MSU LIBRARIES ——. ~— RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES wi11 be charged if book is returned after the date stamped below. TRANSFORMATIONS OF NITROGEN AS OBSERVED IN EXTRACTABLE FRACTIONS OE SOIL AND SLUDGE SYSTEMS BY Mabruk Shtaiwi Turki A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Science 1984 ABSTRACT TRANSFORMATIONS OF NITROGEN AS OBSERVED IN EXTRACTABLE FRACTIONS OF SOIL AND SLUDGE SYSTEMS BY Mabruk Shtaiwi Turki "Active" N fractions were measured at intervals up to 16 weeks in incubating sludge and mixtures with soil or sand. Quick uptake cm'rq by N-deficient oat seedlings was used to reveal short term changes in nutritional or toxic effects. "Active phases" were recovered in tn“) fractions: (1) direct extracts (suspensions) in 2N KCl and (2) the supernatant after autoclaving. Diffusable N83 and distil- lable NH3 were determined in both fractions. Nitrate (N03) and nitrite (NOE) were determined in direct extracts. In one series of experiments, volatile NH3 was trapped in acid, Kjeldahl N was determined in the residue after autoclaving ("inactive phase"), and protein N in the "active phase" was estimated by difference as N not otherwise accounted for. .At the lowest rate of sludge addition (15 T/ha), mineralization and rdtrification proceeded normally and ru was utilized normally by the assay seedlings at all stages Mabruk Shtaiwi Turki of incubation. .At higher rates (30 and 60 T/ha), utiliza- thmi of N was adversely affected by excess NH: during periods when NC; was depleted by reductive processes in the biosphere. During the first 8 weeks, while adequate organic energy sources were present, removal of N03 and N0; appeared to be due mainly to assimilatory reduction by adaptive heterotrophs. .After initially available energy substrates were exhausted, it appeared that nitrification was side- tracked at the N0; stage by reactions of HNO2 with polyphenols, resulting in chemo-immobilization. Exhaustion of organic substrates initially present was evidenced by rapid disappearance (lysis) of proteins, by rapid conversion to exchangeable (diffusable) and alkali labile (distillable) NH3, and by rapid evolution of volatile N83. In initially acid sludge systems, further decreases in pH were evidence that oxidation of NH3 by Nitrosomonas continued without interruption, even though neither NO2 nor N0; accumulated. Diffusable NH3 and non-diffusable but distillable NH3 fluctuated about a 1:1 ratio, indicating a common origin in reversible reactions characteristic of Maillard browning reactions between amino acids and sugars, or of aldol condensations of NH3 with polyphenols. Recoveries of N accounted for indicate that significant losses tn? bio-denitrification etween 11 to 66% of the added NH: -N. These volatilization lxosses decreased as the clay content of the soil increased. \Jolatilization rate increased with increasing rate of sludge iapplication and with repeated application of sludge. Curnoel, in other laboratory studies, reported that increasing temperature or soil pH caused an increase in NH3 volatilization from anaerobically digested sewage sludge lW.E. Curnoe. 1975. Ammonium volatilization from sewage sludge applied to the soil surface. M.S. Thesis. Univ. of Guelph, Guelph, Ontario, NIG2W1. 17 while increasing clay plus silt content in the soil resulted in decreasing PHL3 volatilization. Other factors cm? much lesser importance were the initial moisture content and relative humidity. He concluded that from 5 to 25% of the NHZ-N in applied sludge was lost as N83 in 40 days, depending on the levels of the five factors mentioned above. Lauer et a1. (1976) estimated that 61 to 99% of the ammoniacal N present in manure, surface applied in the field, was lost as NH3 over 5 to 25 days. Most of the losses occurred in the first few days following application. They suggested that the rate of drying was related most closely to the rate of volatilization. Terry et a1. (1978) reported that NH3 volatilization increased from 9 to 35% of added NH: -N as the air flow rate increased from 20 to 511 ml/min. However, incorporating the wastes into the surface 7.5 cm of the soil reduced NH3 losses (King, 1973; Sommers et al., 1979). Beauchamp et a1. (1978) reported that, during a five-day experimental period in May, 60% of the 150 kg aammoniacal N/ha applied in sludge was volatilized, while 56% (If 89 kg ammoniacal N/ha applied was volatilized in a seven-day experimental period in October. These losses from sludge compare favorably with losses of N83 from solid manure of 61 and 99% over time periods of 5 to 25 days in field experiments described by Lauer et a1. (1976). On the other hand, McGarity and Rajaratnam (1973) estimated that only 9.4% of the N applied as urine (118 ng/ha) was 18 volatilized during ea six-day period lJl‘a closed system in the field. Sommers et al., (1979) found that (1% (M5 the applied NHZ-N was lost through NH3 volatilization in Kokomo sludge. They concluded that the loss of added N as NH3 was low, because no air was flowing over the soil surface. However, higher N33 losses were observed where 90 T/ha of sludge was applied all at once or split into two 45 T/ha of sludge applications in soils incubated at 300C and in soils inoculated with fungal mycelium. They suggested that deni- trification and/or immobilization are the major mechanisms for removing N from the nuneral pool in soil treated with sewage sludge. In more recent studies, Feigin et a1. (1981) reported that about 17% of the tagged, mineral 15 N in solution and 124% of the effluent-tagged ammonium-ISN were lost, éapparently through both denitrification and volatilization. They concluded that simultaneous application of C and N in Izhe sewage effluent was probably responsible for the Iincreased losses of N through denitrification in the Gaffluent-tagged ammonium-N treatment. The losses of tagged ifertilizer-N (ammonium-sulfate-ISN) by denitrification were relatively low and the applications of the sewage effluent f N20 were low relative to N not accounted for. In a number of studies, estimates of denitrification losses from sludge applications, based on N not accounted for, have ranged up to 38% of the applied N (Epstein et al., 1978; Kelling et al., 1977b; King, 1973; Ryan and Keeney, 1975). Where volatilized DHL3 was run: measured, these estimates involved a subjective evaluation by the authors of the relative importance of experimental and environmental 23 factors that might have favored denitrification over volatilization of NH3. The above estimates for denitrification losses from sludge compare with estimates of denitrification losses from fertilizer N that range from 0 to 70% (Craswell, 1978; Kissel and Smith, 1978; Kowalenko, 1978; Rolston et al., 1976, 1979). Losses (ME fertilizer DJ from some irrigated California soils, based on evolution of N20, ranged from 95 to 233 kg N/ha/yr (Ryden and Lund, 1980). Average losses of fertilizet' N, based (Ni numerous lysimeter studies, range from 15 to 20% (Nelson, 1982). Chemo-denitrification: In addition to biological cienitrification in soils, some workers have observed :Eormation of gaseous molecular nitrogen and/or gaseous <3xides of nitrogen in soils under conditions not conducive tr) bio-denitrification (Nelson auui Bremner,1969; Reuss and Smith, 1965). These losses of N have often been associated with accu- mUlation of N03, and several studies have provided strong E>rsesumptive evidence that significant gaseous loss of ‘a£>E>lied N can occur through chemical reactions of NO2 formed ‘33? nitrificaticni of NH: and NHZ-forming fertilizers in a5331r to soils treated with a nitrification inhibitor, and the eamount of N20 emitted is related to the NH: -N nitrified. Nelson (1982) has reviewed the evidence concerning CJIemical reactions of NOS as pathways for N loss from soils. fie' summarized major mechanisms that may play a role in Chewe-denitrification as follows: 1) Self-decomposition of nitrous acid in acidic and neutral soils under aerobic and anaerobic conditions to produce nitric oxide (NO) and nitrogen dioxide (N02) (Nelson and Bremner, 1970). 2) Reaction with organic matter groups such as phenolic groups in lignin, lignin degradation products and humic substances to produce nitrous oxide (N O) and DJ. These reactions are most rapid Lelow pH 5.0 but have been observed in soils at pH 7.8. An important consideration is that water films surrounding colloidal surfaces in soils are often 100 times more acidic than in a bulk soil/water slurry. '47-.- r- 1'":- 1"“5—2‘7‘?““'Tf 25 3) Reaction of nitrouszacid WiEQ transition metal cations such as NM] or Fe (Wullstein and Gilmour, 1964). 4) Reaction of nitrite with ammonium or hydro- xylamine (Allison, 1963). 5) Reactions of nitrous acid with compounds containing free amino groups (Smith and Chalk, 1980). 6) Reaction of nitrous acid with clay minerals (Wullstein and Gilmour, 1964; Mortland, 1965). The reactions cited are really reactions of undisso- ciated nitrous acid (HNOZ) and are favored by acid pH. How important they are in nature is not known. The ones that are most likely are those between HNO2 and phenolic compounds (lignin, or its degradation products, and soil riumic substances). Effects Of Sludge On Crop Yields Beneficial Effects The value of sewage effluents, sludges and composts as s<3urces of nutrients for production of crops has been assessed in numerous studies (Barrow, 1955; Bear and Prince, 1947; Day and Tucker, 1958; Duggan and Wiles, 1976; Fraps, 1932; Garner, 1962, 1966; Mitchell et al. 1978a; Muller, 1929; Noer, 1926; Stucky and Newman, 1977; Terman et al., 153'7:3). Beneficial effects of sludge applications are most Often related to N and/or P content (King, 1981; Mays et all ~ , 1973). The ratio of N:P:K in a typical municipal SSJ‘LJCSge is about 11:8:1 (Sommers, 1977). Thus, K is often _ “r-e—“e; Inf-.- ...- ‘r‘ -" a" 26 deficient and may limit crop response, unless supplemental fertilizer is used to give a favorable balance of nutrients (Bunting, 1963; Sikora et al., 1980; Vlamis and Williams, 1961). Much of the N in sewage effluents may be available to crops in the first season (Day and Kirkpatrick, 1973; Day et al., 1962a,b). In sludges, however, only a third to one- half of the N is directly available. The remainder is organic, of which 10 to 30% decomposes the first year after being incorporated into soil. The rate of decomposition and the release of available N decreases in subsequent years (Hinesly et al., 1979; Miller, 1974; Milne and Graveland, 1972; Peterson et al., 1973). When sludges are composted with added carbonaceous wastes, the availability of N may be further reduced by immobilization (Bunting, 1963; Mays et al., 1973; Sikora et al., 1980). Except where the availability of N is reduced by immo- bilization or where nutrient imbalances are not corrected by Supplemental fertilization, the availability of N and P in SGi’woage effluents and sludges compares favorably with COmmercial fertilizers (Boswell, 1975; Day et al., l962a,b; Coker, 1966a,b). Crop responses may be particularly favor- able in soils where soil pH and fertility levels are low (Sheaffer et al., 1979), and during seasons of relatively Llr“favorable weather conditions (Hinesly et al., 1979). Due to continuing release of nutrients as waste organics Gee(Jmpose, residual benefits from sludges or sludge composts 27 continue over longer periods of time than where equivalent amounts of DJ and P are applied as mineral fertilizers (Hinesly et al., 1979; Sikora et al., 1980). Sludges may contain substantial quantities of heavy metals (Berrow and Webber, 1972; Lunt, 1953; Page, 1974). A number of these are essential micronutrients (Cu, Fe, Mn, Zn). Boron, another essential nutrient is also present in varying concentrations, depending upon the source of the sludge. In certain situations, where one or more of these is deficient in the soil, it is possible that specific micronutrient responses may contribute to increased yields (of crops where sludges are applied (Dowdy and Larson, 1975). Eiowever, as is evident from literature cited earlier, most (direct comparisons (HE sludge applications with commercial :fertilizers indicate that the primary yield response is to the N supplied in sludge or, less frequently, to P. Adverse Effects The response of crops to sludge applications varies “'itzh source of sludge, rate of addition, plant species, soil t)?