sludge on soybean and cowpea shoot and root dry weight Compost rates
Soybean
Cowpea
Shoot (g per pot) a
36 DAE 0· 0.5· 1· 2· a
6.2 ± 1.1 5.0 ± 0.9 6.4 ± 0.8 4.1 ± 0.3
Root (g per pot)
63 DAE a* ab a b
19.2 ± 3.1 23.6 ± 2.8 21.5 ± 4.1 20.0 ± 2.2
36 DAE b a ab b
3.8 ± 0.9 1.3 ± 0.3 2.4 ± 1.1 0.9 ± 0.5
Shoot (g per pot)
63 DAE a b ab b
7.9 ± 1.1 9.4 ± 0.9 8.6 ± 1.4 8.0 ± 1.3
36 DAE b a ab b
5.7 ± 0.9 5.5 ± 1.5 5.9 ± 1.1 5.5 ± 1.3
Root (g per pot)
63 DAE a a a a
23.5 ± 3.7 25.1 ± 5.1 20.7 ± 3.5 18.6 ± 3.2
36 DAE a a ab b
1.1 ± 0.3 0.8 ± 0.1 1.2 ± 0.3 1.0 ± 0.2
63 DAE a a a a
7.5 ± 1.2 10.2 ± 2.5 9.4 ± 2.3 4.1 ± 1.4
ab a a b
DAE = Days after emergence. Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test. *
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Table 3 Effect of different rates of composted textile sludge on soybean and cowpea nodule number (NN) and fresh matter (NFM) Compost rates
Soybean
Cowpea 1
1
NN (nr pot ) a
36 DAE 0· 0.5· 1· 2·
63 DAE *
47 ± 12 a 43 ± 9 a 51 ± 15 a 30 ± 11 a
NN (nr pot1)
NFM (g pot )
185 ± 35 182 ± 30 175 ± 31 143 ± 22
a a a a
36 DAE
63 DAE
0.43 ± 0.2 a 0.24 ± 0.1 ab 0.40 ± 0.2 a 0.14 ± 0.09 b
2.71 ± 0.5 3.38 ± 1.1 2.88 ± 0.9 2.85 ± 0.9
a a a a
NFM (g pot1)
36 DAE
63 DAE
71 ± 15 a 35 ± 8 a 48 ± 10 a 21 ± 7 a
42 ± 11 69 ± 21 60 ± 19 45 ± 16
a a a a
36 DAE
63 DAE
0.66 ± 0.1 a 0.82 ± 0.3 a 1.12 ± 0.3 a 0.73 ± 0.07 a
1.43 ± 0.3 2.43 ± 1.0 1.60 ± 0.7 1.43 ± 0.5
a a a a
a
DAE = Days after emergence. Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test. *
Table 4 Effect of different rates of composted textile sludge on nodule GS activity and leghemoglobin content of soybean and cowpea, at 63 DAE Compost rates
0· 0.5· 1· 2·
GS activity (lmoles of glutamyl h1 g1 nodules)
Leghemoglobin content (mg g1 nodules)
Soybean
Soybean
Cowpea
101.3 ± 35.1 126.9 ± 21.0 125.7 ± 23.2 109.2 ± 31.5
a* a a a
176.8 ± 41.1 160.7 ± 32.2 169.8 ± 44.2 165.7 ± 42.6
a a a a
63.4 ± 12.0 91.6 ± 22.8 78.5 ± 15.3 74.3 ± 11.9
Cowpea b a ab ab
37.5 ± 7.1 45.5 ± 6.3 40.9 ± 4.6 38.2 ± 8.2
b a ab ab
*
Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test.
