Microbial activities in soil amended with sewage sludges

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1 Soil Science and Plant Nutrition ISSN: (Print) (Online) Journal homepage: Microbial activities in soil amended with sewage sludges Hiroyuki Hattori To cite this article: Hiroyuki Hattori (1988) Microbial activities in soil amended with sewage sludges, Soil Science and Plant Nutrition, 34:2, , DOI: / To link to this article: Published online: 04 Jan Submit your article to this journal Article views: 139 View related articles Citing articles: 22 View citing articles Full Terms & Conditions of access and use can be found at

2 Soil Sci. Plant Nutr., 34 (2), , 1988 MICROBIAL ACTIVITIES IN SOIL AMENDED WITH SEWAGE SLUDGES Hiroyuki HATTORI The National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan Received April 10, 1987 Microbial populations and enzyme activities in a Light-colored Andosol amended with sewage sludges were studied in relation to the decomposition of the sludges. Six kinds of sewage sludges were mixed with the soil at rates of 5 and 1Yo. Subsequently, the mineralization of C and N, numbers of soil microorganisms, and activities of soil enzymes were determined during an 8-week incubation period at 28~ The mineralization rates of organic C and N increased rapidly and reached maximum values within the first 3 days, but then decreased rapidly. Similar trends were observed for the number of bacteria and proteinase activity in the soil, which showed high correlation coefficients with the mineralization rates of C and N. The numbers of actinomycetes and fungi reached maximum values after 2 or 3 weeks of incubation and remained at the same levels thereafter. The amount of amino acid-n in 6 N HCI extracts of the soil amended with the sludges decreased markedly during the incubation period. These results suggest that proteinase-producing bacteria contribute significantly to the rapid decomposition of sewage sludge in the early days after the application and that actinomycetes and fungi contribute to the gradual decomposition after the end of the rapid decomposition. Key Words: sewage sludge, mineralization, soil microorganisms, enzyme activity. The percentages of sludge C and N mineralized in soil vary with the sludge type applied to the soil (MAGDOFF and CHROMEC 1977; SOMMERS et al. 1979; OBA and NGUEN 1981; PARKER and SOMNERS 1983), as well as with the amount and the composition of the organic matter in the sludges (HATTORI and MUKAI 1986). Since soil microorganisms play an important role in the decomposition of organic matter in or added to soil, the difference in the percentages of sludge C and N mineralized in soil among sludges may be also attributed to the difference in the microbial numbers and enzyme activities in the sludge-amended soil. Though some studies on the effect of the application of sewage sludges on microorganisms and enzyme activities in soil have been carried out (VARANKA et al. 1976; MITCHELL et al. 1978; FuJII and HIROKI 1982; FRANKENBERGER 1983; BONMATI et al. 1985), only few have been dealing with the relationship between the decomposition of sewage sludges and microbial numbers and their activities in soil (KuBoI et al. 1984). We previously studied the mineralization of organic C and N of several sludges in a Light-colored Andosol (HATTORI and MUKAI 1986). In this paper, the microbial populations and enzyme activities in the soil amended with sewage sludges at two 221

3 222 H. HATTORI application rates were determined, and attempts were made to clarify the role of soil microorganisms and enzymes in the decomposition of sewage sludges. The reason for the differences in the percentages of sludge C and N mineralized in soil among the sludges was then discussed in relation to the numbers of microorganisms and enzyme activities. MATERIALS AND METHODS Materials. The sludges and soil used in this experiment were described previously (HATTORI and MUI(AI 1986). The sludges were obtained from domestic (sludges A to E) and industrial (sludge F) sewage treatment plants. These sludges were air-dried and ground prior to use. The surface layer of the Tsukuba Light-colored Andosol was collected from a peanut field (texture, loam; CEC, 19.9 meq/100 g soil). The soil was air-dried and sieved (2 mm). The properties of the sludges and soil are presented in Table 1. Incubation procedure. The incubation procedure was described in detail previously (HATmRI and MUKAI 1986). Each sludge was mixed with portions of the soil at rates of 0, 1, and 5~,~ of dry matter on a dry soil basis. Water was added to the mixtures to reach a moisture equivalent of 60~ of the maximum water holding capacity, and subsequently the mixtures were incubated for 8 weeks in the dark at 28~ During the incubation, the amounts of COs and NHa evolved and the concentration of inorganic N in the soil were periodically measured (HATxORI and MUKAI 1986). The amount of sludge C and N mineralized in soil was calculated from the difference between the amount in the treated and untreated (control) soils. The forms of organic N in the soil with a 5~ sludge application and in the control soil were determined before and after the incubation by Bremner's method (BREMNZR 1965). Microbial and enzyme analyses. Microbial numbers and enzyme activities in the soil were determined at the end of 0, 3, 7, 14, 21, 28, and 56 days of incubation. Table 1. Properties of sewage sludges and soil used in the experiment. Sludge Coagulant ph Org.-C Org.-N (7o) (7o) A None B Ca(OH)2, FeCI C Ca(OH)2, FeCl D Polymer E Polymer F Polymer , 1 Soil