£>e, weather conditions, and management practices. An if“£>cmtant factor is the time allowed for sludge to equili- br‘ate with the soil before a crop is planted. In literature encountered in the present review, OE)‘Zimum yield responses have been reported for rates ranging frOm 7.5 to 180 T/ha (Hinesly et al., 1972, 1979; Kelling et a1 ~ , 1977a,b; Milne and Graveland, 1972; Sheaffer et al., 1979; Vlamis and Williams, 1961). However, the efficiency 28 of N removal from sludge by crops decreases with increasing rate of application (Coker, 1966a; Kelling et al., 1977a; King and Morris, 1972a; Stewart et al., 1975). Also, as sludge inputs are increased, there is an increased possi- bility that crops may be injured during germination or in seedling stages by excess soluble salts, or that excessive uptake of heavy metals may result in toxic effects on the crops themselves or on livestock or humans that consume them. Soluble salts: Adverse effects of very high levels of sludge addition (150 T/ha or more) on yields of crops such as corn and rye have been ascribed to soluble salt effects (Cunningham et al., 1975; Hinesly et al., 1972). No data on soluble salts were presented, but yield reductions were .associated with dry growing conditions. Excessive concentrations of soluble salts are parti- cularly damaging to germinating seedlings. Reductions in fc>rage yields of rye, sorghum and sudan grass were observed if! the first season after application at rates of 30 and 60 IU/Tia (Kelling et al., 1977a). Soil type was a factor, since ‘OEDtLimum yields were obtained with 7.5 T/ha on a silt loam ‘SCDi.1 and at 15 T/ha on a well-drained sandy loam. Soluble salt effects can be reduced by allowing time for leaching and equilibration with soil after addition of S’l-kldge before planting a crop. Reductions in yields of S(Difghum and sudan grass seeded shortly after incorporation C>ff sludge were ascribed to poor germination (Sabey and Hart, 29 1975). Where wheat was seeded three months after similar rates of sludge were incorporated, yields were increased. Crops such as lettuce appear to be particularly susceptible to adverse effects of sludge (John and VanLaerhoven, 1976), whereas coastal bermuda grass appears to be tolerant to high levels of both soluble salts and heavy metals (King and Morris, 1972a; Touchton et al., 1976). Heayy metal toxicity: Much recent work has focused on effects that heavy metals in sludge can have on plant yields (Berrow and Webber, 1972; Page, 1974; Webber, 1972). Under certain conditions, these substances can be taken up in abnormal concentrations by plants and cycled into food <3hains leading to animals and man. The fate of heavy metals in sludges after incorporation in soil is an area of much recent discussion and research (Dowdy and Larson, 1975; I1ecules are often taken up more readily than mineral forms foh the same element. On the other hand, complexes formed “'j;‘:11 organic ligands of high molecular weight will not be aVaIilable, except as the macromolecules are broken down by e"23/me action or by changes in pH, redox potential or other e» . r“’:1ronmental factors. 30 There is a high degree of variability between sludges, not only in total metal content but in form (Sommers, 1977). Silveira and Sommers (1977) found that the distribution of Cu, Zn, (Bi, and ER) in exchangeable, DTPA-extractable, and HNO3-extractable fractions is not similar for different sludges. This variability also occurs over time at a given treatment plant, and seems to be a function of digester efficiency and the composition of the incoming sewage. Further changes will occur after sludges are applied on the land, and these changes will be influenced strongly by soil type, management and weather (John and vanLaerhoven, 1976). There is general agreement that Cu, Ni and Zn pose the greatest threat to crop yield and quality (Cunningham et al., 1975; Lunt, 1953; Merz, 1959; Mortvedt and Giordano, 1975). Of these, Cu appears to be about twice as toxic as Zn, while Ni may be as much as eight times as toxic (Webber, .1972). High concentrations of Cd and Pb may be detrimental tc> plants but, along with Cu and Zn, they are also a LPc>tential hazard to animals, including man, in the food Chain (Chaney, 1973; Haghiri, 1973; Sommers, 1977; Silveira and Sommers, 1977). Several soil variables may influence the toxic effects C)ff trace metals. These include organic matter content, kind Eir‘<3 amount of clay, and pH (Bunzl et al., 1976; Gadd and Griffiths, 1978; Sinha et al., 1978). fl m4A§-’L'Av 3-1]; ‘3‘; l‘ L 31 Interactions of Sludges With Soil Systems Physico-chemical Effects It is generally agreed that sludge applications improve <3hemica1 and physical conditions in soils (Duggan and Wiles, 1976; Evans, 1968; Hinesly et al., 1979; Hortenstine and I%othwell, 1968; Mays et al., 1973; Muller, 1928; Terman et aal., 1973). Increases in moisture holding capacity and (zation exchange capacity have been shown, as well as (Secreases in bulk density and compression strength. These eeffects may be temporary, depending upon the rate of sludge aaddition and the extent to which soil organic matter levels eare increased residually. An important role of the residual humified organic tnatter is to retain and stabilize mineral nutrients and heavy metals and moderate their availability to plants (Bloomfield et al., 1976; Nishita et al., 1956). The stabilizing effect of sludge applications depends upon the nature of complexing organic matter in the sludge and its stage of decomposition (Dowdy and Larson, 1975). Soil pH Relationships Additions of sludge to acid soils may have the effect (of raising pH. In very acid soils this may tn; a primary factor increasing crop yields (Sheaffer et al., 1979; Terman Get al., 1973). In less acid to neutral soils, particularly isoils that are poorly buffered, additions of sludge may lower pH temporarily because of the acidity produced during t1!r-.§.- t 2'. °_—" 'fi- “A o 32 nitrification of DJ introduced with the sludge (Hinesly et al., 1972, 1979; John and VanLaerhoven, 1976). Soil pH is an important factor affecting the availabi- lity and potential toxicity of heavy metals. In general, ssolubility and uptake by plants increases with decreasing pH (Cunningham et al., 1975; Dowdy and Larson, 1975; Mitchell eat al., 1978b; Page, 1974). Increasing pH reduces avail- éibility by favoring reactions that lead to insoluble {precipitates or complexes. Inverse relationships between soil pH and extract- eability and/or plant uptake of Cd, Mn, Ni and Zn from sludge liave been shown (Bloomfield and Prudeau, 1975; John et al., 1972; John and VanLaerhoven, 1976; Mahler et al., 1978). 'The last authors suggest that the solubility of Cd and Zn at higher pH are controlled by their carbonates or phosphates. Many investigators have reported success ix: reducing the solubility and/or toxicity of sludge-applied heavy metals by liming the soil (John and VanLaerhoven, 1976; King and Morris, 1972a,b; Terman et al., 1973; Webber, 1972). Bloomfield and Prudeau (1975) found that liming decreased extractable Ni and Zn, but had no effect on the solubility of Cu. This they ascribed to the higher affinity of Cu for organic matter (cf. Mitchell et al., 1978). Soil Microbial Populations Addition of sludge to soil normally stimulates the rapid development of a large and heterogenous population of Organisms. A number of major groups of microflora are 33 represented (bacteria, actinomycetes, fungi, algae), as well as various fauna, ranging from nanxa to macro forms (e.g. protozoa, nematodes, insects). The majority of soil organisms are heterotrophic, i.e., they derive energy and structural carbon from the decompo- :sition of organic substances. The heterotrophic biomass is {primarily responsible for mineralization and immobilization <>f N in soils (Jansson, 1971). Nitrogen mineralization is (iefined as the transformation of N from the organic state to inorganic forms (NH3 or NH: ). Nitrogen immobilization is defined as the transformation of inorganic N compounds (NHZ, NH3, N03 , NOE ) into the organic state through assimilation into cellular proteins and other nitrogenous products of microbial metabolism. The two processes work in opposite directions, breaking down and building up organic matter. Whenever moisture and temperature are favorable, mineralization and immobilization processes go on simul- taneously. A portion of the nitrogen in available organic substrates is assimilated into the cellular materials of the decomposing population. This recycling of substrate N into microbial cells and products has been referred to as the "internal N cycle" (Jansson, 1958). In later publications by the same author, a more descriptive term, "mineraliza- tion-immobilization turnover" (MIT), has been used (Jansson, 1971; Jansson and Persson, 1982). g ”3411' a... r- 34 If available substrates are high in carbohydrates relative to proteins, NH3 released by deamination of gproteins will be recycled closely. In addition, net immobi- .1ization of N already present in the external mineral N pool nmay occur. On the other hand, if available substrates are relatively high in proteins, NH3 released by deamination V9111 exceed the requirement of the heterotrophic population, earuj net mineralization (ammonification) will occur. The intensity of recycling through the heterotrophic Epopulation will change over time as carbon is respired as (302 from more readily decomposed substrates (e.g. carbohy- ccurs only as soils approach complete saturation. The energy content of raw sewage is reduced substan- tzially during treatment. Nevertheless, the sludge recovered .after treatment still contains a wide range of organic .substrates (Broadbent, 1973). At heavy rates of appli- cation, competition between important heterotrophic and autotrophic populations for mineral forms of hi can be intense. A differential effect on a given N transformation, due to toxicants in the sludge or to soil or management variables, can affect the final result in terms of environ- mental protecthmu or the output of useful plant products. The nature and intensity of probable interactions will be influenced by a number of soil variables, including organic matter content, kind and quantity of clay, moisture level and structural characteristics that affect aeration. Talburt and Johnson (1967) concluded that the sensi- tivity of microorganisms to toxic metal ions may vary. Some species are known to develop resistance by genetic or phenotypic changes, resulting in exclusion or metabolism of the toxic ions (Ashida, 1965). Metal-tolerant. organisms have been isolated from soils where high concentrations 37 occur naturally and from soils that have been polluted by heavy metals (Hartman, 1974). A few studies have investi- gated the influenme of metal in soils or sludge on N transformation and have reported somewhat mixed results. FVilson (1977) found that activity of nitrifiers was inhibited by sludge containing high levels of Cd, Pb, and Zrn Penni and Cornfield (1969) found that ammonification vvas not affected by 1000 ppm Cu or 100 ppm Cr, but 10,000 g>pm Cu greatly reduced ammonification. Quraishi and (Zornfield (1973) reported that addition of up to 10,000 ppm Cu stimulated N mineralization and nitrification during incubation of sandy loam soil treated with 200 ug/gm N as dried blood. The maximum stimulating effect was at 1000 ppm Cu. In contrast to this, Liang and Tabatabai (1977, 1978) found inhibitory effects on mineralization and nitrification from 19 trace elements added at a level of 5 umol/gm soil. Soil temperature, moisture status and pH markedly affect soil biological processes, hence affect sludge decomposition and N transformation. Miller (1974) concluded that soil temperature was (fine major factor affecting the rate (ME sewage sludge decomposition. He stated that the rate of decomposition of sewage sludge was largely independent of differences in soil texture or chemical properties. Soil moisture content did not affect the rate of sludge decomposition in a sandy soil, but saturated conditions reduced it in silt loam soil and almost completely stopped it in a clay soil. ‘ “uh—"l Wfiiinha'fl-m It" | h._ . I l ‘ . . 38 Terry et a1. (1979) found that initial soil pH in the range of 6.3 to 7.5 had little effect on the rate of sludge decomposition. Soil moisture tension in the range of -0.25 to -1 bar also had little effect. However, increasing soil temperature speeded up the decomposition rate of sewage sludge. In a follow-up study, Terry et a1. (1981) found that tzhe nitrification process was faster in sludge amended soil vvith initial pH 7.5 than at pH 6.0 or 6.3. The nitrifica- tion rate of sludge amended soil was increased more at soil Inoisture tensions of -0.25 and -0.5 bar than at -1.0 bar. Temperature had ea strong effect on the nuneralization of sludge-organic N and on immobilization of added inorganic N. Both mineralization and immobilization rates increased as temperature increased from 15 to 30°C. About 40% of added sludge organic N was mineralized in silt loam soil at 21°C after 168 days of incubation, but only up to 26% of added NH;;N was immobilized. '.-.' L- It. .)¢.- A»; h -..-.AA-:"n T MATERIALS AND METHODS The objectives of the research were to evaluate a quick uptake plant assay as a means for following changes in availability of N during the first four months after incor- [poration of sludges in soil, and to relate these changes in availability to changes in forms of mineral and organic N that might be considered to represent actively cycling 1 E"=-- = m ‘7‘, I3 jl ( l I l I 0 4 a 12 16 Figure #1. INCUBATION TIME (weeks) Dry weight of oat seedlings after contact with previously incubated sludge with soil or sand. 2 weeks' mixtures of 54 The yield depression associated with early stages of decomposition occurred also at the lowest rate of addition (15 T/ha) in sand systems, but not in soil. At this rate, the yield response in soil increased to a maximum at four weeks, then declined slowly as incubation time increased. The percent N in tops (Figure 2) was greater in the presence of sludge at all rates than in soil alone or sand alone. .At the beginning of incubation, N content was generally related to the level of sludge addition. However, it appeared that assimilation of N by the seedlings was affected adversely by changes that occurred during the first four weeks CHE incubation 1J1 sludge alone. A similar reduction in N content at four weeks occurred at the 60 T/ha rate in soil. Effects on growth and N content are both reflected in the data for nitrogen uptake (Figure 3). It is apparent that the performance of the assay crop was influenced mainly by inhibitory effects associated with early stages of incu- bathmn. At the lowest rate of sewage addition (15 T/ha), these inhibitory effects were greatly reduced in soil. Forms of N in Rooting Media At the beginning of each two—week plant assay period, a parallel series of similarly incubated samples was extracted with 2N KCl and then autoclaved in 2N KCl for 16 hours. The objective was txa identify "active" forms of N that might influence growth and N uptake by the oat seedlings. As a: procedural variation, direct extracts in 55 SOIL 40 D\ /C)/{3 t\ ’4‘ ;—/—A A if V + E j o &0« A 0 h 0 ’/\.