*3.3. Microbial Parameters*

## 3.3.1. Active Biomass

The response of microbial communities to a readily available supply of substrate can be estimated by the assay of active biomass, which in the present project involved a short-term measurement of respiration following the addition of glucose. The results (Figure 5) show that, when averaged over the entire incubation period, active biomass for residue-treated soil was higher with than without exogenous N, although the difference was not necessarily significant due to limited replication. The importance of an active microbial fraction to soil respiration is also evident from the finding that active biomass was highly correlated (*r* = 0.94, *p* < 0.001) with cumulative CO2 production in the incubation study, as in previous work by Alvarez and Alvarez [84]. A decline in active biomass was expected with the loss of residue C over time, and this was indeed observed when a 2 to 34% decrease occurred during the second month of incubations with residue, as opposed to a corresponding increase of 19 to 119% for treatments without residue (Figure 5). The latter increase, which was most pronounced for the AS treatment, reflects a drastic stimulation of glucose-responsive microbial growth and respiration following an intensifying level of starvation as substrate depletion was exacerbated by aerobic incubation. Under such conditions, the introduction of substrate triggered a microbial shift from the potentially active fraction to an active physiological state, thereby increasing the active biomass pool [85,86] that represented 13 to 35% of the total biomass C.

**Figure 5.** Active biomass measured at five intervals (7, 14, 30, 45, and 60 d) during a 60 d aerobic incubation involving an unamended control and the following eight treatments: potassium nitrate (PN), ammonium sulfate (AS), high N residue (HNR) with or without PN (HNR + PN) or AS (HNR + AS), and low N residue (LNR) with or without PN (LNR + PN) or AS (LNR + AS). Data for each incubation interval shown as a mean from duplicate subsamples with standard error bars. Statistical analyses were performed after averaging data for all five intervals, and treatments do not differ significantly (*p* < 0.05) when bars are accompanied by the same letter.

### 3.3.2. Microbial Biomass C and N

Unlike active biomass, MBC was unaffected by the presence or absence of exogenous N, with significant differences only being observed in comparing some but not all averages for treatments with and without residue (Figure 6A). Similar findings have previously been reported in relevant incubation studies by Muhammad et al. [21], Song et al. [87], and Li et al. [88], and reflect the contrasting effects of residue and mineral N inputs on microbial growth and activity. Organic substrates supply C essential to microbial biomass synthesis, while inorganic N predominantly contributes to extracellular enzyme activities [89]. The view that N can be more important for promoting microbial activities than growth is relevant to the present study because residue treatments with and without inorganic N differed significantly in active biomass and cumulative CO2 production (Figures 3 and 5) but not in MBC (Figure 6A), despite a significant correlation of the latter two parameters (*r* = 0.81, *p* < 0.01).

**Figure 6.** Microbial biomass C (**A**) and microbial biomass N (**B**) measured at five intervals (7, 14, 30, 45, and 60 d) during a 60 d aerobic incubation involving an unamended control and the following eight treatments: potassium nitrate (PN), ammonium sulfate (AS), high N residue (HNR) with or without PN (HNR + PN) or AS (HNR + AS), and low N residue (LNR) with or without PN (LNR + PN) or AS (LNR + AS). Data for each incubation interval shown as a mean from duplicate subsamples with standard error bars. Statistical analyses were performed after averaging data for all five intervals, and treatments do not differ significantly (*p* < 0.05) when bars are accompanied by the same letter.

To better clarify the interacting effects of C and N on total microbial biomass, the MBN fraction was measured at the same incubation intervals utilized in characterizing MBC. The results (Figure 6B) reveal that MBN was significantly greater for the HNR, HNR + PN, and HNR + AS treatments as compared to soils incubated without residue, and with the exception of LNR + AS, significantly lower for the LNR than the HNR treatments. Both findings can be explained by differences in the supply of assimilable N, following similar reports in several previous studies [90–92]. A significant correlation was obtained between MBN and cumulative CO2 production (*r* = 0.78, *p* < 0.05), indicating that organic C mineralization released C and N substrates for microbial biomass synthesis [93]. For the HNR + PN and LNR + PN treatments that supplied ample C and N, MBC was 2 to 25% greater in the second than in the first month of incubation, whereas the corresponding change for the HNR + AS and LNR + AS treatments was a 10% decrease due to soil acidification. Relative to the same temporal periods, a 35 to 64% decrease in MBN was observed for these four treatments, reflecting a decline in microbial activity due to substrate limitation.