4 Microbial Activities and Decomposition of Sewage Sludges 223 Table 2. Enzymes assayed in this experiment. Enzyme Substrate Buffer ph Reaction time Reference Proteinase Casein Tris-HC h LADD and BUTLER 1972 fl-aeetyl- PNP ~-N-acetyl- g- Acetate min KANAZAWA and glueosaminidase D-glucosaminide TAKAI 1976 Urease Urea Phosphate h TABATABAI 1972 Amylase Starch Acetate h ROSEaGE 1978 Cellulase CM b -cellulose Acetate h ROnERGE 1978 fl-glucosidase PNP ~-glucoside Acetate rain HAYANO 1973 Phosphatase PNP ~-phosphate Tris-maleate rain TABATABAI and BR~MNER 1976 Phosphatase PNP ~-phosphate Diethanolamine rain TA~ATABAI and BREMNER 1976 p-nitrophenyl, b carboxymethyl. The numbers of aerobic bacteria, actinomycetes, and fungi were determined by the soil dilution plate method (Soil Microbial Society of Japan 1975). Enzymes were assayed under the conditions shown in Table 2. The enzyme activities were measured by the following procedures. Moist soil (0.5 g) was placed in a test tube and treated with 0.1 ml of toluene. After 5 rain, the treated soil was supplied with 2 ml of buffer (0.2 ~) and 1 ml of substrate and incubated with shaking at 30~ and then the amount of the reaction product was measured. Phosphatase activity was determined at ph 6.5 and at ph 10.0, and the enzyme reaction was stopped by the addition of 8 ml of ethanol (HAYANO 1973) instead of 1 ml of 0.5 M CaC12 (TABA- TABAI and BREMNER 1969). RESULTS AND DISCUSSION 1. Mhwralization of sludge C and N in the soil The amounts of CO2 evolved from the sewage sludges per day changed according to similar patterns irrespective of the sludge type and application rate (Fig. 1). They increased rapidly and reached maximum values within the first 3 days. Then they decreased rapidly over the 2 week period of incubation, and continued to decrease slowly thereafter. The amounts of N mineralized per day were high at 3 days after the incubation, and they decreased markedly at 1 week, except for sludge F. These changes in the pattern were similar to those in the amount of COa. Thus some of the organic C and N present in the sludges was mineralized rapidly during the first 2 weeks. Similar results were obtained by many other researchers (e.g. HSlEH et al. 1981). The total amounts of CO2 evolved and N mineralized over a period of 8 weeks

5 224 H. HATTORI 0 8 SLudge 4 ' A -~ ",',,... B ~' 2 i... C ~1,0 "~ O,,~ 0,6".~,,0,4 SLudge 1% loo" -~0,2 ~ 75[ ~= Z Fig. 1. Mineralization rates of C and N in sewage sludges (A F) applied to soil at the rates of 5 and 1~. varied with the sludges. For example, the total amount of CO2 evolved from sludge F during the incubation period was more than 10 times that from sludge A at both application rates. As described in a previous report (Hha~rom and MU~AI 1986), the percentage of sludge organic C mineralized in soil was negatively correlated to the sum of the inorganic matter and lignin contents of the sludge, and the percentage of N mineralized was positively correlated to the crude protein content of the total sludge organic matter. Therefore the amount of CO2 evolution tended to be higher from the soil amended with sludge which contains a large quantity of organic matter. 2. Microbial nwnbers in soil amended with sewage sludges The number of bacteria in the soil amended with any sludge at 5~o rate reached maximum values at 3 days after the incubation and decreased rapidly thereafter. Then the number increased again at 3 weeks after the incubation (Fig. 2). Since the number of bacteria in the control soil also increased at 3 weeks after the incubation, the increase may be due to other factors than the application of the sludges. The number of bacteria in the soil with lyo sludge application reached maximum values at 3 weeks after the incubation, and decreased thereafter. After 8 weeks of incubation the number was