__ —‘_. a Z 20 g ' SLUDGE 3 O o t/ha I- O 15 (lb: 2 10 A30 t/ha ' A 60 tlha D sludge alone I ‘1 o I g 12 16 INCUBATION TIME (weekS) .. C o O b 0 a. V 2 (“DJ 2.0-1 O) C2 '2 z: 1.0— o I I T I 4 8 12 16 INCUBATION TIME (weeks) Figure #2. Nitrogen concentration of oat seedlings after 2 weeks' contact with previously incubated mixtures of sludge with soil or sand. NITROGEN UPTAKE (mg/pot) 56 104 T I 0 4r 5 12 16 40" SAND INCUBATION TIME (weeks) SLUDGE O 0 tlha O 15 tlha 30 A so tlha A 60 ”In D sludge alone ‘ 20- /e NITROGEN UPTAKE (mg/pot) 1O-T + + I I fi ___l 4 8 12 16 INCUBATION TIME (weeks) 3. Nitrogen uptake by oat seedlings after 2 weeks' contact with previously incubated mixtures of sludge with soil or sand. 57 saturated CaSO4 (approximately 0.01 N) were compared with those in 2N KCl. Sludges alone: Data for NH: and "amino sugar-N" recovered from samples of sludge that had not been diluted with soil or sand are given in Tables 9 and 10 in the Appendix. Significant differences were associated with methods of extraction and interactions with incubation time. The data are presented graphically in Figure 4. Large quantities of NH: (and ”amino sugar-N" were released during incubation in forms that were directly extractable in 2N KCl and in saturated CaSO4. Approximately equal quantities of NHZ-N and amino sugar-N appeared in KCl extracts through the eighth week of incubation, after which N in the amino sugar fraction continued to increase while NH: declined. In CaSO4, quantities of NH: were significantly higher than in KCl in the second sampling and again after 16 weeks of incubation (Table 9), while recoveries of N in the amino sugar fraction were lower in the second and third samplings (Table 10). This indicates that some of the NH: that might otherwise have been diffusable in the presence of MgO was trapped at sites that were occluded by contraction of polymeric structures due to the high ionic strength of the 2N KCl. This trapped NH: was released by subsequent distillation in NaOH and thus appeared in the amino sugar fraction. mmoeeu EXTRACTED (ug/g) NITROGEN EXTRACTED (Halo) 58 alone. KCI A Autoclaved KCI a 7000. B 0 3 700 7 6000- o 6000; w y— 5000‘ 2 5000‘ c 4000' I): 4000‘ w 3000-1 2 3000.. m 2000- 0 2000- fl ---—"2 J. o d ” . c ,r 500 L- 500 ,x' z I O I I I I l I T ‘ 4 8 12 16 ° 4 8 12 16 INCUBATION TIME (weeks) INCUBATION TIME (weeks) 0.804 70001 8000‘ 5000- 4000- ...-.. NHI-N 3000‘ ,..-v".’ o—-——o -N 2 0 0 O- r Amho suger «5 7 500 / ’I T r I 1 ° 4 a 12 1s INCUBATION TIME (weeks) Figure *9 KHZ-N and amino sugar N fractions in sludge 59 At zero time, a much larger proportion of total NH and amino sugar-N was released by autoclaving than could be extracted directly with KCl. As incubation progressed, how- ever, the proportion directly extractable increased. After 16 weeks, approximately three-fourths of time N associated with amino sugars was directly extractable (Table 10). After four weeks of incubation, two-thirds of the N hydrolyzed by autoclaving (Figure 4) appeared in the amino sugar fraction. In later samplings, the hydrolysates contained similar quantities of N as NH: and in the amino sugar fraction (cf. Tables 9 and 10). Analyses for N0; and N0; were performed only in direct KCl extracts. Small amounts were found. These data will be discussed in a later section. Sludge mixtures: Data for diffusable N (NHZiQQ—9»NH3) in direct extracts of sludge mixtures with soil or sand are presented in Tables 11 and 12 in the Appendix. The results for soil systems are plotted in Figure 5 and for sand systems in Figure 6. As was the case for sludge alone, NH: directly extractable in CaSO4 was frequently higher than in KCl (Tables 11 and 12). Quantities released during incubation were directly related to rates of sludge addition and reached peak values at four to eight weeks (Figures 5 and 6). At the 60 T/ha rate, the maximum recovery in KCl at eight weeks was 550 ug/g in soil and 480 ug/g in sand. These values compare with a maximum at this time of 2700 + 4 60 70°“ KCI 500 - °°l /\ 100 80- 604 40.. 20- INCUBATION TIME (weeks) NH} (MN/0) 0 ‘d Q d N ‘ a 700-, CaSO, 500-1 ’3 a ‘ 100 \ 3 :l 30 SLUDGE V E? 0‘ 0 t/ha Z 60 015 t/ha A30 t/ha ‘°" A60 t/ha ZOdA o I l I I 4 8 12 10 INCUBATION TIME (weeks) Figure #5. Directly extractable NHZ-N in soil/sludge mixtures. 61 700 ‘ 500 ‘ 300 " NH: (HON/0) KCI I 4 s 12 INCUBATION TIME (weeks) 3? s g '00- SLUDGE 3 O 0 Ilhs Mr O 15 tlhs :2: A 30 ”be 16.60 t/ha o‘# I 700 - CaSO, 500 '- 300-1 m INCUBATION TIME (weeks) Figure #6. Directly extractable NHI-N in sand/sludge mixtures. 62 ug/g in sludge alone (cf. Figure 4). After this peak, recoveries declined more rapidly and to lower values at 16 weeks in soil systems (Figure 5) than in sand (Figure 6), or in sludge alone (Figure 4). Data for non-diffusable, alkali-distillable N (amino sugar fraction) in direct extracts are given in Tables 13 and 14 in the Appendix. The data are plotted in Figures 7 and 8. In general, patterns of change in this fraction were similar to those for directly extractable NH: (cf. Figures 5 and 6), but peak quantities at eight weeks were very much less in most systems (cf. Tables 13 and 14 with 11 and 12). Recoveries of the amino sugar fraction in CaSO4 were frequently very different than in KCl, sometimes more, some- times less (Tables 13 and 14). These erratic differences between the two extractants may reflect dynamic changes in polymeric or particulate fractions formed during decomposi- tion, and ill their susceptibility Ix) flocculathmi at the high ionic strength of the KCl. Flocculated materials removed by centrifugation might be expected to appear in the hydrolysate obtained later by autoclaving the residue. Data for NH: and the amino sugar fraction in hydrolysates obtained by autoclaving are tabulated in the Appendix (Tables 15 and 18). They are presented graphically in Figures 9 and 10. The materials subjected to autoclaving 1J1 @i’KCI had previously been extracted with 2N KCl. Thus, the NH: re- leased by autoclaving from soil systems (Figune 9) may be 63 300 KCI SLUDGE 1 O O t/ha I3 200 O 15 1111a 10 3 L F A. A30 I/ha < \ : A 60 I/ha 1- z 100 J (g a g 3 h z: 8°“ x < “‘3 .0. ). :1” O .. oz 40 . m - 55 20« e A 01' ‘ D I I I I I ° 4 6 12 1s INCUBATION TIME (weeks) 5001 C3304 m 0 .1 .. 3“ L A m 0 AP \ :5 z 1001 o a < 3 m .n l- : so A :K 3 A I.“ a 301 >' U) / .4 1—‘0 40« 0 .2. ~ I“ 2 E < 02:11Ill'li'rr r 4 8 12 INCUBATION TIME (weeks) soil/sludge mixtures. . Directly extractable amino sugar N 10 fractions in 64 3201 KCI 0.1 .1 A m a \ 40d :5 z 2 0 Q < 3 c: l- a: ><“ 160‘ m (D a >- (0 A I-‘O o z I.” 5 80-1 5 < o 0 1 1' 4 s 12 1s INCUBATION TIME (weekS) 300-1 320. CaSO‘ m -| A m a \ :E 2 g 3 SLUDGE ,_ m X ( . 0 t/ha 10 g C>15 Ilha >. a: “0‘ A 30 I/ha :l O A 60 t/ha o E I“ 2 E < a 80“ INCUBATION TIME (weeks) Figure #8. Directly extractable amino sugar N fractions in sand/sludge mixtures. 6S SOIL SLUDGE O 0 ”ha ‘ O 15 t/ha A 30 (lbs A 60 Ilha 700 - 500 -: 300 .1 100 , ' AUTOCLAVED 1111;010:110) ° 4 6 1'2 16 INCUBATION TIME (weeks) 500. SAND 300 . AUTOCLAVED NH:(00N/0) INCUBATION TIME (weeks) Figure #9. NHZ-N released by autoclaving from soil/sludge and sand/sludge mixtures. Figure #10. AUTOCLAVED AMINO SUGAR (”QM/9) AUTOCLAVED AMINO SUGAR (LION/9) 600' 300 100 75- 50 25 66 SO“. SLUDGE O O t/ha O 15 t/ha A 30 Ilha A 60 t/ha 100- 75-1 50- 25‘ 4 0 1'2 16 INCUBATION TIME (weeks) SAND A A ‘. O 4‘. O I :T 4 8 12 16 INCUBATION TIME (weeks) Amino sugar N fractions released by autoclaving from soil/sludge and sand/sludge mixtures. 67 + 4 (Figure 5). Patterns of change were essentially similar in compared directly with NH extracted directly in KCl that recoveries before and after autoclaving reached maximum values at four to eight weeks of incubation. The same was true for sand systems (cf. Figures 6 and 9). The autoclaved amino sugar fraction (Figure 10) peaked sharply at four weeks of incubation at the higher rate of sludge addition, as it had in sludge alone (cf. Figure 4). Autoclaved NH: (Figure 9) tended to peak 4 weeks later. This suggests that some of the "amino sugar N" in autoclaved fractions was converted sequentially to diffusable NHZ. It should be noted that no N was recovered from sand alone in any of the fractions of Figures 6, 8, 9, or 10. Nitrite plus nitrate: Both N03 and N0; were determined in direct extracts in 211 KCl. At zero time, NC; was encountered at concentrations ranging from traces to 12 ug N/g. This maximum concentration was found in soil amended with 60 T/ha sludge. Initial concentrations declined quickly, and only trace quantities ranging up to 3 ug N/g were found in any system during subsequent incubation. For this reason, N0; is not reported separately. The sum of N recovered as NOS plus N02 is reported for sludge and its mixtures with soil or sand in Tables 19 and 20 in the Appendix. The data are presented graphically in Figures 11 and 12. Nitrification was rapid in soil at the lowest rate of sludge addition (15 T/ha). In the first four weeks of 68 600 q SOIL SLUDGE O 0 tin: 50° " O 15 tlha A 30 t/ha ‘00 ‘ A 60 t/ha E] sludge alone 300 - 200 q 4’ «r 100 d N05+Nog (ugN/g) INCUBATION TIME (weeks) Figure #11. Nitrate plus nitrite in soil/sludge mixtures and sludge alone. 69 141 SLUDGE O 0 t/ha 12— O 15 t/ha A 30 I/ha A 60 t/ha D sludge alone A U) \ Z O) 2 V IN 0 Z q. 11') 0 Z 4... ‘ A 2d 0 O f O A T I A 4 8 12 16 INCUBATION TIME (weeks) :- “’3 \Y:-O-y\ 7 1...“ -lgur’e : (.0 lli¢v-ate DAVIS V1-5- q ‘ a h \ HR - V“ D¢J&Se 6.513148. 70 incubation, NOS increased to a maximum 308 ugN/g, after which it declined slowly to 190 ugN/g at 16 weeks (Figure 11). Nitrification proceeded more slowly, but continuously in soil alone, reaching a maximum of 98 ugN/g as N03 at 16 weeks. By contrast, N03 plus NOE ixutially present in soil containing 30 (n: 60 T/ha of sludge largely disappeared in the first four weeks. Nitrate accumulation was seriously interfered with through the eighth week of incubation. During the last eight weeks, NOS accumulated again at rates similar to those observed during the first four weeks at the 15 T/ha sludge addition. Nitrification was strongly inhibited in sludge alone (Figure 11). However, significant increases in N0; did occur between the fourth and 16th weeks of incubation, reaching a maximum value of 13 ugN/g (Figure 12 and Table 19). Levels of N03 plus NOE in mixtures of sludge with sand were lower in all samplings than in sludge alone. In these mixtures, significant increases occurred during the first eight weeks of incubation and significant decreases thereafter. It should be noted that NC; was the principle anionic form of N encountered at all times in all systems except sand alone, where none was found. Nevertheless, the presence of N0; even in trace amounts, indicates that the levels of N0; encountered represent equilibria controlled by nitrification and opposing reactions in which N0; is an 71 intermediate. Thus, periods when net losses of NOS + N0; occurred in the systems of Figures 11 and 12 may be ascribed to one or more of several processes which consumed N03 or I“); more rapidly than they were being formed by nitrifying bacteria. Probable consuming reactions include biological immobilization, biodenitrification, chemodenitrification and chemical fixation of N0; (Firestone, 1982; Nelson, 1982). Considerations Regarding Toxic Effects In Figure 11, the apparent inhibition of nitrification between the fourth and eighth weeks of incubation at 30 and 60 T/ha of sludge coincides with a period when diffusable NH: in KCl extracts of the same soil/sludge systems was extremely high (Figure 5). The pH of the soil was initially 7.2 and, as will be seen in data from the second experiment, may have been increased by as much as one pH unit by ammoni- fication at these high rates of sludge addition. + H Thus, the equilibrium NH3‘____ NHZ,would have favored NH3 concentrations that might have inhibited oxidation of N05 to N03 by Nitrobacter (Aleem and Alexander, 1960). However, NO; did not accumulate -- only trace quantities ranging up to 3 ug N/g were found. It is possible that Nitrosomonas types (NHZ—eNOE) were also inhibited. However, systems were already present that had removed DMD} and N05 initially present by the fourth week of incuba- tion (Figure 11). Also, net nitrification resumed abruptly after eight weeks and proceeded at the same high rate observed earlier in tflme 15 T/ha system. Conversion of NH .1. 