Table 5 Effect of different rates of composted textile sludge on shoot accumulation of nitrogen (SAN) by soybean and cowpea Compost rates
Soybean
Cowpea
SAN (mg per plant)
SAN (mg per plant)
a
36 DAE 0· 0.5· 1· 2·
52.1 ± 15.9 38.8 ± 12.1 49.2 ± 15.0 37.1 ± 11.5
a* a a a
63 DAE
36 DAE
63 DAE
84.5 ± 21.3 b 91.1 ± 20.6 b 111.2 ± 18.4 a 38.5 ± 9.4 c
32.2 ± 9.2 b 31.5 ± 11.0 b 68.8 ± 14.2 a 27.4 ± 8.7 b
56.1 ± 11.8 b 52.4 ± 9.4 b 99.6 ± 14.3 a 32.4 ± 10.2 c
a
DAE = Days after emergence. Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test. *
inhibited nodulation (Abd-Alla et al., 1999). Our findings show that none of the parameters examined were negatively affected by the heavy metals in composted textile sludge. When short-term adverse effects of sludge on symbiotic parameters or plant growth were observed, the effects were probable related to salts added to the soil with the sludge (Angle et al., 1992). Since salts are easily leached from soils, these adverse effects should diminish with time as rainfall or irrigation water leach salts from the root zone.
to examine the long-term effect of composted textile sludge in soils, particularly in relation to biological indicators of soil quality. Acknowledgements The authors are thankful to Dr. J.A.G. Silveira, UFC, Brazil, for providing the laboratory facilities, Bioland Co. for providing the textile sludge compost, and Dr. M.V.B. Figueiredo, IPA, Brazil, for her suggestions. R.T.R. Monteiro is supported by CNPq personal grant.
4. Conclusions References Composting can be an alternate technology for the management of solid textile mill sludge. This study verifies that the composted textile sludge was not harmful to growth, nodulation and nitrogen fixation of soybean and cowpea; however, proper consideration should be given to the metal content of textile wastes. Further field studies are required
Abd-Alla, H.M., Yan, F., Schubert, S., 1999. Effects of sewage sludge application on nodulation, nitrogen fixation and plant growth of faba bean, soybean and lupin. J. Appl. Bot. 73, 69–75. Angle, J.S., Madariaga, G.M., Heger, E.A., 1992. Sewage sludge effects on growth and nitrogen fixation of soybean. Agri. Ecosys. Environ. 41, 231–239.
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Appleby, C.A., 1984. Leghemoglobin and Rhizobium respiration. Ann. Rev. Plant Physiol. 35, 521–554. Arau´jo, A.S.F., Sahyoun, F.K., Monteiro, R.T.R., 2001. Evaluation of toxicity of textile sludge compost on seed germination and root elongation of soybean and wheat. Ecossistema 26, 117–120. Arau´jo, A.S.F., Monteiro, R.T.R., 2005. Plant bioassays to assess toxicity of textile sludge compost. Sci. Agri. 62, 286–290. Balan, D.S.L., Monteiro, R.T.R., 2001. Decolorization of textile indigo dye by lignolytic fungi. J. Biotechnol. 89, 141–145. Brookes, P.C., 1995. The use of microbial parameters in monitoring soil pollution by heavy metals. Biol. Fert. Soils 19, 261–279. Canellas, L.P., Santos, G.A., Rumjanek, V.M., Moraes, A.A., Guridi, F., 2001. Distribuic¸a˜o da mate´ria orgaˆnica e caracterı´sticas de a´cidos hu´micos em solos com a adic¸a˜o de resı´duos de origem urbana. Pesq. Agropec. Bras. 36, 1529–1538. CETESB, 1999. Aplicac¸a˜o de biosso´lidos em a´reas agrı´colas: crite´rios para projeto e operac¸a˜o. Norma P4230, Sa˜o Paulo, p. 32. Eivazi, F., 1990. Nitrogen fixation of soybean and alfafa on sewage sludge-amended soils. Agri. Ecosys. Environ. 30, 129–136. Farnden, K.J.F., Robertson, J.G., 1980. Methods for studying enzymes involved in metabolism related to nitrogenase. In: Bergensen, F.J. (Ed.), Methods for evaluating biological nitrogen fixation. John Wiley & Sons, New York, pp. 265–314. Freire, J.R.J., 1992. Fixac¸a˜o do nitrogeˆnio pela simbiose rizo´bio/leguminosas. In: Cardoso, E.J.B.N., Tsai, S.M., Neves, M.C.P. (Eds.), Microbiologia do Solo. SBCS, Campinas, pp. 121–140. Gonnet, S., Diaz, P., 2000. Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp. Rev. Bras. Fisiol. Veg. 12, 195–202. Kaushik, P., Garg, V.K., 2003. Vermicomposting of mixed solid textile mill sludge and cow dung with the epigeic earthworm Eisenia foetida. Biores. Technol. 90, 311–316.