6 Microbial Activities and Decomposition of Sewage Sludges 225 o 12[~ SLudge 5% 2 =~ iole J~"~X... B -----_---- c Studge 1%..... a A -----,,,..11,, \...@,)~,~ x : Conic0[ O0-89 ' 4 " 8 O o 6 4 E 2 o 0 y 9 x...a..~ i,~:-~k ~. ',.:f-/ "" ;~-_ 4 i',,tl'4~/ \\ \ -~--:T~-... '4 '8 o~ / / ~ = :... RZ-?-: ; 2 " 4 8 9[,,, 4,0 F /x o 7t /" X IUI- r'~" "~-'--.~= i,, " o,sy/,7~0,4 2 4 ~O ~ Z 2 _ T_ =~ g Fig. 2. Changes in microbial numbers in soil amended with sewage sludges (A-F) at the application rates of 5 and 1 ~. almost equal to that in the control soil. The change in the number of bacteria in the soil amended with sludges at 5~ rate was similar to the change in the amounts of COs evolution and N mineralization except for the increase after 3 weeks. Furthermore the bacterial numbers after 3 days were larger in the soil with higher rates of CO~ evolution than in the soil with lower rates of CO~ evolution. The number of actinomycetes and fungi increased only slightly in the first 3 days of incubation when the mineralization rates and bacterial number showed maximum values. But the number increased to reach maximum values after 2 or 3 weeks of incubation, and did not decrease significantly thereafter9 The number of actinomycetes in the soil amended with sludge F at 5~ rate decreased markedly after 3 weeks of incubation, while the number for the soil with 1 ~o application remained high. The amount of NH4-N increased rapidly in the soil amended with sludge F at 5~ rate due to the decomposition of organic N, and a quantity of 3.2 mg NH4-N/g soil accumulated in the soil after 2 weeks of incubation. Therefore the ph value of the soil increased above 8.0 after 2 weeks of incubation (Fig. 3). The decrease in the number of actino-

7 226 H. HATTORI ph 9,0 SLudge 5% ph 9,0 SLudge I%.... A 8,0 7, C.... E) E *, F.... 7,0 x: Control 6,0 " -"-?.~ "~ "" = =-"-"... 2"-'2"5.'_" 5,D 510 4,0 0 Fig. 3. Changes in ph values in soil amended with sewage sludges (A-F) at the application rates of 5 and 1~. mycetes may be related to the increase of the ph value. The ph value in the soil amended with sludge F at 1~ rate decreased after 4 weeks of incubation because nitrification followed the accumulation of NH4-N. The increase in the number of actinomycetes and fungi following the increase in the number of bacteria was observed in the Light-colored Andosol amended with limed sewage sludge in a lysimeter experiment (FuJII and HIROKI 1982). The results suggest that the succession occurred in the soil amended with sludges, regardless of both the sludge type and application rates. The succession suggests that bacterial populations in soil contribute to the rapid decomposition of sewage sludge in soil during the early period of incubation and that the populations of actinomycetes and fungi contribute to the gradual decomposition after 3 days of incubation. KUBOI et al. (1984) also observed in a lysimeter experiment that the rapid increase of soil CO, level was coupled with that of the bacterial number. 3. Enzyme activities in soil amended with sewage sludges Enzyme activities are generally considered to be a more direct expression of soil biological activity, or of the activities of specific processes of nutrient cycling and organic matter turnover, than measurements of microbial numbers (LADD 1986). The changes in the activities of 8 enzymes in the soil amended with sludge at 5~ application rate are shown in Fig. 4, while those at 1 ~ application rate, which were similar to those at 5~, are not shown. Proteinase activity increased rapidly after sludge application, and after 3 or 7 days the activity reached maximum values in all the treatments. Thereafter the activity continued to decrease. Since the newly synthesized proteinase is considered to be lost rapidly (NANNIPIERI et al. 1979), it is suggested that the proteinase was produced by microbial populations which proliferated, possibly bacteria, immediately after the application of sewage sludges and would be short-lived in soil. The changes in the