4 72 to NO3 between eight and 16 weeks was essentially stoichiometric (cf. Tables 11 and 19 in the Appendix). These results suggest that oxidation of NH: by Nitrosomonas proceeded without interruption during the period when neither N03 or NO; accumulated, but NC; was removed as rapidly as formed, by reactions leading to denitrification and/or to immobilization in organic forms. The data collected in this experiment do not permit further speculation on this point. With regard to inhibitory effects on growth and assimilation of N by oat seedlings (Figures 1 to 3), it does . + . . appear that adverse effects of dominantly NH4 nutrition were involved. In both soil and sand systems, toxic effects on + oats at four and eight weeks occurred where diffusable NH I h in the rooting media was very high (Figures 5 and 6) and NO U) was very low (Figures 11 and 12). Incubation Experiment No attempt was made in this second experiment to repeat tflue plant assays. Incubations were repeated, with the following variations: soil, sand, and sludge alone were compared with mixtures at only the 60 T/ha rate of sludge; incubations were carried out in sealed glass containers rather than in polyethylene bags; 5N H2804 was used to trap volatilized NH3 in one set of containers but not in the other: the incubation temperature was ea little lower (250C rather than 30°C). Incubated samples were extracted with 2N KCl, then autoclaved in 2N KCl, as in the first experiment, 73 and the same forms of N were determined. In addition, changes in rfll were followed, auuj Kjeldahl N was determined in the residue after autoclaving. Changes in pH The addition of sludge (initial pH 4.62) caused the initial pH to decrease from 7.38 and 6.80 in soil and sand, respectively, to 6.85 and 5.23 (Table 1). After four weeks of incubation, the pH for all treatments had increased. These increases were associated with large increases in diffusable NHZ: in direct KCl extracts of sludge alone and the two mixtures, but not in soil or sand alone. In fact, in sand alone, no N was found at any time during the incubation. These initial pH increases in soil and sand cannot be ascribed to ammonification or to additions of alkalinity in the deionized water (pH 6.2) used to activate the samples prior to incubation. It appears that the deionized water acted as a mild extractant and favored equilibria that led to dissociation of cations in excess of anions. Trapping of ambient free N83 had no consistent effect on pH changes during incubation. As will be pointed out in a later section, periods of net nitrification in individual systems were accompanied by decreased pH. Decreases in 2 change in pH values. N0; plus NO were accompanied by increases in pH or no 74 0 .21 :z ucmwnam m>oamu Ou amuu 5000030 000m .mmHQEmm mumowaasuvmov How mommz u 0 cm nuHB 00000:o:« mumB mmaqsmm ummamwe u + 5H.0 00.0 00.0 00.0 00.0 0H.0 00.0 11 00.0 Hz NH.m 00.0 mm.0 no.0 mm.0 00.0 mn.m 11 No.0 macaw mwesdm a 00.0 00.0 00.0 00.0 00.0 00.0 00.5 11 00.0 .. .. 92 00.0 H0.0 00.0 00.0 00.0 00.0 00.0 11 00.0 0003Hm\0cm0 H 00.5 00.5 00.5 00.5 00.5 00.5 00.5 11 00.0 .. .. 52 00.5 00.5 00.5 00.5 0H.5 00.5 00.5 11 00.0 ecoam 0:00 B 50.0 00.0 05.0 05.0 50.0 00.0 00.0 11 00.0 .. .. 92 00.0 00.0 00.0 H0.0 00.0 00.0 00.0 11 00.0 000:Hm\afiom H 00.5 05.5 00.5 H0.5 00.5 00.5 00.5 11 00.5 .. .. 92 00.0 00.5 00.5 05.5 00.5 00.5 00.5 11 00.5 mCOHm 0000 H 0H 0H NH 0H 0 0 0 N 0 nmaamuu uoc 5:000: .m> mxmmz CH mafia cowumnsocH + woaamue mwnoam .0cmm .Haom 0cH>Ho>CH meumxm nmaamuu uo: 0cm wmammuu mo +.mmu=uxHE 0cm coaumnsocfi mcauzv m0 :« mmwcmsu .0 00008 7S Immobilization y§ Denitrification Nitrogen recoveries for trapped samples are presented in Tables 21 to 24 of the Appendix. At zero time, the sum for all measured forms of N, including volatilized NH3, represented 92 to 95% of calculated inputs, based on total N in soil, sand, or sludge and the quantities used in each system. Percentage recoveries decreased to values as low as 62% (soil/sludge, Table 22) at four to eight weeks and then increased to values that were at times equal to or greater than at the beginning of incubation. Quantities of N (ug/g) not accounted for are given in Table 2. Negative values for soil alone at 12 to 16 weeks might suggest that N fixation occurred to replace N lost earlier by denitrification. However, these negative values could be accounted for by no more than a 6% error in deter- mining N in the original soil (cf. Table 21). Considering the data for all systems, there is no evidence that important net losses of N by denitrification occurred. Rather, ii: appears that nuufli of the 11 not accounted for at various times during incubation was retained (immobilized) in forms that were not measured. Forms of N Not Measured Forms of N that were not measured were those that were resistant 1x3 alkaline hydrolysis when direct extracts and autoclaved hydrolysates were distilled in NaOH. These would have included OC-amino DJ in microbial cells and cellular debris, and structural N in fulvic and humic acids 76 0500 5500 000 0050 5000 0000 0000 0000 0000 mcon wwcsam 000 0.00 00H 0.50 0.00 000 000 000 00a 000:Hw\0cmm 000 H00 000 H00 000 000 0000 000 000 000:Hm\afiom 0.001 0.001 0.001 000 0.50 000 000 5.00 0.00 mcoam Hwom 11 111111111111111111111111111111111111 0\zm 1 11111111111111111111111111111111111111 0H 0H 0H 00 0 0 0 0 0 8:000: mxmm3 :0 cofiumnsocfi mo mafia .mmuauXHE wwvsam cam .mwvsam .000m :0 vasoM mfiuom HmGOwuomuw How HmuOu ecu :0 you vmucsooom uo: cmmouufiz .0 00008 77 (Stevenson, 1982). Both the direct extracts and the hydro- lysates were colored by humic substances and also undoubtedly contained microbial cells and other particulates of colloidal size that were not removed by centrifuging for eight minutes at 400 xg. Diffusable NH: and Amino Sugar Fractions Totals for extracts plus hydrolysates: In Figures 13 and 14, changes in N not accounted for are compared with diffusable NH: or amino sugar fractions as the sums recovered in direct extracts plus the hydrolysate after autoclaving. In soil alone and in the soil/sludge system, N not accounted for increased quickly to maximum values at four weeks (Figure 13). In the sand/sludge mixture and in sludge alone (Figure 14), peaks occurred at six or eight weeks, and sharp increases occurred again in the last sampling at 16 weeks. The patterns of change in N not accounted for are consistent with an expected initial increase in microbial numbers and ac-amino N 1J1 cellular proteins (Miller, 1974; Broadbent, 1973). The much earlier increase in numbers indicated for soil alone and the soil/sludge mixture reflect their higher pH (Table 1). Also, it is likely that the soil provided an inoculum of zymogenous organisms with a wider range of enzymatic capabilities than were present in the sludge. Increases near the end of incubation might reflect: a. growth of a successional population; b. conversion of labile N to alkali-stable struc- tural N in fulvic and humic acids; 200 1 ugN/g 78 (a) H N-not accounted for H Amino Sugar-N o—o NHz-N o ' U ‘r U 7 I r T ' 2 4 6 6 10 12 14 16 INCUBATION TIME (weeks) 1,100 ' 1.0001 9CD" 700- ugN/a (b) Figure #13. V I I ‘ I r I 1 2 4 6 6 10 12 14 16 INCUBATION TIME (weeks) Summed recoveries (extracts plus hydrolysates) of diffusable NHZ and amino sugar fractions compared with N not accounted for in soil alone (a) and soil/sludge mixtures (b). Data for trapped samples only. 79 8006 (a) 600-1 ugN/g 8 9 200-1 I V I 6 16 12 14 16 r W o N u. 01 INCUBATION TIME (weeks) 6.5001 (b) 0.000-1 H N-not accounted for H Amlno Sugar-N O—-O NH;-N 5.500- 50%-1 4,500- 4.000« 3,500 «1 . ugN/g 3,000.1 2,0004 1,500-1 1 1.00011 . I I I I I T r r 0 2 4 0 8 10 12 14 16 INCUBATION TIME (weeks) Figure #14. Summed recoveries (extracts plus hydrolysates) of diffusable NH4 and amino sugar fractions compared with N not accounted for in sand/sludge mixtures (a) and sludge alone (b). Data for trapped samples only. 80 c. losses of N by denitrification. Amino sugars are important constituents of microbial cell walls and extracellular mucopolysaccharides (microbial "gums" or "mucilages"). The N in this fraction (Figures 13 and 14) increased to peak values considerably later than N not accounted for. Mature or senescent microbial popula- tions might be expected to continue producing extracellular polysaccharides for a period after active growth in numbers had ceased. However, the indicated maximum quantities of amino sugar N appear unreasonably high in relation to the indicated earlier levels of microbial protein. Also, major increases in this fraction appear to have coincided with rapid and extensive lysis of microbial proteins. The intimate mixture of enzymes, amino acids, sugars and phenols in cellular autolysates is highly reactive. It is likely that much of the alkali labile N that appeared during and after lysis was in the form of amides and amines in humic acid precursors formed by Maillard reactions between amino acids and sugars, and by analogous condensa- tion and degradation sequences involving amino acids or NH3 and polyphenols (Nommik and Vahtras, 1982; Stevenson, 1982). Diffusable NH: :h1 direct extracts is simply NH: ex- changeable to 2N KCl. It represents net release of NH: by ammonification, which ii; the first step ixilnineralization. Diffusable NH+ in the autoclaved supernatants probably 4 represents ammonified N that was retained initially by 8l electrovalent exchange also, tun: at sites that were later covered up by hot water soluble products of metabolism. Increases 1J1 diffusable NHZ' reflect increases 1J1 surface area and number of oxidized acidic sites capable of retaining NH: by electrovalent exchange. Increases in the total for categories of N depicted in Figures 13 and 14 were derived largely from forms of N that were initially resistant to hydrolysis by autoclaving ("residual N" in Tables 21 to 24 of the Appendix). Thus, decomposition resulted 1J1 the transfer (mobilization) of N from "inactive" to "active" organic fractions. Some of this N was further mineralized to NH+, N0; and NO; recovered in the direct KCl extract. Some N may have been lost by denitrification. How- ever, such losses would appear to have been small compared with immobilization in microbial cells and products in "active" fractions extractable with KCl or released by autoclaving. Extracts and hydrolysates compared: Data for the amino sugar fractions 1J1 direct extracts and autoclaved hydroly- sates are presented in Tables 25 to 30 of the Appendix. Data for diffusable NH: appear in Tables 31 to 36. Data for samples where ambient NH3 was removed by acid trapping are presented in Figures 15, 16, and 17. Fractional recoveries for soil alone (Figure 15a, b) may be compared with their sums in Figure 13a. In both the + extract and the autoclaved hydrolysate, NH4-N and N in the 82 A a \ Z a a V 1.. O < 1: 1- x In 1. O m 0 V I I T I j r If 95 o 2 4 6 8 1o 12 14 16 O INCUBATION TIME (weeks) 1; Anuno Sugar-N 2 NHg-N >-13 03 0 (0 ul (0 E (> I.I.I< 4.4 IMO C(D .— D < O 'f 1 I I r I I I 1 o 2 4 6 6 10 12 14 16 INCUBATION TIME (weeks) Figure #lS. Amino sugar N fractions and NHZ-N in direct extracts (a) or released by autoclaving (b) from soil alone (data for trapped samples only). RELEASED BY :- 1. l 83 (a) A 4.000” O \ Z O 3 3.000« .- 0 < I: 2.000-1 '- X I.“ S 1.000< H Amino Sugar-N 3:1 O_.O NH;-N a o I I I 0 2 4 5 8 10 12 14 16 INCUBATION TIME (weeks) A (b) O 3.000‘ \ 2 O 3 2.000“ (D E i .1 1.000- (D '0 I'- 3 0 T v I T r I I I I ‘ 0 2 4 6 6 10 12 14 16 INCUBATION TIME (weeks) . . + . . ure #16. Amino sugar N fractions and NH4-N in direct extracts (a) or released by autoclaving (b) from sludge alone (data for trapped samples only). 00 RELEASED BY AUTOCLAVING (HON/0) DIRECT EXTRACT (ugN/g) 84 500- (a) 4mr- . O O umd . O O 200‘ . H Amino Sugar-N 1001 H "Hz-N 0, r f I I I I I n 0 2 4 e a 10 12 14 1s INCUBATION TIME (weeks) 400.. (b) 300-1 200-1 100- of I T I F r I T 7 O 2 4 e a 10 12 14 16 INCUBATION TIME (weeks) .. . . . . + . . :igure #17. Amino sugar N fractions and NH4-N in direct extract (a) or released by autoclaving (b) from soil/sludge mixtures (data for trapped samples only). 85 amino sugar fraction were recovered periodically in about equal quantities. A similar tendency for these two categories of N to return periodically to equal quantities is apparent 1J1 the direct extract (NE sludge alone (Figure 16a) and the soil/sludge mixture (Figure 17a). Reciprocal increases and decreases in these two forms of N in direct extracts of the sand/sludge system were very similar to those for soil/sludge in Figure 17a. (cf. Figure 19a,b). This tendency for diffusable NE: and N in the amino sugar fractions to fluctuate about a 1:1 ratio suggests a basic stoichiometry between sites active in electrovalent exchange of NH: ayui sites capable of condensing reversibly With NH3 (Lindbeck and Young, 1965; Mortland and Wolcott, 1965; Nommik and Vahtras, 1982). Effects of Acid Trapping The above results for trapped samples probably represent rather well what might be expected under well aerated field conditions where ambient NH3 could diffuse out of the soil. On the other hand, results with samples where ambient NH3 was not removed by acid trapping indicate that surface interactions can be influenced significantly by local concentrations of NH3 in microenvironments. Probable sources of volatile NH1: Very small amounts of volatile NH3 were recovered from sludge alone (Figure .1. H 18). This reflects the effect of low pH on the NH3';—_2 NH: equilibrium (cf. Table 1). Significantly larger quantities of NH3 were recovered from soil alone (Figure 18 and Table 86 (a) 300- A 3 2004 H soll/sludge a O——O sand/sludge :1 v e I") I 100. 2 o I I I I 70 2 4 6 6 10 1‘2 14 15 INCUBATION TIME (weeks) 20- A O 154 \ g O—-O soll alone :1 v” 101 0—0 sludge alone I Z 5 . 0‘ I INCUBATION TIME (weeks) Figure #18. Volatilized ammonia collected from trapped systems. 