Martinelli, U.A., Peron, A.P., Martins, E.P., Scharf, M., Budag, N., Barcellos, L.O., 2002. Lodo Teˆxtil: um problema ou uma soluc¸a˜o? Quı´mica Teˆxtil, vol. 69, pp. 16–23. Ndegwa, P.M., Thompson, S.A., 2001. Integrating composting and vermicomposting in treatment and bioconversion of biosolids. Biores. Technol. 76, 107–112. Reddy, G.B., Cheng, C.N., Dunn, S.J., 1983. Survival of Rhizobium japonicum in soil-sludge environment. Soil Biol. Biochem. 15, 343– 345. Tedesco, M.J., Gianello, C., Bissani, C.A. 1995. Ana´lises de solos, plantas e outros materiais. Porto Alegre: UFRGS, Boletim Te´cnico, p. 174. United States Department of Agriculture, 1980. Manual for composting of sewage sludge by the Beltsville aerated-pile method, USDA-EPA, p. 65. United States Environmental Protection Agency, 1986. Test method for evaluating solid wastes. Report Number SW-846, Washington, DC, p. 255. Verma, D.P., Long, S., 1983. The molecular biology of Rhizobium-legume symbiosis. Int. Rev. Cytol. 14, 212–245. Vieira, R.F., 2001. Sewage sludge effects on soybean growth and nitrogen fixation. Biol. Fert. Soils 34, 196–200. Vieira, R.F., Tsai, S.M., Teixeira, M.A., 2004. Efeito do lodo de esgoto no crescimento e fixac¸a˜o simbiotica de N2 em feijoeiro (Phaseolus vulgaris L.). Jaguariu´na: Embrapa Meio Ambiente, Boletim de Pesquisa e Desenvolvimento. 18p. Viser, S., Parkinson, D., 1992. Soil biological criteria as indicator of soil quality: soil microorganisms. Am. J. Alternative Agri. 7, 33–37. Wetzel, A., Werner, D., 1995. Ecotoxicological evaluation of contaminated soil using the legume root nodule symbiosis as effect parameters. Environ. Toxicol. Water Qual. 10, 127–133. Wilson, D.O., Reisenauer, H.M., 1963. Determination of leghemoglobin in legume nodules. Anal. Biochem. 6, 27–30.
Effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea A.S.F. Arau´jo a
a,*
, R.T.R. Monteiro b, E.M.S. Carvalho
c
Universidade Estadual do Piauı´, Av. Nossa Senhora de Fa´tima, S/N, Parnaı´ba, PI 64202-220, Brazil b Centro de Energia Nuclear na Agricultura, P.O. Box 96, Piracicaba, SP 13400-970, Brazil c Universidade Federal do Piauı´ – Campus da Socopo, Teresina, PI 64049-550, Brazil Received 28 December 2005; received in revised form 13 April 2006; accepted 13 April 2006 Available online 19 June 2006
Abstract The effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea was evaluated in a greenhouse experiment. The compost was incorporated into soil at 0, 9.5, 19 and 38 t ha1 (bases upon the N requirement of the crops, i.e., 0, 50, 100 and 200 kg available N ha1). Growth, nodulation and shoot accumulation of nitrogen were evaluated 36 and 63 days after plant emergence. Nodule glutamine synthetase (GS) activity and leghemoglobin content were evaluated 63 days after emergence. Composted textile sludge did not show negative effects on nodule number and weight, nodule GS activity and leghemoglobin content. Nitrogen accumulation in shoot dry matter in soybean and cowpea was higher than other treatments with application of 19 t ha1 of compost. Composting can be an alternate technology for the management of solid textile mill sludge. This study verifies that the composted textile sludge was not harmful to growth, nodulation and nitrogen fixation of soybean and cowpea. 2006 Elsevier Ltd. All rights reserved. Keywords: Industrial wastes; Nitrogen fertilization; Symbiosis; Glutamine synthetase