8 0 E =L >; Microbial Activities and Decomposition of Sewage Sludges 227 /~ Proteinase --- BA 4... C I;% ~2;_.~.&\. - - F Z [\. o, i ~, ~. 0 ~ 40[ Urease o = I CetLutase ~ " p-acetytgtucosaminidase "',\. \ z ~ a o 2~j e /" ~. --~ 4 8 zs L/~'~, P~osphatase (ph 6.5)zs L Phospllotase (ph 10.0~ S..... / r, ~ O L,,, o z 4 8 o Fig. 4. Changes in enzyme activities in soil amended with sewage sludges (A-F) at the application rate of 5~. pattern of the proteinase activity were similar to those of C and N mineralization and of the numbers of bacteria. The sludges contained 40-50~ amino acid-n out of the organic N content (HATTORI and MUKAI 1986), and the amount of amino acid-n in the soil amended with the sludges decreased markedly during the incubation period (Table 3), particularly in the case of sludge F which showed a high proteinase activity. These results suggest that proteinase contributes significantly to the decomposition of the protein in the sludges. Cellulase, /~-glucosidase, /3-acetylglucosaminidase, and amylase activities were highest in the soil amended with sludge E. Cellulase complex can be induced in many microorganisms and be synthesized in the presence of cellulose or carbohydrates (ALEXANDER 1977). The high activity of ceilulase in the soil amended with sludge E is compatible with this assumption since sludge E contained more cellulose than the

9 228 H. HATTORI Sludge Table 3. Changes in the amounts of different forms of hydrolyzed N during 8-week incubation of soil amended with sludges at 5~o application rate. ('in mg/100 g dry soil) Amino acid-n Amide- and NH4-N Hexosamine-N Unidentified-N X Y X Y X Y X Y A B C D E F Control X, N content before the incubation; Y, N content after the incubation. other sludges (HAa-rORI and MUKAI 1986). I-IAYANO and TUBAKI (1985) indicated that fungi are the primary source of/3-glucosidase in tomato field soil. Since the number of fungi increased considerably in the soil amended with sludge E, it is likely that /3-glucosidase activity would be highest in this soil. In spite of the increase of the p-acetylglucosaminidase activity, the amount of hexosamine-n in the soil tended to increase during the incubation period (Table 3). Presumably hexosamine may be synthesized as a component of the fungal and bacterial cell wall after sludge application. Urease and phosphatase (ph 10.0) activities were high in the soil amended with limed sludges (B and C). On the other hand, phosphatase (ph 6.5) activity was high in the soil amended with the other sludges. EIVAZI and TABATABAI (1977) indicated that acid phosphatase was predominant in acid soils and that alkaline phosphatase was predominant in alkaline soils. Urease activity in soils can be increased by the addition of liming materials (BREMNER and MULVAI~rEY 1978). The ph values in the soil amended with sludges B and C at 5% application rate were above 7.0 immediately after the application (Fig. 3). Urease and phosphatase activities in the soil may become high due to the high ph value. Organic phosphorus of sludge F was appreciably mineralized in the Light-colored Andosol incubated for 4 weeks at 28~ whereas the amount of organic phosphorus in the soil amended with the other sludges remained unchanged during the incubation period (HATTORI 1986). Thus the increase of the phosphatase activity was not necessarily related to the decrease of the content of organic phosphorus in the soil. All the enzyme activities measured increased after sludge application, and the degree of the increase is considered to be affected by the organic matter composition and ph values of the sludges. But the role of the enzyme activities in the decomposition of the sludge organic components was not clear except for proteinase.