87 37). The rate of release increased sharply after eight weeks. Much larger quantities of volatile NH3 were produced in the In“) mixtures, and variance was analyzed separately (Table 38). Again, rates increased sharply after eight weeks (Figure 18). ZLf one compares the data in Figure 18 with those in Figures 13a, b and 14a, it will be seen that major releases of volatile NH3 from soil alone and the two mixtures occurred after major decreases in size of microbial popula- tions (as inferred from N not accounted for). Rapid release of NH3 began as diffusable NH: and alkali-labile N (amino sugar fraction) approached maximum values. Later releases were accompanied by large reciprocal fluctuations of alkali-labile N and diffusable NH: in the hydrolysate from soil alone (Figure 15b) and in the direct extract from soil/sludge (Figure 17a) and sand/sludge (Figure 19a, b). In autoclaved hydrolysates of the soil/sludge mixtures (Figure 17b), the two forms of N tended to accumulate in parallel fashion, but much larger quantities were retained as diffusable NHZ. The same was true for trapped samples of the sand/sludge mixture (cf. Figure 20a and 20b). In sludge alone, N that might have come off as NH3 at higher gfll was apparently retained mainly 1J1 alkali-labile forms in both the KCl extract and the hydrolysate (Figure 16a, b). 5001 400- 300‘ 200 -1 100‘ AMINO SUGAR (ugN/g) NHfi (uoNlo) 100... 88 Figure #19. (a) e e e e e e 2 4 6 67 10 12 14 16 INCUBATION TIME (weeks) (b) O-——O trapped O—O not trapped I I I I I I I 4 6 8 10 12 14 16 T 2 INCUBATION TIME (weeks) . . ‘,,,+ , . . Amino sugar N fractions (a) and uH4-N (b) in direct extract (2NKC1) of sand/sludge mixture. 89 ’3 \ i; 200T 3 2‘ (9 100.. D (D 0 r) 5f 0 .2 < INCUBATION TIME (weeks) 300. (b) A g H trapped 3 20°. O——O not trapped a V +v 1GP I 2 OF— T T l I I l T I 0 2 4 6 8 10 12 14 16 INCUBATION TIME (weeks) Figure #20. Amino sugar N fractions (a) and MHZ-N (b) released by autoclaving from sand/sludge mixture. 90 The above relationships indicate that rapid release of volatile NH3 occurred only after saturation of sites capable . . + of retaining NH4 by electrovalent exchange (n: NH3 by reversible condensation reactions. The rate (ME volatili- zation would appear to have been controlled primarily by equilibria involving reversibly condensed Duh; (the "amino sugar" fraction). Reciprocal variations ix] this fraction and NH: cjiffusable in the presence of MgO probably reflect cyclic changes in oxidation state of organic functional groups and/or changes imi degree (Hf protonation associated with periods of net nitrification or net reduction of nitrite (Alexander, 1977; Nommik and Vahtras, 1982). Effects of trapping on DHf+ and "amino sugar" frac- 4 tions: In sludge alone, average recoveries in fractions obtained by direct extraction (Tables 29 and 35) or by autoclaving (Tables 30 and 36) were very similar for trapped and not trapped samples, although significant differences in patterns of accumulation did occur. In particular, the sharp reciprocal fluctuations of NH: and the amino sugar fraction at four weeks in the direct extract of trapped samples (Figure 16a) was less marked where ambient NH3 was not removed (Tables 29 and 35). Later increases in extractable NH: were greater in trapped samples (Table 35), whereas alkali-labile N reached higher maximum values where ambient NH3 was not removed (Table 29). In the two mixtures, the large reciprocal fluctuations observed at four weeks in direct extracts of trapped samples 91 did not appear in not trapped samples (Figures 17a and 19a, b; cf. Tables 27 and 33). Later reciprocal changes occurred more or less simultaneously in trapped and not trapped samples. Materials released by autoclaving were apparently less directly influenced by changes in the active biosphere (Figures 17b and 20a, b). Except for sharp dips at 14 weeks, violent fluctuations did not occur. In trapped samples of soil/sludge, N accumulated less rapidly in hydro- lyzable fractions during the first half of the incubation than in not trapped samples (Tables 28 and 34). Early accumulations were similarly retarded in both trapped and not trapped samples (n5 sand/sludge. In later samplings, hydrolyzable NH: (Table 34) fluctuated over a higher range of values IJ] trapped than IJI not trapped samples. Ranges for hydrolyzable "amino sugar" N (Table 28) were similar in trapped and not trapped samples during the latter half of the incubation. In soil alone, hydrolyzable fractions were apparently associated with surfaces directly accessible to active microbial populations. Hydrolyzable bnfi: initially present was utilized similarly ix} trapped and not trapped samples during the first four weeks (Figure 15b and Table 32). Later reciprocal fluctuations with hydrolyzable "amino sugar" N (cf. Table 26) were similar to those observed in direct extracts of soil/sludge (Figure 17a) and sand/sludge (Figure 19a” t3), and were observed in both trapped and not 92 trapped soil. The principal effect of trapping was in the direct extracts, where alkali-labile N (Table 25) and NH: (Table 31) tended to decrease during the latter half of incubation in not trapped samples but continued to increase in trapped samples. In summarizing the above observations, it would appear that an early effect of the N83 retained in not trapped systems was to delay the development of alkali-labile sites at surfaces accessible to direct extraction. This suggests that NH3 initially adsorbed by the reaction NH3 + HX;;£!NH4X may have served to buffer surface sites against oxidative changes leading to condensation reactions with NH3 (Mortland and Wolcott, 1965; Nommick and Vahtras, 1982). As will appear in the next section, fluctuations of NH: and alkali-labile N in extracts and hydrolysates may have involved interactions between heterotrophic and autotrophic organisms. Effects of acid trapping on N03 plus N0; and on residual N: As noted earlier, NC; was encountered in all samples, except sand alone, but usually in trace amounts (Tables 21 to 24 in the Appendix). In soil alone, the sum of NOS plus NOE was very much higher throughout incubation in samples where ambient NH3 was not removed by trapping (Figure 21a and Table 39). This differential was established during the first two weeks and can be accounted for by reciprocal changes in residual N (Figure 23a and Table 41). At the same time, the pH of 93 (a) 90 1 80‘ 701 e N05+NOé-(uoNlo) 10" I r ' T I '1 0 2 4 6 8 1O 12 14 16 INCUBATION TIME (weeks) 6001 (b) 400« H trapped O——O not trapped 200‘ 80-4 .. \l' - N05+NO§-(ugng) w I T O 6 8 1O 12 14 16 INCUBATION TIME (weeks) Figure #21. Nitrate plus nitrite in soil alone (a) and soil/ sludge mixture (b), incubated with and without an acid trap to remove ambient NH3. 94 3- (a) A a \ Z a 4‘ O 3 e I.“ . . . . z d + 2 '0 0 Z ofi I I I I r I r ‘1 O 2 4 6 8 10 12 14 16 INCUBATION TIME (weeks) 121 (b) H‘ trapped 1; C}——() nottrapped \ E. a 3“ V IN 0 s .. 11') (0 2 o I I I T f T r 1 0 2 4 6 8 1o 12 14 16 Figure #22. INCUBATION TIME (weeks) Nitrate plus nitrite in sand/sludge (a) and sludge alone (b), incubated with and without an acid trap to remove ambient NHB' 9S (a) A a \ a a V Z I J < D Q (I) LIJ m V I 00 i 3 a a 10 12 14 1e INCUBATION TIME (weeks) (b) 5,600 3 H trapped 3 5.5001 O—‘O not trapped : v 2. I 5,400- ..I .< i: 9 i a, 5.200 In C: 5.000 I a: 1 I v I V I r ‘ O 2 4 6 6 10 12 14 16 INCUBATION TIME (weeks) Residual nitrogen after autoclaving in soil alone (a) and sludge alone (b), incubated 'ith and without an acid trap to remove ambient NH3. 96 (a) 2,200- A O H not trapped Einmo- Z: I ..l < 3 01,4004 0) III I l ‘3’ a I a I. ah 1'2 14 *6 INCUBATION TIME (weeks) A a \ a a v z I .4 1< D .9 1m u: m: as» :L' I I I 0 3— 2 1 3 5 1'0 12 14 1e INCUBATION TIME (weeks) I“ ' I e e e n e -igure #24. ReSidual nitrogen after autoclaVing in 8011/ sludge (a) and sand/sludge (b), incubated with and without an acid trap to remove ambient NH3. 97 trapped samples increased and remained higher than in not trapped samples in most later samplings (Table 1). This is consistent with the release of alkalinity that occurs when N0; is reduced biologically (Alexander, 1961; Broadbent 1973). Thus, it would appear that removal of ambient NH3 may have reduced the availability of N to organisms‘ requiring NH3 or INTI, thereby favorimg the early enrichment of the heterotrophic population with types capable of reducing N0; and N05. It is apparent from Figures 21 and 22 that active nitrifying populations 'were [present 1J1 all. systems. Increasing CO2 concentration in the sealed incubation containers would have been increasingly favorable for these autotrophic organisms (Bremner and Douglas, 1971; Eno, 1960). It is possible that the removal of ambient NH3 reduced the availability of N83 or NH: to Nitrosomonas types responsible for the first step of nitrification, thereby lowering the rate of the overall transformation in trapped samples. In any case, it is apparent that the quantities of nitrified N found reflect a cyclically changing equilibrium’ between rates of nitrification and rates of reduction of N0; and/or NOE. Dissimilatory reduction (denitrification) may have occurred in systems involving sludge (Tables 22 to 24). However, such losses would appear to have been minor relative to processes leading to incorporation of reduced N 98 into solid phase components. It is likely that No; and N0; were first reduced assimilatively and incorporated into microbial cells. The very low pH values that developed in sludge alone after six weeks (Table 1) would have been favorable for chemical fixation of N0; (Nelson, 1982). However, the patterns of change in N not accounted for in Figures 13 enui 14 indicate that major transfers of N among solid phase components involved first an early rapid uptake by microbial cells that could be suspended by shaking in the direct extract and/or by autoclaving in the hydrolysate. It is clear from Figures 23 and 24 that much of the nitrogen that appeared as microbial cells and extractable or hydrolyzable fractions came from materials that were ini- tially resistant to hydrolysis by autoclaving. In the case of soil alone, a substantial additional source was NH: sus- ceptible to release by autoclaving (Figure 15b). Sludge apparently contained substantial quantities of extractable or hydrolyzable N in alkali-stable substrates (N not accounted for in Figure 14b). These "active phase" substrates were metabolized before any major mobilization of more resistant forms in the "inactive phase" occurred (Figure 23b). The electrically neutral IML3 molecule is; transferred across cell membranes more readily than charged species such as NHZ, N03 or NOE. Removal of NH3 would account for differentially greater utilization of N03 or No; in trapped samples (Figures 21 and 22). It would have also influenced 99 surface interactions an: the interface between the solution phase and accessible surfaces in the solid phase. Preferen- tial utilization of NH3 over NH: undoubtedly contributed to differential effects (NE trapping (N1 the distribution of N . + . . . among the various N84 and "amino sugar" fractions in Tables 25 to 36. SUMMARY Systems responsible for the extensive changes in forms of N observed in this study are obviously complex. Trans- formations and equilibria that may have been involved are outlined in Fig. 25. The dynamic nature of the observed transformations derives from readily available energy materials present initially in soil and/or sludge. Estimating levels and probable effects of available energy in soils or in organic materials added to soils is a major unsolved problem in predicting the fate of N and other nutrients or toxicants in field situations (Broadbent, 1973; Jansson and Persson, 1982). The soil in this study had been air dried, and the sludge had been dried by heating. Before use, both were ground to pass a 2 mm sieve. Drying and grinding would have exposed energy substrates to support the flush of microbial growth evidenced by increases in N not accounted for during the first several weeks of incubation (Figs. 13 and 14). In the soil (C/N 3 9/1) and in the sludge (C/N 3 3.4/1), removal of structural C as CO2 by heterotrophic respiration would have led tx) net mineralization of N (Jansson and Persson, 1982). Mineralized N appears first as 100 lOl .meumzm wwvsa com Hfiom mo Am:0Huumum wflnmuemuuxov mommza 0>Huum ca 2 w=H>Ho>cfi maunwafisvw was macaumEHOmemuH .mN .wfim 2 .MA Mm "mmummcmvcoe ofisss magnum :a z Hmumawxm seem cu macaummcwvcoezdoa “sneeze a z AEOfiuMucwewmuu .mcoaumcmnmwv umxumuumv N N Amcofiummcmvcou Houfim .chHuumeu mcacsoumv mflocwzazdom z .o z mcfium o:HE< G a « mumwsm K cowumoaw 4 % me4093< I. Ifiuuacwc % Ioawzo :ofiumNfiH zo IanoeeH mwcaa< waees< W ouzuzuza mloewzo N =zum =ZIonm m +zmzuca =z-;m .Iuiir .nnllliv. =zuzaII/ir u\\\\\\\\ .. Lumv I 7 O W S as A vmzz =o|oum seesaw 4// \\ma mole muuuuvw Iowa :1 a z Ouzusm :AIIIIIIIII =Olzm magmu A N N A szz z .o z 102 NH3 in the aqueous phase. As shown in Fig. 25, aqueous phase equilibria can lead to losses of NH3 by volatili- zation, or 1x3 protonation and electrovalent adsorption of NH: at acid sites in mineral or organic colloids (Nelson, 1982). Condensation of N83 with dienols or polyphenols to form alkali labile amides and amines may occur also (Broadbent and Stevenson, 1966; Nommik and Vahtras, 1982). As long as available energy substrates are present, NH3 will be closely cycled by immobilization in successive hetero- trophic populations (referred to by Jansson and Persson, 1982, as mineralization-immobilization turnover, or MIT). The above processes were effective in preventing losses of NH3 by volatilization through the first eight weeks