1. Introduction Large volumes of organic waste is generated by the textile industry and released into the environment (Kaushik and Garg, 2003). Concerns about environmental quality have led to the introduction of alternative disposal methods such as the use as nutrient source for plants and as soil conditioners. Canellas et al. (2001) reported that the use of industrial organic matter in agricultural lands can be justified by the need of finding an appropriate destination for waste recycling. Textile sludge has a variable composition (Balan and Monteiro, 2001) and normally contains high organic matter, N, P, K and micronutrients contents (Martinelli et al., 2002). Additionally, dyes, heavy metals and pathogenic microorganisms may be present, and composting is
*
Corresponding author. Tel.: +55 86 321 1800; fax: +55 86 321 1825. E-mail address: [email protected] (A.S.F. Arau´jo).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.04.028
often required for utilization and transformation of these wastes into a soil amendment. During composting, the nutrients present in the wastes are converted by microbial action into soluble forms available to plants (Ndegwa and Thompson, 2001). Composting can also decrease or eliminate the toxicity of textile sludge (Arau´jo et al., 2001; Arau´jo and Monteiro, 2005). Kaushik and Garg (2003) evaluated the composting of textile sludge using worms, and concluded that the waste could be converted into a stabilized product for agricultural use. Little information about toxic effects of composted industrial wastes on soil microbiology and biochemical processes is available. Biological nitrogen fixation is a biochemical process that has been suggested as important measure of the effects of soil disturbances (Viser and Parkinson, 1992). Brookes (1995) recommended the measurement of biological nitrogen fixation as an indicator of soil stress resulting from pollutants, and Wetzel and Werner (1995) reported that nodulation is an important parameter related to toxic effects of pollution due to
A.S.F. Arau´jo et al. / Bioresource Technology 98 (2007) 1028–1032
compost application. Furthermore, reduction and assimilation of atmospheric nitrogen can be affected by contaminated compost. According to Gonnet and Diaz (2000), nodule glutamine synthetase (GS) (E.C.6.3.1.2) is crucial in the assimilatory process of atmospheric nitrogen, as it catalyzes the first step in the conversion of inorganic nitrogen (ammonium) into its organic form (glutamine). Therefore, reduction of GS activity, resulting from the application of toxic compounds to the soil, leads to a decrease in biological nitrogen fixation by legumes. The leghemoglobin content of the nodules also influences biological nitrogen fixation rates, since leghemoglobin provides a flux of O2 for rhizobial respiration and maintains O2 at a concentration that does not render the nitrogenase complex inactive (Appleby, 1984). Some research has been conducted on the effect of sewage sludge on biological nitrogen fixation in soybean (Angle et al., 1992; Vieira, 2001) and common bean (Vieira et al., 2004), but none on the effect of composted textile sludge on soil microbiology. The aim of this work was to evaluate the effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea. 2. Methods 2.1. Textile sludge compost The compost was produced with sludge obtained from the wastewater treatment plant of a textile mill located at Americana city, Sa˜o Paulo State, Brazil, and a structuring material (wood wastes) mixed in the ratio 1:1 (sludge to wood). The composting process was the Beltsville aerated-pile method (USDA, 1980) using 94,690 kg of mixture (60% humidity and C/N ratio 30:1) and was conducted for 25 days on a cemented pavement. The triangular pile was sieved and allowed to mature for another 65 days. Twenty single samples were collected at several sites of the stabilized compost to produce a composite sample. The chemical characteristics of the stabilized compost were determined by EPA 3051 method (USEPA, 1986).