10 Microbial Activities and Decomposition of Sewage Sludges Role of soil microbial activities in C and N mineralization of sewage sludge To evaluate the relationships between the mineralization rates of C and N, numbers of microorganisms and enzyme activities, correlation coefficients were calculated using the data of the soil amended with sludges at the rate of either 5Yo or 1 ~ (Table 4). The mineralization rates of C and N showed high correlation coefficients with the proteinase activity at both application rates, and also with the number of bacteria at 5Yo level. The correlation coefficient between the number of bacteria and proteinase activity was also high (r=0.71 and 0.49 at 5 and lyo rates, respectively). These results suggest that proteinase excreted by bacteria significantly contributes to the rapid decomposition of sludges in the early days after the application. The decrease of amino acid-n in the soil during the incubation period supports this assumption. Though phosphatase (ph 6.5) itself does not participate in the mineralization of organic C and N directly, it showed a high correlation coefficient with the mineralization rates of C and N. Phosphatase (ph 6.5) exhibited a high correlation coefficient with the number of bacteria (r=0.44 and 0.41 at 5 and lyo rates, respectively). Presumably the phosphatase originated mainly from bacteria, and as a result, the activity would be related to the C and N mineralization rates. The large variation of the organic matter content among the sludges examined may have affected the microbial numbers and enzyme activities in the soil. But the Table 4. Correlation of mineralization rates of sludge organic matter (C and N) with microbial populations and enzyme activities in soil amended with sewage sludges at application rates of 5 and 1%. Sludge 5Yoo (n=36) Sludge lyoo (n=36) Mineralization rate Mineralization rate C N C N Bacteria 0.91"** 0.65*** 0. 35* 0. 39*** Actinomycetes * Fungi Proteinase 0.87*** 0.78*** 0.78*** 0.84*** /LAcetylglucosaminidase Urease * Cellulase 0.49** /LGlucosidase ** 0. 39* Amylase * 0.26 Phosphatase (ph 6.5) 0. 51" 0.52** 0.52** 0.64*** Phosphatase (ph 10.0) *, **, *** Significant at 5, 1, and 0.1~ levels, respectively. The data on mineralization rates, microbiap populations, and enzyme activities in soil amended with sewage sludges (A-F) after 3 days, 1, 2, 3, 4 and 8 weeks of incubation were used.

11 230 H. HATTORI 500 "O 7,0 "o 4,0 g o 300 i00 ~, 3d e A 0 D. o,s > cd o 0,25 oom~ ~o" A O o 9 9 [] A O 1,5 ; Proteinose oetlvity, #mot tyrosine equiv./g soil/h O.Qo~ N 20 2~ O L.. C E z 1D. o 0 A 0,5 ~ i',5 ~ ~ Proteinose octivi,y, /~mol tyrosine equiv./g soil/h o Fig. 5. Relationship between proteinase activity and mineralization rates of C and N in soil amended with sewage sludges at 5,~ application rate. Sludge: zx, A; I,, B; El, C; E, D; 9 F. number of microorganisms in the soil amended with sludge F at 1~ rate (organic-c: 4.9 mg/g soil) was higher than that in the soil amended with sludge A at 5~ rate (organic-c: 7.1 mg/g soil). Similar results were obtained for the proteinase activity. Therefore, it appears that the quality as well as the amount of organic matter in sewage sludge applied to soil affects the number of microorganisms and proteinase activity in the soil. 5. Conclusion The number of microorganisms and the enzyme activities measured increased by the application of sewage sludges. Among them, the bacterial number and proteinase activity changed according to a similar pattern to that of the mineralization rates of C and N. As shown in Fig. 5, the amount of CO2 evolved and N mineralized from the sewage sludges in soil tended to increase when the proteinase activity in soil increased irrespective of the sludge type. The amount of amino acid-n also decreased markedly during the incubation. These results suggest that the protein in the sludges is decomposed rapidly by proteinase-producing bacteria after the application. We reported that the rate of sludge N mineralized in soil for 8 weeks was related to the crude protein content of the total sludge organic matter (EIVAZI and TABATABAI 1977). The difference in the amount of crude protein in sewage sludge may have affected the number of bacteria and the proteinase activity in the soil amended with the sludge, hence the difference in the mineralization rate among different types of sewage sludges.