of incubation (Fig. 18). Comparison of Fig. 18 with Figs. 13 amd 14 indicates that significant volatilization of NH3 occurred only after energy substrates had been depleted and extensive lysis of microbial cells had occurred. As noted in Fig. 25, degradative reactions that follow the autocatalytic condensation of amino acids with sugars or with polyphenols would be expected to give rise to NH3 or NH;' in. both. diffusable (exchangeable) and .alkali-labile condensed forms (Nommik and Vahtras, 1982; Stevenson, 1982). Early sequences 1J1 the Maillard browning reactions between amino acids and sugars are reversible (Mortland and Wolcott, 1965). The same is true for aldol condensations between NH3 and polyphenols (Lindbeck and Young, 1965). The reversibility of these early reaction sequences may have 103 contributed to the large reciprocal fluctuations between diffusable NH: and the "amino sugar" fraction observed in all incubated systems (Figs. 15, 16, l7, 19). The alkali labile amides and amines visualized as early condensation products in Fig. 25, are subject to extensive further polycondensation to form high molecular weight polymers. ‘The skeletal II in these humic condensates would not be released by distillation with NaOH. Transformations of N were further complicated by inter- actions involvimg the autotrophic nitrifying bacteria. A significant aspect of these interactions was their marked sensitivity to removal of ambient NH3 by trapping (Figs. 21 and 22). Jansson and Persson (1982) note that heterotrophic and autotrophic organisms compete for mineralized N83, not only with each other, but also with physical and chemical processes which adsorb or fix_NH3 in combinations that cover a wide range (ME bonding energies. Equilibria among these forms shift quickly in the direction of more stable, less available combinations. For this reason, newly mineralized NH3 or NH: in the close vicinity of the active biomass will be used preferentially. Data obtained in the first experiment indicate that utilization of N by oat seedlings was adversely affected by dominantly NH: nutrition during periods when N03 levels were low. Thus, from the standpoint of plant nutrition, the critical biosphere interactions at high rates of sludge 104 addition involved nitrification on the one hand and processes that removed No; or No; on the other. Net removals of N0; during the first weeks of incuba- tion were most likely due to assimilatory reduction by adaptive heterotrophs. Removal of freshly mineralized NH3 by acid trapping apparently favored an earlier adaptation to assimilative use of NO3 as an alternative N source. Dissimilatory reduction to N20 or N2 by facultative anaerobes did not appear to have occurred during the flush of microbial activity supported by energy substrates initially present. At the acid gfii of the sludge and time sand/sludge mixture (Table ll), N0; produced by Nitrosomonas was apparently "side-tracked" by non-enzymatic reactions with polyphenols originally present in soil or sludge or released by lysis of microbial cells. These side-tracking reactions involve undissociated nitrous acid (HNOZ) and are, there- fore, favored by low pH (Mortland and Wolcott, 1965; Nelson, 1982). As outlined in Fig. 25, initial products of nitrosation are readily reduced to aromatic amines‘that are entirely analogous to the labile amines formed in browning reactions or by aldol condensations of NH3 (Nelson, 1982; Nommik and Vahtras, 1982; Stevenson, 1982). Thus, in acid systems, NOE-N may be side-tracked and retained in a cycle involving Nitrosomonas and labile products of chemical fixation of HNOZ. Such a cycle would not depend on organic energy 105 sources. It would be sustained by the energy derived from oxidation of NH3 and the catalytic effect of protons produced in the oxidation. This cycle could continue as lomg as appropriately reactive polyphenols were present to trap N02- and prevent its further oxidation to No; by Nitrobacter. This proposition is supported by the very large accumulations of alkali labile N ("amino sugar" fraction) in sludge alone and the associated decreases in pH (cf. Fig. 14 and Table 1). A similar tendency for pH to decline as alkali labile II increased was apparent in sand/sludge but not at the alkaline pH of soil or soil/sludge. Neverthe- less, side-tracking reactions could have proceeded in microenvironments closely' adjacent 133 active Nitrosomonas populations and could, therefore, have interfered with normal accumulations of NO3 in soil or soil/sludge also. Side-tracking of N02— can lead also to chemodenitri- fication (Broadbent and Stevenson, 1966; Mortland and Wolcott, 1965; Nelson, 1982). However, losses of N20 or N2 by side-tracking are observed only during cycles of drying. No losses of moisture occurred during incubation. Thus, increases in N not accounted for near the end of incubation (Figs. 13 and 14) do not appear to have been due to chemodenitrification. It is nmnna likely that II in measured forms declined at this time because of increased synthesis of proteins by successional microbial populations, 106 or because of accelerated polycondensation of alkali-labile amides and amines to form stable residual humic condensates (Fig. 25). CONCLUSIONS From the standpoint of theory, two potentially signifi- cant concepts are derived from this study: 1. Lysis of microbial cells may be an important aspect of biosphere transformations in soils. The possible significance of lysis in humifi- cation has been alluded to (Swaby and Ladd, 1966), but its implications do not appear to have been studied systematically. Data reported here suggest that the availability of N can be influenced dramatically by non- enzymatic reactions among products of autolysis. The probable nature of reactive species leads to the expectation that the availability of other nutrients, including potentially toxic heavy' metals, may be influenced also. 2. "Side-tracking" of nitrification at the N02 stage has been recognized as a probable mechanism for chemodenitrification in acid soils. Results of the present study suggest that, in the absence of drying, the side- tracked N may be retained in an active cycle involving Nitrosomonas and labile products of reaction of HNO and polyphenols. This cycle would be sustained by the energy from oxida- tion of NH and the catalytic effects of protons produced by the Nitrosomonas reaction. From the standpoint of methodology, several obvious deficiencies in the data should be noted. Microbial proteins were estimated by differences as N not otherwise accounted for. This interpretation was supported by the plotted data. However, confirmation of the indicated autolysis of microbial cells will require specific measure- ment of proteins or ofCJC-NH2 after acid hydrolysis of extracts and autoclaved supernatants. 107 108 The difference between distillable and diffusable NH3 is not specific for amino sugars. A more specific determination for amino sugars would have helped to support interpretations based on the probable role of alkali-labile condensates. Chromatographic and spectrophotometric criteria for characterizing postulated humic condensates according to molecular size will be needed in future studies to test the propositions presented here. It is likely that incubations longer than 16 weeks will be necessary to estimate the length of time before normal net release of N03 might be expected. Nevertheless, the use of extraction and autoclaving to focus on important components in the active biosphere appears promising. Also, the quick uptake plant assay appears useful to reveal rapidly changing short term nutritional effects associated with decomposition of sludges or other organic amendments. APPENDIX 109 APPENDIX ‘17a1t31e 3. Dry weight of oat seedlings after two weeks' contact with previously incubated soil/sludge mixtures and sludge alone. Incubation time in weeks Mean for 55321.nidge rate 0 4 -8 16 rates T/ha mg/pot 0 605 700 570 608 621 ef T de efg ef B T 15 800 924 855 745 831 bed ab bc ed A 30 819 569 516 701 651 bed efg fg de B 60 748 564 537 834 671 cd efg fg bcd B SSiludge alone 1000 483 462 557 625 a ‘ fg g fg B ‘ '1‘ a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 110 ZZable in Dry weight of oat seedlings after 2 weeks' contact with previously incubated sand/sludge mixtures and sludge alone. Incubation time in weeks Mean for S ludge rate 0 4 8 l6 rates T/ha mg/pot O 714 752 626 581 '663 cde 1 bed defg efgh A T 15 867 543 475 738 656 b fgh gh bcd AB 30 818 513 467 621 605 be fgh h defg AB 60 722 477 541 637 594 cde gh fgh def B E331udge alone 1000 483 462 557 625 a gh h fgh AB 7F a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 111 ‘IIEitale 5. Nitrogen concentration of oat seedlings after 2 weeks' contact with previously incubated soil/sludge mixtures and sludge alone. Incubation time in weeks Mean for S 1 ud ge rate 0 4 8 l6 rates T/ha ZN O 2.45 2.74 2.55 2.59 2.58 g T efg fg fg D t 15 3.49 3.49 3.41 3.28 3.41 bcd bcd cd cd B 30 3.18 3.01 3.15 3.17 3.13 de de de de C 60 3.75 3.04 3.29 3.45 3.38 abc def cd bcd B Sludge alone 3.92 3.18 3.74 4.00 3.71 ab de abc a A 'T’ a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 112 T?znt>]_e 6. Nitrogen concentration of oat seedlings after 2 weeks' contact with previously incubated sand/sludge mixtures and sludge alone. Incubation time in weeks Mean for S ludge rate 0 4 8 16 rates T/ha — ZN 0 1.32 1.18 1.48 1.75 1.48 hi I 1 hi h D T 15 2.77 2.76 2.95 2.81 2.82 fg fg efg efg C 30 3.06 2.71 2.67 3.14 2.89 defg g g defg C 60 3.10 3.28 3.32 3.49 3.29 defg cdef cde bcd B Sludge alone 3.92 3.18 3.74 4.00 3.71 ab defg abc a A T a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). ' 113 Table 7. Nitrogen uptake by oat seedlings after 2 weeks' contact with previously incubated soil/sludge mixtures and sludge alone. Incubation time in weeks Mean for S ludge rate 0 4 8 l6 rates 'I/ha — 1c/pot 0 14.8 18.1 14.5 15.6 15.7 h fgh h h D t 15 28.0 31.7 29.3 24.3 28.3 bcd b be cde A 30 25.7 17.0 16.4 22.7 20.5 cde gh h def C 60 27.9 17.1 17.7 28.7 22.8 bcd gh fgh bc B ‘pure sludge 39.1 15.3 17.2 22.2 23.5 a h gh efg B T a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 114 1:21t31e 8. Nitrogen uptake by oat seedlings after 2 weeks' contact with previously incubated'sand/sludge mixtures and sludge alone. Incubation time in weeks Mean for S ludge rate 0 4 8 16 rates 'I/ha . mg/pot 0 9.4 8.8 9.1 10.1 9.4 ij T j ij hij C t 15 24.1 14.8 14.0 20.7 18.4 bc efghi fghij bcde B 30 25.1 13.8 12.4 19.4 17.7 b fghij ghij bcdef B 60 22.7 15.7 18.4 22.2 19.7 bcd efgh cdefg bcd B pure sludge 39.1 15.3 17.2 22.2 23.5 a efgh defg bed A t a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 115 Table 9. Ammonium-N in sludge alone. Incubation time in weeks Mean for Extractants 0 4 8 l6 Extractants new 3 -- 291 KCl 196 1973 2697 2289 1781 h T e b c B T 55 E“::§33d 267 213 2259 3039 1924 4 h d b a A autoclaved ZN.KC1 528 1396 1945 2132 1500 S f e d C ‘ T a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 116 Table 10. Amino sugar-N fractions in sludge alone. Incubation time in weeks Means for IEisctractants O 4 8 16 Extractants ugN/s 2N_KC1 77 1858 2665 5696 2574 e t c b a A" Saturated 65 469 197 5624 2033 CaSO 4 e d C a B a“‘°°la"ed 523 2943 1865 1897 1807 2N K01 - d b c c C T a,b,c...;A,B,C...mean values accompanied by the same letter are not significantly different at P(05). 117 122113143 11. Directly extractable NHZ-N in soil/sludge mixtures. Incubation time in weeks Sludge Means for Ext ractant rate 0 4 8 16 rates T/ha ugN/g t 0 2.7 4.1 5.4 2.7 3.6 n t m 1 n D i 15 43.5 49.3 59.6 13.5 36.2 i h g k C ZN KCl 30 84.3 241 395 27.2 121 f d c j B 60 128 496 549 51.7 205 e b a h A 0 4.3* 8.6* 16.9* 4.3*’ 7.2* Q / - ) '1 ' * '9 / Saturated 13 43.2 14 * 171* 10.9 63.0* C3504 30 56.7* 285 * 373* 34.1* 119 60 116 513 802* 98.5* 262 * t geometric means (antilog of mean log transform) 3 a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). means for saturated CaSOa extract are significantly different from corresponding means for 2N_KC1 extract. 118 + Table 12. Directly extractable NH4 N in sand/sludge mixtures. Incubation time in weeks Sludge Means for Extractant rate 0 4 8 16 rates T/ha ugN/g t 0 = 0 0 0 0 15 15.5 194 140 81.6 67.6 2N KCI k S e 3 h L 3 30 36.7 365 381 177 173 j be c f B 60 44.9 412 478 326 231 i b a d A 0 0 O O 0 15 25.7* 276*‘ 298* 59.9* 106* Saturated CaSOA 30 38.4 360 458 * 176 183* 60 59. 9* 608 * 720 * 248 * 284 * t geometric means (antilog of mean log transform) Values for the rate zero are not included in ANOVA. w; a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). * means for saturated CaSOA extract are significantly different from corresponding means for 2N_KCL extract. 119 Table 13. Directly extractable amino sugar-N in soil/sludge mixtures. Incubation time in weeks Sludge Means for Extractant rate 0 4 8 l6 rates T/ha ugN/g I O 16.9 16.3 17.6 10.9 15.4 ef * ef ef g C e 15 5.2 19.5 38.9 13.3 15.1 2N_KC1 h de c fg C 30 13.1 65.8 83.2 18.9 34.1 fg b b e B 60 25.9 193 189 21.5 67.1 d a a de A O 4.3* 4.3* 42.4* 7.8* 8.8* - - 97 7 7 Saturated 13 5.8 17.5 -4.9 10.0 l-.6 casoa 30 24.6* 83.0 390 * 24.9 66.7* 60 38.8 74.2* 170 64.2* 74.9 Ir 3? geometric means (antilog of mean 10g transform) a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). means for saturated Ca804 extract are significantly different from corresponding means for 2N_KC1 extract. 