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respectively. The available N of compost was calculated according to the formula: Nc ¼ 0:1ðNK NAm Þ þ NAm þ NNit ðCETESB; 1999Þ Nc = available N in the compost (kg kg1); NK = total N in the compost (Kjeldahl method); NAm = NH4–N; NNit = NO3–N. The rate of applied compost (dry weight) was calculated: Rate of compost (kg ha1) = Recommended N (kg ha1)/Available N in the compost (kg kg1). Pots of 5-kg capacity were filled with the amended soils, wetted with distilled water to 40% of water holding capacity of the mixture and incubated for one week before sowing. Soybean and cowpea seeds were inoculated with a commercial inoculum at a level of 1 kg per 50 kg of seeds. Six seeds per pot were sowed at 0.5 cm depth. After emergence, seedlings were thinned to two plants per pot. Plants were harvested at 36 and 63 days after emergence to measure growth, nodule number, nodule fresh matter and shoot nitrogen accumulation. Glutamine synthetase (GS) activity and leghemoglobin content were determined at 63 days. The shoots and roots were dried at 60 C for 72 h for dry weigh determination. A randomized complete block design with six replications of all treatments was used and data were analyzed by analysis of variance at the 5% level of probability by Duncan’s multiple range test. 2.3. Preparation of nodule crude extracts A composite sample of five representative nodules from each treatment was used for the preparation of nodule extracts. Nodule samples (0.3 g) were ground with liquid N2 in a chilled mortar with a pestle and extracted with 2 ml of 25 mM Tris–HCl buffer, pH 7.6, containing 5 mM EDTA, 10 mM 2-mercaptoethanol, polyvinylpyrrolidone (PVP) 5% (w/v), and 1 mM MgCl2 Æ 6H2O, for 5 min. The suspension was centrifuged at 15,000g at 4 C for 15 min, and the supernatant was used to determine GS activity.
2.2. Greenhouse study
2.4. Glutamine synthetase (GS) activity and leghemoglobin content
A yellow Podzol soil sample was selected to be amended with the composted textile sludge. Yellow Podzol soils are very permeable, have a low carbon content, poor water retention and low fertility and therefore allow for the differentiation of the effect of the organic compost. The soil sample presented the following chemical characteristic (Tedesco et al., 1995): pH, 5.7; organic matter, 19 g dm3; P, 12 mg dm3; K, 9; Ca, 7; Mg, 5 and CEC, 47 mmolc dm3. Yellow Podzol soil were collected at a depth of 0–20 cm and amended into soil at four rates: 0· (0 t ha1), 0.5· (9.5 t ha1), 1· (19 t ha1) and 2· (38 t ha1). The 1· (recommended rate) rate was applied to supply 100 kg available N ha1. The 0.5· and 2· application rates supplied 50 and 200 kg available N ha1,
GS activity (EC 6.3.1.2) was determined by the hydroxamate biosynthesis method (Farnden and Robertson, 1980). The reaction mixture consisted of 600 ll of 250 mM Tris– HCl buffer, pH 7.0, 200 ll of 30 mM glutamate, 200 ll of 30 nM ATP, 200 ll of 500 mM MgSO4 Æ 7H2O, 500 ll of enzyme extract and 200 ll of 1 M hydroxylamine. The mixture was incubated at 30 C and measured spectrophotometrically at 540 nm. Hydroxylamine was omitted from the control. The GS activity was expressed as lmoles of glutamyl-hydroxamate h1 g1 fresh nodules. Nodule leghemoglobin was extracted with Drabkin reagent and its concentration determined by the cyanmethemoglobin method (Wilson and Reisenauer, 1963) using human hemoglobin as standard.
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3. Results and discussion The chemical properties of the stabilized compost are given in Table 1. From the data it is clearly evident that the composted sludge presents a neutral pH and contained appreciable quantities of N, P and K. Additionally, the total metal content was below the limits permitted by CETESB (1999). The total dry matter production by soybean and cowpea at 36 and 63 days after emergence (DAE) are given in Table 2. Toxic effects on soybean plants were observed at 36 and to cowpea at 63 DAE for the 2· rate of composted textile sludge application, compared to soil control, while no difference was observed among any of the other treatments. The decrease in shoot and root dry weight at the first harvest of soybean supplied with 2· compost suggests a possible toxic effect to soybean plants. These results are in agreement with the findings of Eivazi (1990) who observed a decrease in the dry weight of soybean after application of sewage sludge at high rates to the soil. Except for the roots of soybean which showed a lower nodule weight at the 2· rate of compost application at 36 Table 1 Chemical composition of stabilized compost
pH (CaCl2) Organic matter (%) Total N (g kg1) Ammonium N (g kg1) Nitrate N (g kg1) Total P (g kg1) Total K (g kg1) Sodium (mg kg1) Iron (%) Mn (mg kg1) Mo (mg kg1) Cu (mg kg1) Zn (mg kg1) Cr (mg kg1) Pb (mg kg1) Cd (mg kg1) Ni (mg kg1) Co (mg kg1) a
Stabilized compost
Limits of heavy metal permitteda
6.8 29.27 1.31 1.61 51.51 8.70 3.40 0.29 5.61 700.0 <4.0 110.8 397.0 73.9 33.4 <0.3 30.1 14.6
– – – – – – – – – – – 4300 7500 3000 75 85 420 840
CETESB (1999).