12 Microbial Activities and Decomposition of Sewage Sludges 231 REFERENCES ALEXANDER, M. 1977: Introduction to Soil Microbiology, 2nd ed., p. 160, John Wiley & Sons, Inc., New York BONMATI, M., PUJOLA, M., SANA, J., SOLIVA, M., FELIPO, M.T., GARAU, M., CECCANTI, B., and NAN- NIPIER L P. 1985: Chemical properties, populations of nitrite oxidizers, urease and phosphatase activities in sewage sludge-amended soils. Plant Soil, 84, BREMNER, J.M. 1965: Organic forms of nitrogen. In Methods of Soil Analysis, Part 2, Ed. C.A. Black et al., p , American Society of Agronomy, Inc. Publishers, Madison BREMNER, J.M. and MULVANEY, R.L. 1978: Urease activity in soils. In Soil Enzymes, Ed. R.G. Burns, p , Academic Press, New York EIVAZI, F. and TABATABAI, M.A. 1977: Phosphatases in soils. SoilBiol. Biochem., 9, FRANKENBERGER, W.Z., Jr., JOHANSON, J.B., and NELSON, C.O. 1983: Urease activity in sewage sludgeamended soils. Soil Biol. Biochem., 15, FtJJii, K. and HIROKI, M. 1982: Microflora in soils amended with sewage sludge. In Proceedings of International Symposium on Land Application of Sewage Sludge, p , Association for Utilization of Sewage Sludge (ed.), Japan HATTORI, H. 1986: Forms of phosphorus in sewage sludges and their transformations in soils. Res. Rep. Natl. Inst. Environ. Stud., Jpn., 93, (in Japanese with English summary) HATTORI, H. and MUKAI, S. 1986: Decomposition of sewage sludges as affected by their organic matter composition. Soil Sci. Plant Nutr., 32, HAYANO, K. 1973: A method for the determination of fl-glucosidase activity in soil. Soil Sci. Plant Nutr., 19, HAYANO, K. and TUBAKI, K. 1985: Origin and properties of/~-glucosidase activity of tomato-field soil Soil Biol. Biochem., 17, HSIEH, Y.P., DOUGLAS, L.A., and MoTxo, H. 1981: Modeling sewage sludge decomposition in soil: I. Organic carbon transformation. J. Environ. Qual., 10, KANAZAWA, S. and TAKAI, Y. 1976: A method for the determination of fl-acetylglucosaminidase activity of soil. J. Sci. Soil Manure, Jpn., 47, (in Japanese) KUBOl, T., HIROKI, M., HATTORI, H., and Fuji1, K. 1984: Relationship between soil air composition and microbial populations in a soil amended with sewage sludge, Res. Rep. Natl. Inst. Environ. Stud., Jpn., 68, (in Japanese with English summary) LADD, J.N. 1985: Soil enzymes. Iu Soil Organic Matter and Biological Activity, Ed. D. Vaughan and R.E. Malcolm, p , Martinus Nijhoff/Dr W. Junk Publishers, Netherlands LADD, J.N. and BUTLER, J.H.A. 1972: Short-term assays of soil proteolytic enzyme activities using proteins and dipeptide derivatives as substrates. SoilBiol. Biochem., 4, MAGDOFF, F.R. and CHROMEC, F.W. 1977: Nitrogen mineralization from sewage sludge. J. Environ. Sci. Health, A12, MITCHELL, M.J., HARTENSTEIN, R., SWIFT, B.L., NEUHAUSER, E.F., ABRAMS, B.I., MULLIGAN, R.M., BROWN, B.A., CRAIG, D., and KAPLAN, D. 1978: Effects of different sewage sludges on some chemical and biological characteristics of soil, J. Environ. Qual., 7, NANNIPIERI, P., PEDRAZZINI, F., ARCARA, P.G., and PIOVANELLI, C. 1979: Changes in amino acids, enzyme activities, and biomass during soil microbial growth. Soil Sci., 127, OBA, Y. and NGUEN, Q.L : Decomposition of sewage sludge in soil (I) Evolution of carbon dioxide from seven soils with two kinds of sludge added under aerobic condition. J. Sci. Soil Manure, Jpn., 52, (in Japanese) PARKER, C.F. and SOMMERS, L.E. 1983: Mineralization of nitrogen in sewage sludges. J. Environ. Qual., 12,

13 232 H. HATTORI ROBERGE, M.R. 1978: Methodology of soil enzyme measurement and extraction. In Soil Enzymes, Ed. R.G. Burns, p , Academic Press, New York Soil Microbial Society of Japan (ed.) 1975: Methods in Soil Microbiology (Dojo Biseibutsu Jikkenho), 469pp., Yokendo Co., Tokyo (in Japanese) SOMMERS, L.E., NELSON, D.W., and SILVIELA, K.J. 1979: Transformations of carbon, nitrogen, and metals in soils treated with waste materials, d. Environ. Qual., 8, TABATABAI, M.A. and BREMNER, J.i. 1969: Use ofp-nitrophenyl-phosphate for assay of soil phosphatase activity. Soil Biol. Biochem., 1, TABATABAI, M.A. and BREMNER, J.M. 1972: Assay of urease activity in soils, Soil Biol. Biochem., 4, VARANKA, M.W., ZABLOCKI, Z.M., and HINESLY, T.M. 1976: The effect of digested sludge on soil biological activity, d. Water Pollut. Control Fed., 48,