*2.3. Incubation Procedure*

For preincubation, 252 samples of field-moist soil (50 g dry weight equivalent) were weighed into 120 mL polypropylene specimen containers and adjusted to 40% WHC by the addition of deionized water using a Metrohm 665 Dosimat (Metrohm, Herisau, Switzerland). To maintain moisture content and aerobic conditions, the containers were each sealed with Parafilm (Alcan Packaging, Neenah, WI, USA) that was punctured by forming 8–10 holes with a syringe needle. Preincubation was carried out for 7 d under dark conditions in a constant-temperature room maintained at 25 ◦C.

After preincubation, the following treatments were randomized among nine sets of replicate soil samples: (1) no amendment (control), (2) high N residue (HNR), (3) HNR + KNO3, (4) HNR + (NH4)2SO4, (5) low N residue (LNR), (6) LNR + KNO3, (7) LNR + (NH4)2SO4, (8) KNO3, and (9) (NH4)2SO4. Residues were added at the rate of 10 mg dry weight g−<sup>1</sup> soil, which was selected to roughly represent modern corn production in the Midwestern USA, followed by thorough mixing for uniform incorporation. Nitrogen as NO3 − or NH4 <sup>+</sup> was applied as uniformly as possible using the Dosimat to dispense a solution that supplied 0.1 mg N g−<sup>1</sup> soil. All soil samples were treated with sufficient deionized water to bring the moisture content to 50% WHC by using the Dosimat, three specimen containers from each treatment (total of 27) were weighed for monitoring soil moisture content, and every specimen container was transferred to a 1.9 L wide-mouth Mason jar. Three jars from each treatment-specific set of samples, and three additional jars with no soil, were sealed using lids equipped with a pair of ball valves for atmospheric sampling, and the remainder were sealed using standard jar lids. All jars were returned to the constant-temperature room for 60 d of incubation in the dark at 25 ◦C.

After incubation for 7, 14, 30, 45, and 60 d, five moist soil samples per treatment were thoroughly mixed, and the composite sample was analyzed for soil pH, active biomass, microbial biomass C and N, cellulase and protease activities, and gross N mineralization and immobilization.

#### *2.4. Atmospheric Analyses*

At 1 d intervals during the first week of incubation, and at 2 or 3 d intervals thereafter, the Mason jars equipped with gas sampling lids (Figure 1) were each connected to a sampling tube and circulating pump for atmospheric sample collection following the

technique described by Horgan et al. [38]. After sampling, lids were removed from all jars in use for incubation and also from the three used for background atmospheric sampling, and the jars were left open for one hour of aeration. If necessary, deionized water was added to replace evaporative losses before reattaching jar lids, and incubation was resumed in darkness at 25 ◦C.

**Figure 1.** Unit used for incubation with atmospheric sampling by the system of Horgan et al. [38], consisting of a specimen container with soil (1) in a 1.9 L Mason jar equipped with a lid having inlet (2) and outlet (3) ball valves (item # 38EF92, Grainger, Lake Forest, IL, USA) connected to 6.4 mm O.D. brass tubing (4).

Analyses for CO2 and O2 were performed using a Hewlett-Packard Model 5790A gas chromatograph (GC) (Agilent Technologies, Santa Clara, CA, USA) equipped with an eight-port sampling valve (Valco Instruments Co., Houston, TX, USA) employing dual 0.5 mL sample loops, a Tracor U-90 ultrasonic detector (Tracor, Austin, TX, USA), and a Hewlett-Packard Model 3390A reporting integrator. This instrument used ultra-high purity He as the carrier gas, Porapak Q for separation of CO2 at 50 ◦C, and molecular sieve 5A for separation of O2 + Ar at 25 ◦C. Calibration was carried out for every set of analyses using certified mixtures of CO2 in He (Matheson, Joliet, IL, USA) and of O2 in N2 (Airgas, Radnor, PA, USA).

#### *2.5. Soil pH*

Duplicate 5 g (dry weight) samples from each treatment were mixed with sufficient deionized water to obtain a 1:1 soil:water suspension, and pH was measured using a glass electrode [27].

#### *2.6. Microbial Biomass Parameters*

The active component of microbial biomass was estimated by a modified version of the biokinetics method described by Van de Werf and Verstraete [39]. In the modified method,

5 g (dry weight) of soil was adjusted to 60% WHC with or without the addition of glucose medium and then incubated (25 ◦C, 6.67 h) in a 250 mL straight-sided glass jar equipped with a gas-tight lid having a ball valve for CO2 analysis using the GC system previously described. Active biomass was calculated using the equation given by Van de Werf and Verstraete [39] from the increase in CO2 production observed for glucose-treated samples.

The chloroform fumigation/extraction procedure described by Vance et al. [40] was utilized to measure soil microbial biomass C (MBC) and N (MBN). Following K2SO4 extraction, organic C was determined by dichromate oxidation [28] for calculation of MBC (*Bc*) using the equation proposed by Vance et al. [40], *Bc* = *Fc*/0.45, where *Fc* = [(organic C extracted by K2SO4 from fumigated soil) − (organic C extracted by K2SO4 from non-fumigated soil)], and the value in the denominator represents the proportion of biomass C mineralized to CO2.

Microbial biomass N was estimated from the difference between fumigated and unfumigated extracts when analyzed for total N by Kjeldahl digestion [29] and diffusion [30]. A value of 0.54 was assumed as the correction factor for calculating biomass N (BN) by an equation that follows the same form as the one given in the previous paragraph for biomass C.