120 Table 14. Directly extractable amino sugar-N in sand/sludge mixtures. Incubation time in weeks Sludge Means for Extractant rate 0 4 8 l6 rates T/ha -- ugN/g t 0 i O 0 0 O 15 13.6 24.5 66.3 16.0 24.4 ZE KCl h 3 fg c h C 5 30 22.3 45.0 127 29.7 44.2 g d b ef B 60 36.6 110 331 109 110 de b a b A 0 0 0 0 0 15 4.3* 31.4 49.2* 98.3* 28.4* Saturated C3°°4 30 17.5 311 343 4 72.6* 108 * 60 12.8* 321 300 124 111 t geometric means (antilog of mean log transform) II- S a,b,c.. significantly different at P(05). Values for the rate zero not included in ANOVA. .;A,B,C...mean values accompanied with the same letter are not * means for saturated CaSOA extract are significantly different from corresponding means for ZN,KC1 extract. 121 Table 15. Ammonium—N released by autoclaving from soil/sludge mixtures. Incubation time in weeks Means for Sludge rate 0 4 8 16 rates T/ha — — IJgN/g t --— O 57.0 111 59.2 93.9 77.0 k=‘= i k j 1).-:- 15 95.2 260 149 115 143 i e gh i C 30 107 363 321 159 211 i c d fg B 60 136 433 634 174 284 h b a f A T geometric means (antilog of mean 10g transform) 4 a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). 122 Table 16. Ammonium-N released by autoclaving from sand/sludge mixtures. Incubation time in weeks Means for Sludge rate 0 4 8 16 rates T/ha u gN/g i'---- 0 _() i 0 0 0 15 36.7 97 5 93.4 25.7 54.1 3 5 f f k C § 30 54.4 186 224 46.9 101 h d c i B 60 64.3 426 493 120 200 g b a e A f geometric means (antilog of mean log transform) # Values for the rate zero not included in ANOVA. 5 a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). 123 Table 17. Amino sugar-N released by autoclaving from soil/sludge mixtures. Incubation time in weeks Means for Sludge rate 0 4 8 16 rates T/ha UgN/g 1' 0 26.7 18.9 26.4 26.2 24.4 fg é g fg fg D 3 15 29.8 40.8 48.3 31.5 36.9 efg def cde efg C 30 39.0 151 67.5 25.2 56.3 ef b cd fg B 60 37.3 643 72.1 40.6 91.6 ef a c def A f geometric means (antilog of mean log transform) # a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). 124 Table 18. Amino sugar-N released by autoclaving from sand/sludge mixtures. Incubation time in weeks Means for Sludge rate 0 4 8 16 rates I/ha ugN/g + 0 0 i 0 0 0 0 15 1.9 27.4 7.2 20.5 9.4 d § b c b C§ 30 17.9 62.2 16.9 18.5 24.3 b a b b B 60 20.6 95.0 79.9 31.9 47.3 b a a b A f geometric means (antilog of mean log transform) i Values for the rate zero not included in ANOVA. § a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). 125 Table 19. Nitrogen as N03 + N02 in soil/sludge mixtures and sludge alone. Incubation time in weeks Means for Sludge rate 0 4 8 l6 rates T/ha —- 4 ugN/g + _____ 0 43.4 58.1 86.9 98.1 68.1 eféfi e d d B 3 15 35.4 308 295 190 153 f b be c A 30 37.5 2.6 4.2 296 11.6 f k ij b D 60 30.9 3.5 5.4 563 23.9 f j hi a C Sludge only 5.0 4.7 6.2 13.2 6.68 hij hij h g E T geometric means (antilog of mean log transform) $ a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). 126 Table 20. Nitrogen as NO_ + N0 in sand/sludge mixtures and sludge alone. 3 2 Incubation time in weeks Means for Sludge rate 0 4 8 16 rates T/ha ---- ugN/g 'f —— 0 0 :* 0 0 0 15 0.86 1.68 2.06 1.31 1.47 b § gh gh gh D § 30 0.94 2.61 3.86 1.29 2.18 h efg de gh C 60 0.71 3.49 5.85 3.64 3.42 h def bc de B Sludge only 5.02 4.73 6.30 13.3 7.33 bcd cd b a A + geometric means (antilog of mean log transform) Values for the rate zero not included in ANOVA. a,b,c...;A,B,C...mean values accompanied with the same letter are not significantly different at P(05). l0} 127 .U:Q:H mm commasoamo Zlacmpamnx w\w1 c.meH mo ucmouma mm How vmussooom Znamuoe .m:«>mao0u:m umumm cwwouuw: H:MpammM Hmuoe n z Hmspfimmm II .fi. ll 4.. 00H moH Hos Ha Ha om mm mm ms + emnm>oomn ucmonmm NoNH mead mmaa wmod Heed aNoH mma “Nod macs now umucsouum annoy mam «mo mmm cam mom Mme saw was ewe + z Hmsenmmm ao.o~ so.mfl Hm.m ow.m H©.H qm.~ mk.o om.o .. znmmz emuaafiumao> om.o NH.o NH.o mq.q aa.H mm.a oo.m m~.o afl.o Azumozv munnuaz N.H© m.m~ H.NH H.NH ~.N~ m.a~ w.mm N.oH H.5m Azumozv mumuufiz mHH s.mo mma NmH “.0w m.km «.mm 5.2m m.ma m>mH00usm kn emmmmamm m.mw m.mm m.mm H.mm o.aa o.m~ H.m~ o.m~ “.ms mozzN an eunumuuxm IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII w\zqu IIIIIIIsullllnllullllllnltuIIIIIIII OH «H NH 0H m o c N o coonUfi: mo mspom mxmmB cw cofiumnaocfi mo mafia .Ammaoamm pmaamuuv ocoam Hfiom you mmaum>ooou cowouufiz .HN magma 128 .usch mm pwumasuqmo Zlanmpaonx w\w1 m.qmnm mo ucmouoa mm How nmucsooom anmuoH .w:«>m~u0u:m Houmm cowouufic HsmpamnM Hmuow u z Hmapfimmm II .a. ll 4. mxmo3 ca mafia coaumnsocH om om no ca am On me am mm + emnm>oumn nemunmm ooqm seem Hmmm scam oNNN woos «men oNNN «omw pom emuasouom annoy mama NoNH mama moan mmma mmma HmNH oaks NNNN + z Hmaewmmm mom mom oNN cam m.mm m.qm o~.qa OH.Q -- z-m=z emufiasumao> km.o mH.o OH.o Ha.o Na.o Hq.q mN.H «N.o mm.o AZIWOZV muduunz ow.H ken mm.a mm.o a.oa om.m m.me ¢~.m H.om Aznmozv msmnufiz ofim mmm mmm cam “on man can mma Nam m>mHUOS=m ea emmmmamm cam cam no“ NNN ode mmq HON mmm m.ma HumzN an emuumunxm uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu wxzw: uuuuunuun:ItunnunnnnunnunlununuluIn: 0H 4H NH on m o a N o awwouunc I «o mauom .Amwaaamm pomdmuuv ouSuxflE mwcaam\afiom How mmfium>owmu cowouuwz .NN manna 129 .uijH mm pwumazoflmo znanmoamnx w\w: o.o~o~ mo ucmouma mm How nonsmooom ZlamuOH n + .w:«>mH60u=m umumm :mmouuwc azmpamflx Hmuoe n z Hmspwmmm n.+ mm mo om ma Nm Nm «w mm Nm * pmum>oomu ucmoumm MONH cwma omqa mama onH HNMH quH qua mmqa HOW pmucsooom Hmuoe «mm mac com NNq mam Noe com «moa «mma + z Hmavwmmm N.mmH N.<©H N.qu m.am m.HN w.mH m.o N.q In Zimmz vmmwawumao> oN.o No.H mo.H om.o mN.o mm.o NN.o mH.o No.0 Azamozv mufluuwz ma.a om.o mm.a mo.a on.a mm.o qw.H NN.N mq.o Azumozv mumuuwz mNN QMH mam com mom 0.0m m.0e a.qm k.mw wcn>mauou=m kn emmmmamm qu coo «mm me omm on Nmm HON o.oN Husz cw pmuomuuxm nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn w\zw1 uuulullaauulunllnluuluIIIIIuIIIIII OH «H NH od w o q N o cowouufic mo 950m mxmma Ca :OHumnaoca mo mBHH .Ammadamm vmadmuuv muauxwa mwpaam\pGMm How mmfium>oomu cowopufiz .MN manme .u:QCH mm pmumHsono ZlempHMnM w\w: o.oooNo mo ucwouma mm now pmucsooom ZIHmuoH n % .w:H>mHoou:m umumm cowouuHc HampHmnM Hmuoe u z HmapHmom u + co mm mm om Nm mm No om ma * pmum>oomu osmoumm qumm mNmoo quHo mwNom mmmom qumm owooc meqmm NNomm ROW wouasooum Hmuoe Nmmom qHNNm «HHNm mmHHm OHHHm MONmm Nmmmm wmoNn comma + z Hmstmom AU Nm.N mo.N Hm.H qm.H ow.o NN.o 00.0 NN.o In Zlmzz pmNHHHumHo> 3 1 3.0 are 8.0 $5 .38 «To 3.0 3.0 35 Azumozv 32:2 o~.m oH.q ow.¢ Om.m om.c om.q oo.m oH.N o~.o Azumozv wumuuwz Nmom mwom mmNm NmmN meN wqu omoH quH NNNH wcH>mH00uom >2 pmmmmHmm owmm MHmm Hmmm NmHm NHNm NNNH oqu moHH oQN Hosz :H monomuuxm IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII w\zw1 IIIIIlltlllIlnllullllllllllllIIlull 0H «H NH 0H m o c N o cowouuH: mo mason mxwm3 :H coHumnao:H mo mEHH .AmmHaEmm cmaamuuv mcon mmnsz you mmHuo>oomu cowouUHz .qN mHnme 131 .Amovm um pmmdmuu How momma mchcoammuuoo Eoum ucmuoWMHp >HucmoHMchHm mum woodman uoc How memo: u * .Amovm um ucmumwan xHucmoHMHome nos mum umuumH mamm mnu :uHs pchmanoom momma...o.n.m n + .<>oz< CH popsHUCH uo: muo3 oumu msHu you mmsHm> n + «o~.m *oH.© *om.o H.MH mm.“ q.qH mm.a n.0H SH.o a.q emaamps “oz m n on no no no u nupu m.sH w.om o.HN m.OH m.OH mH.o mq.k Nq.o H.HH m.q emanate “ llllllllllllllllllllllllllllllllllllllllllllllll w\zw: lllll II III: pmadmuu uo: 0H «H NH oH w o q N .Ho pmaamuu uoc pom pmammuu .m> “0m momma mxmmz :H mEHu GOHumnsocH cmaamue .mcon HHom :H :OHuomuw Human ooflam mHnmuomuuxm xHuoman .mN mHnma 132 .Amovm um pmaamnu HOW momma wcHwooammuuoo Eoum ucmumwpr zHucmoHWchHm mum pmeamuu uo: HON memo: I « .Amovm um ucmummep >HucmonchHm no: mum umuumH mamm mcu nuH3 pchmaBOUUm m:mmE...o.n.m u_% .<>OZ.< CM UQVDHUC“ uOC 0H0») OHMN QEHU HOW mMDHm> H + m.0m H.NN «m.oq m.wo o.wo «0.0N m.mN m.¢N w.qH w.m pmadmuu uoz m o m m n o o %“o N.©q m.mo w.mN N.Hw ¢.©N N.Hm o.mN N.mH m.NH m.m mundane lllllllllllllllllllllllllllllllllllllllllllllllll w\zw1 Illnlulllllllnltu: In unit: 838“ nos 2 3 NH 2 N o a N .3 8&8“. no: can pmaamuu .m> u0m mono: mxmmz :H maHu COHumnaocH pmaamue Hmwam OCHE< .oN mHnme .mcon HHom Eoum wcH>mH00u=m an pmmmmHmu chHuomuu 133 .Amovm um pmoamuu how momma wchcoammuuoo .Amovm um newnmmmNn NHusm0NmNamNm .<>OZ.< C.“ UfiUDHUCfi HOG mums» OHQN NEHU HOW mMSHQ> Eoum ucmpmmeu hHucmoHMchHm mum pmaamuu uo: you mammz no: mum umuum mamm m u uHB magmanoom m:moE...o. .m . p . a I! -K II +0- u -s {NNN mmN qu *NNN NNq Nam NNN *H.Nm m.aq NN.N awesHm\ncmm NNN .NNN qu *QNH mos *qu NNN «N.Nm H.NN a.m mmest\NNom emaamtn 2oz xHNgwmm :Nmm meg m an stHN we EH oNN «NN qu NNN mam Nae «NH NNN N.oq NN.H ewest\ncmm New stN *unm pm owe xHNsm meszm EH qu NNN NNN NHq qu NNN NON moN m.am N.N mwenHm\HNom emaamne lllllllllllllllllllllllllllllllllllllllllll w\zw1 IIInIIIIIIuIIIIIIIIaIIIIII:II emaamuu soc 0N 4N NH oN w o a N +o emaampu no: new pmaamuu Eanmz .m> pow mono: mxmos cH maHu :OHumnsocH pwaomua .mmu3uxHE mwpsHm\©cmm pom ownsHm\HHom scum coHuomuw unwom onwam oHnmuomuuxm >HuomuHQ .NN mHan 134 .Amovm um pmdmmuu no“ momma wchCOQmmuuoo scum uanmNWHp >HucmonchHm mum vmaamuu uoc pom memo: .Amovm um ucmuowpr >HucmoHWchHm uoc mum umuumH mEmm mnu suHB vasquooom momma...xw.< m...o.n.m .<>OZ< CH fiUfiDHUCH HOS OHGB OHMN QEHH HON mQJHm> ll -K II .fl. II ..p. «.mm NHH N.qq o.mm «mmH m.mn N.mN m.wN w.qH m.m ownsHm\ncmm m.qm «ooH w.Nm qu NNH «wHH «m.mq «m.mm «m.mH o.mN mwanm\HHom nomamuu uoz m nun N; can meg zwmm New N N N.m© mHH N.om mHH HQH m.mo H.mm N.HN w.qH m.¢ mwpaHm\pcmm < as New a was New owe was *ewum N.Nm NNN N.HN qu OHH N.HN m.Nm N.mw o.mo o.mN owesHm\HNom emaamue u: IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII w\zw: llllllllllllllllllllllllllllll cmadmuu uoc OH «H NH 0H m o c N +.o vmaamuu no: new pmdamuu EsHpoz .m> now mammz mxmmz :H mEHu coHumnsoaH vmmamuh .mmusuxHE wwpaHm\ocmm cam mwpaHm\HHom Eouw wcw>mHUOusm >n pmmmmHmu :oHuomum Hmwom OCHE< .wN MHAmH 13S .Amovm um pmadmpu no“ momma wchcoammpuoo Eoum ucmumMWHp >HucmuHMchHm mum pmaamuu uo: pom memo: u « .Amovm um ucmumNMHp >Hucmonwstm uoc mum umuumH msmm mcu :uHB pchmQEouom momma...o.n.m u * .<>oz< :H pmpaHooH no: mumz oumm mEHu pom mmnHm> u + omNN oomN «mHNm won «once NHmN on on mmm m.o~ poodmpu uoz m m e... u w a .1 u: NoNN NHmN mmmN Homm mmmm HoHN Now «Na 0mm m.om umdomue lllllllllllllllllllllllllllllllllllllllllllllllll w\zw1 IIIIIIIIIIII:IIIIIIIIIIIIIIIIuIIII e383 no: 2 i NN S N o q N t. o 832“ no: new cmaamuu .m> HON memo: mxmmz cH mafia COHumnoocH pmaamua .mcon ownsHm 50pm COHuomnm umwsm ocHEm mHnmuomuuxm xHuomuHQ .mN mHnme 136 .pmdamuu new mmsHm> Eouw ucoummev no: mum pomqmuu uoc MOM mmsHm> u m .Amovm um ucmummep >HucmonHcmHm no: mum umuuMH mamm mnu sow: pchdeooom momma...o.a.m n"% .<>oz< :H pmpoHocH no: mums oumn QaHu How mmaHm> n + «oMH mmmH mHoN MNMN NNoN mme HHN ohm MNm mmq mandamuu uoz am an m a o p mop + w NNNN momH ome mNmN oomH ooNN NNN mmm NNN was emanate llllllllllllllllllllllllllllllllllllllllllllll m\zw1 IIIII lulunuuulnnnnlnull emaamnu soc OH «H NN ON m o s N .Ho emaamnu Soc cam pmmamuu .m> no“ mommz mxmoz CH mEHu coHquSUCH pmaamue .mcon mwpaHm Eoum wcH>mHUOuzm up pmmmmHmu :oHuomum umwsm OCHE< .om mHan 137 .Amovm um nmaamuu pow memos wchcoammuuou Eoum ucmummme mHucmoHMchHm mum wmammuu no: you mono: ll '1‘ .Amovm um ucmHQMMHu >HucmonchHm uoc mum umuumH 05mm mcu :uH3 ochmQEooom momma...o.n.m u % .<>oz< :H pmpDHocH nos mama oumu oEHu How mmon> u + «NN.¢ .am.m *m«.« «Nm.N m.oH N.CH N.«H ««.oH m.«H m.«H emaampu uoz m m m mp on on a “two N.oN m.NN m.0m m.NN n.oH w.OH o.mH N.HN m.«H w.«H pmaamue IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII m\zw1 Illallnulllnluluu II IIIII pmadmuu no: OH «H NH 0H m o « N .Ho pmmamuu no: new pmammuu .m> now mama: mxmma cH msHu :oHumnaocH pmaamue .ocon HHom « aouw :z mHAmuomuuxm zHuomuHQ .Hm mHan + 138 .Hmovm um pmaamuu now momma wchcoamwuuoo Eoum ucmummme mHucmoHMchHm mum pmammuu no: MOM mama: .Amovm um ucwummep hHucmoHWchHm no: mum umuuvH oEmm mcu :uH3 pchmanoom m:mmE...u.n.m .<>OZ.< CH UOQSHUCH HOG 0Hm3 OHQN mEHu HOW mmSHm> II ll %- t II + «N.o« 4N.Nm N.oo N.om o.Nm H.«m N.NN N.NN N.mm o.mo emaamnu uoz cu m up a owe we ; “was m.«« H.m« N.HN N.Hm H.mm o.mm N.Hm N.HN «.mm o.mo ameamne llllllllllllllllllllllllllllllllllllllllllllllll w\zw1 lulllllllillllllIIIIIIIIIIIIIIIIII pmadmuu no: oH «H NH oH w o « N .Ho voaamnu no: pom nonamnu .m> NON mammz mxmm3 :H mEHu COHumnsoaH cmaamua .QGOHm HHom Eouw wcH>mH00usm xn pommmHmu Zlachan< .Nm mHnme 139 .Amovm um vmmdmuu pow momma wcHucoammuuoo Boom ucmummep hHucmonHcmHm mum pmadmuu no: HOw mono: .Hmovm um ucmumHmHn xHucmonchHm no: mum uOOOOH 05mm mnu nqu pchmOEooom .¢>oz< :H pmpOHucH no: mums ll -K memos. o oma ll .1... .«mm NON com mqq NNN NNm mam «qu «HN N.NN mwesHm\ecmm room NHN OH« «NHm NHN NNN HNN *HmH NNH «.mm mNEHEHHom emaamnu “oz < Hammm mean eunm Hewcm eunm momma H xH HNN NHN NH« «ms NNN «Nq «NH «.mq NHN N.NN mwenHm\ecmm m xHH; «can womau xHH; Newmm xHH; H 4.1 NSN NoN NH« mmm OQN NNN qu m.om HmH «.am mmesHm\HHom emanate IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII w\szHIIIIIIIIIIIIIIIItlulullllllllll emaamnu no: OH «H NH 0H m o a N .Ho emaamuu no: can vmammuu EOHme .m> pow mama: mxmm: OH maHu OOHumnaocH pmaamue .mOHSOxHa mwpaHm\pcmm pom mwpaHm\HHom aoum :2 mHanomuuxm hHuumuHo .mm mHnmE 140 .Amovm um pommmuu How momma wchcoammuuoo Eoum ucwummep zHucmOHchme mum pmmmmuu no: how memo: .Hmovm um ucmummew xHucmOHchme no: mum nmuuoH mEmm man nqu pchmOsooom .<>OZ¢ CH UOGDHUGfi uOfl 0Hm3 OHQN mEHu HOW mwfiHHw> momma...m. you mammz mxmm: OH mEHu OOHOOOOOOH pmadmua .mmusuxHE wwpan\p=mw pom ownsHm\HHom aoum wOH>mHOOOOm xn vmmmmHmu ZIEOHOOEE< .