DAE, there were no differences in either the number or weight of root nodules of soybean and cowpea among the treatments (Table 3). The composted textile sludge caused no negative effects on the formation of nodules, contrary to the common view that nodulation is suppressed in soil high in N (Angle et al., 1992). However, it is possible that nodule formation on the compost-amended soil occurred only after much of the readily available N had been depleted. These results are similar to those of Angle et al. (1992) and Vieira (2001) for sewage sludge applied to the soil. Nodule glutamine synthetase (GS) activity and leghemoglobin content of the soybean and cowpea were not affected by the application of compost (Table 4). Moreover, there was a higher nodule leghemoglobin content of plants grown in soil amended with 0.5· compost, compared plants grown in control soil. These results confirm that there were no toxic effects of the compost on nitrogen fixation by soybean and cowpea. Additionally, the initial stage of Bradyrhizobium-legumes symbiosis was favored by the application of compost at a low rate. All nodules presented a pink-colored interior due to leghemoglobin pigment. Leghemoglobin is a symbiotic product whose globin part is synthesized by the plant in response to the bacterial infection (Verma and Long, 1983), and leghemoglobin formation occurs before the beginning of nitrogen fixation (Freire, 1992). At 36 DAE no significant differences were observed in the accumulation of N by soybean shoots, while cowpea accumulated more N when the compost was applied at the 1· rate (Table 5). At 63 DAE shoots of soybean and cowpea presented higher accumulated N than other treatments when the compost was applied at the 1· rate. The highest amount of N in shoots, compared to control soil, may be related to the uptake by the plants of nitrogen derived from compost. In contrast, the decrease in the shoot-accumulated N at the 2· rate of composted sludge may be due the excess of N and may also be partially responsible for reducing the plant dry weight. There are conflicting reports on the effects of sludge on soil population of rhizobia, N fixation and yield of legumes (Angle et al., 1992). Some papers reported that sludgeborne heavy metals significantly reduced soil population of Bradyrhizobium japonicum (Reddy et al., 1983) and
Table 2 Effect of different rates of composted textile sludge on soybean and cowpea shoot and root dry weight Compost rates
Soybean
Cowpea
Shoot (g per pot) a
36 DAE 0· 0.5· 1· 2· a
6.2 ± 1.1 5.0 ± 0.9 6.4 ± 0.8 4.1 ± 0.3
Root (g per pot)
63 DAE a* ab a b
19.2 ± 3.1 23.6 ± 2.8 21.5 ± 4.1 20.0 ± 2.2
36 DAE b a ab b
3.8 ± 0.9 1.3 ± 0.3 2.4 ± 1.1 0.9 ± 0.5
Shoot (g per pot)
63 DAE a b ab b
7.9 ± 1.1 9.4 ± 0.9 8.6 ± 1.4 8.0 ± 1.3
36 DAE b a ab b
5.7 ± 0.9 5.5 ± 1.5 5.9 ± 1.1 5.5 ± 1.3
Root (g per pot)
63 DAE a a a a
23.5 ± 3.7 25.1 ± 5.1 20.7 ± 3.5 18.6 ± 3.2
36 DAE a a ab b
1.1 ± 0.3 0.8 ± 0.1 1.2 ± 0.3 1.0 ± 0.2
63 DAE a a a a
7.5 ± 1.2 10.2 ± 2.5 9.4 ± 2.3 4.1 ± 1.4
ab a a b
DAE = Days after emergence. Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test. *
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Table 3 Effect of different rates of composted textile sludge on soybean and cowpea nodule number (NN) and fresh matter (NFM) Compost rates
Soybean
Cowpea 1
1
NN (nr pot ) a
36 DAE 0· 0.5· 1· 2·
63 DAE *
47 ± 12 a 43 ± 9 a 51 ± 15 a 30 ± 11 a
NN (nr pot1)
NFM (g pot )
185 ± 35 182 ± 30 175 ± 31 143 ± 22
a a a a
36 DAE
63 DAE
0.43 ± 0.2 a 0.24 ± 0.1 ab 0.40 ± 0.2 a 0.14 ± 0.09 b
2.71 ± 0.5 3.38 ± 1.1 2.88 ± 0.9 2.85 ± 0.9
a a a a
NFM (g pot1)
36 DAE
63 DAE
71 ± 15 a 35 ± 8 a 48 ± 10 a 21 ± 7 a
42 ± 11 69 ± 21 60 ± 19 45 ± 16
a a a a
36 DAE
63 DAE
0.66 ± 0.1 a 0.82 ± 0.3 a 1.12 ± 0.3 a 0.73 ± 0.07 a
1.43 ± 0.3 2.43 ± 1.0 1.60 ± 0.7 1.43 ± 0.5
a a a a
a
DAE = Days after emergence. Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test. *
Table 4 Effect of different rates of composted textile sludge on nodule GS activity and leghemoglobin content of soybean and cowpea, at 63 DAE Compost rates
0· 0.5· 1· 2·
GS activity (lmoles of glutamyl h1 g1 nodules)
Leghemoglobin content (mg g1 nodules)
Soybean
Soybean
Cowpea
101.3 ± 35.1 126.9 ± 21.0 125.7 ± 23.2 109.2 ± 31.5
a* a a a
176.8 ± 41.1 160.7 ± 32.2 169.8 ± 44.2 165.7 ± 42.6
a a a a
63.4 ± 12.0 91.6 ± 22.8 78.5 ± 15.3 74.3 ± 11.9
Cowpea b a ab ab
37.5 ± 7.1 45.5 ± 6.3 40.9 ± 4.6 38.2 ± 8.2
b a ab ab
*
Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test.
Table 5 Effect of different rates of composted textile sludge on shoot accumulation of nitrogen (SAN) by soybean and cowpea Compost rates
Soybean
Cowpea
SAN (mg per plant)
SAN (mg per plant)
a
36 DAE 0· 0.5· 1· 2·
52.1 ± 15.9 38.8 ± 12.1 49.2 ± 15.0 37.1 ± 11.5
a* a a a
63 DAE
36 DAE
63 DAE
84.5 ± 21.3 b 91.1 ± 20.6 b 111.2 ± 18.4 a 38.5 ± 9.4 c
32.2 ± 9.2 b 31.5 ± 11.0 b 68.8 ± 14.2 a 27.4 ± 8.7 b
56.1 ± 11.8 b 52.4 ± 9.4 b 99.6 ± 14.3 a 32.4 ± 10.2 c
a
DAE = Days after emergence. Mean ± standard error. In each column the means followed by the same letter do not differ statistically (P < 0.05) from each other, according to Duncan’s test. *
inhibited nodulation (Abd-Alla et al., 1999). Our findings show that none of the parameters examined were negatively affected by the heavy metals in composted textile sludge. When short-term adverse effects of sludge on symbiotic parameters or plant growth were observed, the effects were probable related to salts added to the soil with the sludge (Angle et al., 1992). Since salts are easily leached from soils, these adverse effects should diminish with time as rainfall or irrigation water leach salts from the root zone.
to examine the long-term effect of composted textile sludge in soils, particularly in relation to biological indicators of soil quality. Acknowledgements The authors are thankful to Dr. J.A.G. Silveira, UFC, Brazil, for providing the laboratory facilities, Bioland Co. for providing the textile sludge compost, and Dr. M.V.B. Figueiredo, IPA, Brazil, for her suggestions. R.T.R. Monteiro is supported by CNPq personal grant.
4. Conclusions References Composting can be an alternate technology for the management of solid textile mill sludge. This study verifies that the composted textile sludge was not harmful to growth, nodulation and nitrogen fixation of soybean and cowpea; however, proper consideration should be given to the metal content of textile wastes. Further field studies are required
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