«m mHan 142 .Hmovm no Umaamuu new momma wCHUOOOmmHnOO Scum acoummep xHucmunchHm mum pmaemnu no: nOu mccoz I ! .Amovm um ucmummuHc xHucmOHuchHm no: mum HouumH mEmm mzu LUHB pchmOEOoum m:mmE...o.O.m n % .<>Oz< OH popOHucH no: mnoz OHmN mEHu no“ mmon> u + NNNN NmoH HocH o«m «moH «mN «HNN «NNN NNN NNN pchmnH ucz cm a on m ONO mu m % 61o HHN NmOH oHHH Nmm NooH N«N HNN N«m mNN NoN mundane IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII w\zw1 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 3.5m: nos NH «H NH NH N N a N +0 Exam: soc pom pwaasuu .m> .HOu mammz 9395 :H 9:: coHumnsocH pmoamue .mcon mwpsHm Eoum wcH>mHO0u=m an pmmmmHmu zuEOHOOEE< .om oHan 143 .Hmovm Om uOmummeO >HOOOOHchme OOO mum umoumH msmm OOH >3 OmHOmOEooum mOmma...m. .Nm mHnme 144 .Hmovm um uOmummeO >HuOmOHwHOme uOO mum HOOHOH mamm mOu >3 OOHOOOEOOOO mOmoa...m. .NN «Hams 145 .Hmovm um pmaamuu How mOme wOHpOOOmmHuoo Eouw HOOOONWHO NHOOOOHMHOme mum OOOOOHO uOO pom mOmmz u « .Hmovm um uOmewwHO zHuOOOHwHOmHm uOO mum HmuumH mamm man OOHB OOHOOOEOOOO mOmwB...m.oz¢ OH OOOOHOOH HOO muoz OHON mEHu Now mmOHm> n.H «N.«H N.N *NHN «N.N N.N *«.«N N«.NN N.NN N.N H.NN NNNNHN\HHON «N.«N ««.ww NH.wN «N.No NN.NN «m.mm «N.Nm ««.HN ««.wm H.Nm OOOHO HHom OOOOOHO uoz N NH N N NHH H: NHHN NNN HHHN «.N N.H ««H m.H N.N m.m «.m N.m« N.N H.0m mwOOHm\HHOm < wmmp mm mm m mum mm wmm mHN N.NH H.NN N.NH N.NH N.NH «.HN «.NH N.NN N.N H.NN Naon HHON NNNNNNH llllllllllllllllllllllllllllllllllllllllll + w\zw1 IlluunlulullnulIlllunnvnlulluIn NNNNNNN Nos NH «H NH NH N N « N N.N NNNNNNN Nos OOm pmmampu SOHOoz .m> How mOmmz mxmma OH mEHu OOHOOOOOOH OOOOOHH .mHOuxHE mwcsHm\HHom OOm wOon HHOm How muHHuHO mOHO mumuqu .mm OHOOB 146 .Amovm um pmaamuu you mOmme wOHOOOOmmHHou Eouw NOONOHMHO zHuOOOHMHOme mum OOOOOHO uOO How mOmmz II -K .Hmovm um uOmumwwHO zHuOmoHMHOme uOO mum HauumH mamm mxu OOH3 OOHOOOEOOOO mOmmE...m.oz< OH OOOOHOOH uOO mums OHmN oaHu you moaHm> II +3. ll 4. «NN.N N«.« NN.N NN.« «NN.« «NN.NH «HN.N NNN.N HN.N NN.N NOOHN mNHSHN NNN.N NN.N «NN.N «NH.N «NH.« «NH.N «NN.N NNN.N No.N N«.o NNN=HN\NNNN NNNNNHN Noz N NHHNN HHNNN NNNN NHHNN No NNN me u NN.N NN.N NN.N NN.« NN.N NN.N NN.« NN.N NN.N NN.N NNNHN mNNsHN m OO O no OOOE OOO O OOOB mOEme NN.H «H.H NN.o NN.H NN.H NN.H Nm.o NN.H NN.N N«.o NNNsHm\NcmN NNNNNNH IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII % m\zw1 InIIIIIIIIIIIIIIIIIInIIIIIIIII NNNNNNN No: NH «H NH NH N N « N + o NNNNNHN No: OOO pmaamuu MOM mOmm: mxmmz OH mEHu OoHumnsoOH EOHOOZ .m> tonnage .mOon mwOOHm pOm OHOOXHB mwpaHm\pOmw HOW ONHHuHO OOHO mumuqu .o« OHOOH 147 .Amovm um pmaamuu How mOmmE wOHOOOOmmHuoo Eouw uOOHONMHO zHuOOOHMHOme mum pmadmuu uOO How mOmmz .Hmovm Om uOmummeO AHHOOOHNHOme uOO mum umuumH mamm OOu OuH3 OOHOOOEOoom mOmmE...u.m.< .<>oz< OH OOOOHOOH uOO who: OHON maHu Mom mmsHm> o coconnnm ll '3‘ ll -H- II 4. «Nwm NN« mmm Nm« HH« No« owm «wmo HHOH mNmH mwOOHm\OOmm *o«NH «ONHH «NNQH HoHH «on NmNH mHNH NoNH mHNH NNNN owOOHm\HHOm «NNN NNN NNN N«N NHN N«N NNN NNN NHNN NNN NHNO HHoN NNNNNNN Noz o OOE OEH go no OOO Hx flHOw mum Hmo mmm mHo oom Nm« mHm NON owm mum mmmH mwOOHm\pOmm < n n on to O on on m HNmH «mNH NONH mmNH mcHH NNmH NNNH omNH NmNH NNNN mmpaHm\HHom N HNNN NNN HNNN HHN HNNN HNNN HHN N NNN NHN NHN NNN NNN N«N NNN NNN N«N NNN NNN NHeo HHoN NNNNNNH IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII m\zw1 IaulcnnnunIqlutluunlualialnluun pmaamuu uOO NH «H NH 9H m o « N +.o OOOOOHO uOO OOm Omaamuu EOHOOZ .m> NON mOmmz mxmmz OH OoHumnaoOH mo msHB OOOOOHH .OOmm no HHom OOH: OmOOHm mo mounuxHe OOm HHOm NON wOH>mHOOuOm unumm z Hmszmmm .H« OHOOB 148 .prOmnu NON mmsHm> EoOw uOmpmwwHU Ho: mum Omammuu uOO OOH mmsHm> ll Lm .Amcvm um ucmumwNHO NHOOOOHNHOme uOO mum cmuumH mEmm msu :NHB OOHOOOEOOOO mOmmE...O.n.m u % .<>Oz< OH OOOOHoOH no: mumz Oumw oEHu NOW mwOHm> u + NN.N NN.« NN.N «N.N NH.N NH.N N«.N «N.N NN.N NN.N N NNNNNNN Noz mm mono wowu mm mm onm m «.Om NN.N cH.m NN.N HN.N HH.N HH.N NN.N NN.N HN.N NN.N NNNNNNH lllllllllllllllllllllllllllllllllllllllllllllllllll ZN IIIIIIIIIllllllllllllllllllllllIllll poaamuu NCO 0H «H NH OH w o « N +HN Omaamuu uOO Ocm OOOOOOO -sll .m> now mcmmz mxmo3 OH mEHu OOHOOQOOOH OOOOOOH .OOOHO mwszm NON wOH>OHOON2m umeO z HmOpHmmm .N« mHOmH BIBLIOGRAPHY BIBLIOGRAPHY Aleem, M.I.H., M. Alexander. 1960. Nutrition and physiology of Nitrobacter agilis. Applied Microbiol. 8:80-84. Alexander, M. 1961. Introduction txa Soil Microbiology. p. 272-308. John Wiley & Sons, New York. Allison, F.E. 1963. Losses of gaseous nitrogen from soils by chemical mechanisms involving nitrous acid and nitrites. Soil Sci. 96:404-409. Allison, F.E. 1965. Evaluation of incomimg and outgoing processes that affect soil nitrogen. NHL. W.V. Bartholomew and F.E. Clark (ed.) Soil Nitrogen. Agronomy 10:573-606. Amer. Soc. of Agron., Madison, Wisconsin. Ashida, J} 1965. Adaptation of fungi to metal toxicants. Annu. Rev. Phytopathol. 3:153-174. Barrow, \hln 1955. Use of activated sewage sludge as a fertilizer. World Crops 7:435—437. Bear, F.E., and A.L. Prince. 1947. Agriculture value of sewage sludge. New Jersey Agr. Exp. Sta. Bull. No. 733, New Brunswick, N.J. 12p. Beauchamp, E.G., G.E. Kidd, and G. Thurtell. 1978. Ammonia volatilization from sewage sludge applied in the field. J. Environ. Qual. 7:141-146. Berrow, M.D., and J. Webber. 1972. Trace elements in sludges. J. Sci. Fed. Agr. 23:93-100. Bloomfield, C., W. I. Kelso, and G. Pruden. 1976. Reactions between metals and humified organic matter. J. Soil Sci. 27:16-31. 149 150 Bloomfield, C., and G. Purden. 1975. The effects of aerobic and anaerobic incubation on the extractability of heavy metals in digested sewage sludge. Environ. Pollut. 8:217-232. Bollag, J.M., and G. Tung. 1972. Nitrous oxide release by soil fungi. Soil Biol. Biochem. 4:271-276. Boswell, F.C. 1975. Municipal sewage sludge and selected element application to soil: Effect on soil and fescue. J. Environ. Qual. 4:267-273. Bremner, J.M. 1965a. Total nitrogen. In C.A. Black (ed.) Methods of soil analysis. Agronomy 9, Part II: 1149-1178. Amer. Soc. of Agron., Madison, Wisconsin. Bremner, J.M. 1965b. Inorganic forms of nitrogen. £3 C.A. Black (ed.) Methods of soil analysis. Agronomy 9, Part II: 1179-1237. Amer. Soc. of Agron., Madison, Wisconsin. Bremner, J.M. 1965c. Organic forms of nitrogen. In C.A. Black (ed.) Methods of soil analysis. Agronomy 9, Part II: 1238-1255. Amer. Soc. of Agron., Madison, Wisconsin. Bremner, J.M., enui A.M. Blackmer; 1978. Nitrous oxide: emissions from soils during nitrification of ferti- lizer nitrogen. Science 199:295-296. Bremner, J.M.. and L.A. Douglas. 1971. Use of plastic films for aeration in soil incubation experiments. Soil Biol. Biochem. 3:289-296. Bremner, J.M., and F. Ehflun 1966. Tracer studies of the reaction of soil organic matter with nitrite, p. 337- 346. _£Q_the use of isotopes in soil organic matter studies. Pergamon Press, Inc., Elmsford, N.Y. Bremner, J.M., and K. Shaw. 1955. Determination of ammonia and nitrite in soil. J. Agr. Sci. 46: 320-328. Bremner, J.M., and K. Shaw. 1958. Denitrification in soils II. Factors affecting denitrification. J. Agr. Sci. 51:40-52. Broadbent, F.E. 1973. Organics. p. 97-101. In Recycling municipal sludges and effluents on land. National Assoc. of State University and Land-Grant Colleges. Library of Congress Catalog no. 73-88570. Broadbent, EBEL, and E2. Clark. 1965. Denitrification IE; W.V. Bartholomew and F. Clark (ed.) Soil Nitrogen. Agronomy 10:344-359. Amer. Soc. Agron. Madison, Wisconsin. 151 Broadbent, F.E., and F.J. Stevenson. 1966. Organic matter interactions. p. 169-187. £1_H.N. McVickar et al. (ed.) Agricultural Anhydrous Ammonia. Technology and Use. Proc. Symp. St. Louis, Mo., 29-30. Sep. 1965. Agric. Ammonia Inst., Memphis, Tenn., Am. Soc. of Agron., and Soil Sci. Soc. of Am., Madison, Wisconsin. Bunting, A.M. 1963. Experiments on organic manures, 1942-49. J. Agri. Sci. 60:121-140. Bunzl, K., W. Schmidt, and B. Sansonik. 1976. Kinetics of ion exchange in soil orga 'c ma er: V. Adsor ion and desorption of Pb2+, CJ&+, Cdai, ZnE+, and CaBE by peat. J. Soil Sci. 27:32-41. Cady, E.B., and W.V. Bartholomew. 1960. Sequential products of anaerobic denitrification in Norfolk soil material. Soil Sci. Soc. Am. Proc. 24:477-482. Chaney, R.L. 1973. Crop and food chain effects of toxic elements in sludge and effluents. p. 129-144. NHL Proc. of the Joint Conf. on Recycling Municipal Sludges and Effluents on Land. Champaigne-Urbana, Ill. Coker, E.G. 1966a. The value of liquid digested sewage sludge: I. The effect of liquid sewage sludge on growth and composition of grass-clover swards in southeast England. J. Agric. Sci. 67:91-97. Coker, E.G. 1966b. The value of liquid digested sewage sludge: III. The results of an experiment on barley. Craswell, EXT. 1978. Some factors influencing denitrifi- cation and nitrogen immobilization in clay soils. Soil Biol. Biochem. 10:241-245. Cunningham, J.D., D.R. Keeney, and J.A. Ryan. 1975. Yield and metal composition of corn and rye grown on sewage sludge amended soil. J. Environ. Qual. 4:448-454. Day, B.D., and T.C. Tucker. 1958. Production of small grains pasture forage using sewage effluent as a source of irrigation and plant nutrients. Agron. J. 51:569-572. Day, A.D., T.C. Tucker, and M.G. Vavich. 1962a. City sewage for irrigation and plant nutrients. Crops and Soils 14(8):7-9. Day, A.D., T.C. Tucker, and M.G. Vavich. 1962b. Effects of city sewage effluent on the yield and quality of grain from barley, oats, and wheat. Agron. J. 54: 133-135. 152 Day, A.D., and R.M. Kirkpatrick. 1973. Effects of treated municipal wastewater on oat forage and grain. J. Environ. Qual. 2:282-284. Dement, J.D., G. Stanford, and C.M. Hunt. 1959. A method for measuring short term nutrient absorption by plants: III. Nitrogen. Soil Sci. Soc. Amer. Proc. 23:371-374. Dowdy, R.M., and W.E. Larson. 1975. Metal uptake by barley seedlings grown on soils amended with sewage sludge. J. Environ. Qual. 4:229-233. Duggan, J.C., and C. Wiles. 1976. Effects of municipal compost and N fertilizer on selected soils and plants. Compost Sci. 17:24-31. Eno, C.F. 1960. Nitrate production 1J1 the field by incubating the soil in polyethylene bags. Soil Sci. Soc. Amer. Proc. 24:277-279. Epstein, E., D.B. Keane, J.J. Meisinger, and J.O. Legg. 1978. Mineralization of nitrogen from sewage sludge and sludge compost. J. Environ. Qual. 7:217-221. Evans, J.D. 1968. Usimg sewage sludge (n1 farmland. Compost Sci. 9(2):16-17. Feigin, A., S. Feigenbaum, and H. Limoni. 1981. Utiliza- tion efficiency from sewage effluent and fertilizer applied to corn plants growing in a clay soil. J. Environ. Qual. 10:284-287. Fewson, C.A., and D.J.D. Nicholas. 1961. Utilization of nitrate by microorganisms. Nature (London) 190:2-7. Firestone, M.KN 1982. Biological denitrification In F.J. Stevenson (ed.) Nitrogen in Agricultural Soils. Agronomy 22:289-326. Fraps, (L. 1932. The composition and fertilizer value of sewage sludge. ‘Dexas Agr. Exp. Sta. Bull. No. 445, College Station, Tex. 23 p. Gadd, G.M., and A.J. Griffiths. 1978. Microorganisms and heavy metal toxicity. Microb. Ecol. 4:303-317. Garner, H.V. 1962. Experiments with farmyard manure, sewage sludges, and two refuses on microplots at schools, 1940-1949. Empire J. Exp. Agr. 30(120): 295-304. 153 Garner, H.V. 1966. Experiments on the direct, cumulative and residual effects of town refuse, manures, and sewage sludge at Rothamsted and other centres, 1940-47. J. Agr. Sci. 67:223-233. Guenzi, W.D., W.E. Beard, F.S. Watanabe, S.R. Olsen, and L.K. Porter. 1978. Nitrification and denitrification in cattle manure-amended soil. J. Environ. Qual. 7:196-202. Haghiri, F. 1973. Cadmium uptake by plants. J. Environ. Qual. 2:93-96. Hartman, L.M. 1974. .A preliminary report: Fungal flora of the soil as conditioned by varying concentrations of heavy metals. Am. J. Bot. 61:23. Hashimoto, A.G., and D.C. Ludington. 1971. Ammonia desorp- tion from concentrated chicken manure slurries in livestock. waste: management. and. pollution abatement. Proc. Intl. Symp. Livestock Waste. Am. Soc. Eng., Columbus, Ohio. Hinesly, T.D., R.L. Jones, and E.L. Ziegler. 1972. Effect on corn of application of heat-dried anaerobically digested sludge. Compost Sci. 13:26-30. Hinesly, T.D., E.L. Ziegler, and G.L. Barrett. 1979. Residual effects of irrigating corn with digested sewage sludge. J. Environ. Qual. 8:35-38. Hoagland, D.R., and D.I. Arnon. 1950. The water-culture method for growing plants without soil. Calif. Agr. Expt. Sta. Circ. 347. Hortenstine, C.C., and D.F. Rothwell. 1968. Garbage compost as a source of plant nutrients for oats and radishes. Compost Sci. 9(2):23-25. Hsieh, Y.P., L.A. Douglas, and R.L. Motto. 1981. Modeling sewage sludge decomposition in soil: II. Nitrogen transformation. J. Environ. Qual. 10:59-64. Jackson, M.L. 1960. Soil chemical analysis. Prentice-Hall, Inc., Englewood Cliffs, N.J. Jansson, S.L. 1958. Tracer studies on nitrogen transforma- tions in soil with special attention to minerali- zation-immobilization relationship. Kungliga Lantbru- shogskolans Annalar. 25:101-361. Jansson, S.L. 1971. Use of 15N in studies of soil nitrogen. p. 129-166. In A.D. McLaren and J. Skujins (ed.) Soil biochemistry II. Mercel Dekker Inc., New York. 154 Jansson, S.L., and .1. Persson. 1982. Mineralization and immobilizathmw of soil nitrogen. In F.J. Stevenson (ed.) Nitrogen in Agricultural Soils. Agronomy 22:229-252. John, M.K., H.H. Chuah and C.J. VanLaerhoven. 1972. Cadmium contamination of soil and its uptake by oats. Environ. Sci. Technol. 6:555-557. John, M.K., and C.J. VanLaerhoven. 1976. Effect of sewage sludge composition, application rate, and lime regime