*2.2.1 NH3 recovery*

Our process design principle is to keep the process as simple as possible: our NH3 recovery system consists of two columns: the NH3 stripping by aeration in one column and the NH3 dissolution into the water in another.

**Figure 1** illustrates the experimental setup for the NH3 recovery. Namely, our process strips the NH3 gas from manure/digestate liquid by aeration, using a low-cost chemical, specifically a BrØnsted base, in Column a and then dissolves the NH3 gas into an acidic aqueous solution in Column b to produce a highly concentrated N solution.

#### **Figure 1.**

*Experimental setup for the NH3 recovery: (a) the NH3 stripping column, (b) the NH3 recovery column, (c) an air pump, (d) an air diffuser, (e) a pipe, and (f) discharge of the NH3 solution.*

Our process is based on the following reactions for the NH3 stripping, eq. 1, and the NH3 dissolving into the water for recovery, eq. 2:

$$\text{NH}\_4^+ + \text{B}^- \rightarrow \text{NH}\_3 + \text{BH} \tag{1}$$

$$\text{NH}\_3 + \text{AH} \rightarrow \text{NH}\_4^+ + \text{A}^- \tag{2}$$

where B� and AH refer to an anion of a BrØnsted base and acid, respectively. There is no membrane and no evaporator involved in our process. Our process can produce highly concentrated N solutions from which liquefied NH3 can be obtained. Liquefied NH3 has an energy density twice as much as liquefied H2 and is receiving increasing attention as the next generation of zero-carbon energy storage or fuel [20].

The formulated sample was first introduced into Column a, the stripping column, while an acid solution was poured into Column b, the recovery column. The acid solution was prepared by mixing 550 g of 99.99% sulfuric acid with 1 L of distilled water. Stone air diffusers were located at the bottom of each column for aeration. Before aeration of the formulated sample, 7 g of Na2CO3 was added to Column a which triggered the following reaction:

$$2\text{NH}\_4^+ + \text{Na}\_2\text{CO}\_3 \rightarrow 2\text{Na}^{2+} + 2\text{NH}\_3 + \text{CO}\_2 + \text{H}\_2\text{O} \tag{3}$$

The stoichiometric amount of Na2CO3, given 2100 mg/L of NH4 + , was 6.18 g. The excess amount of Na2CO3 was added to ensure the completion of eq. 3. According to eq. 3, one mole of Na2CO3 produces two moles of ammonia, and CO2 is produced as a by-product. The subsequent aeration stripped ammonia produced by eq. 3. The NH4 <sup>+</sup> concentration was monitored by a UV-vis photo spectrometer (DR 6000 by Hach) over time. The NH4 <sup>+</sup> removal rate, *<sup>η</sup>NH*<sup>4</sup> *remove*, was defined by the following equation:

$$\eta\_{remove}^{NH4} = \mathbf{100} \times \left\{ \mathbf{1} - \left( [\mathbf{NH}\_4^+]\_{i} - [\mathbf{NH}\_4^+]\_{f}^{\mathrm{strip}} \right) / [\mathbf{NH}\_4^+]\_{i} \right\} \tag{4}$$

where [NH4 + ]i and [NH4 + ]f strip refer to the initial and the final NH4 <sup>+</sup> concentration in Column a, respectively.

The NH3 gas stripped in the stripping column was sent to Column b, along with other gases, N2, O2, and CO2, through a pipe and dissolved into the acid solution through a stone diffuser. When the NH3 gas contacts the acid solution, the following reaction occurred:

$$\text{H}2\text{NH}\_3 + \text{H}\_2\text{SO}\_4 \to (\text{NH}\_4)\_2\text{SO}\_4\tag{5}$$

This is an acid-base reaction continuing until all sulfuric acid is consumed. The NH4 + concentration in the recovery column was monitored over time. The NH4 <sup>+</sup> recovery rate, *ηNH*<sup>4</sup> *recovery*, was defined by the following equation:

$$\eta\_{\text{recovery}}^{\text{NH4}} = \mathbf{100} \times \left\{ \mathbf{1} - \left( [\text{NH}\_4^+]\_{\text{i}} - [\text{NH}\_4^+]\_{\text{f}} \right) / [\text{NH}\_4^+]\_{\text{i}} \right\} \tag{6}$$

where [NH4 + ]f rec represents the final NH4 <sup>+</sup> concentration in Column b.

The above operation was continued by replacing the spent formulated sample in Column a with a new one and adding the same amount of Na2CO3 into the Column a after each batch aeration until the NH4 <sup>+</sup> concentration no longer increased in the Column b. No additional H2SO4 was added to Column b. The original amount of

H2SO4 added to Column b, 550 g, was determined by the maximum solubility of (NH4)2SO4 in water, 744 g/L. When all H2SO4 was consumed in Column b, the operation was stopped.

### *2.2.2 Protein recovery*

We have developed a two-heating step process for the extraction of protein from DS by THP. The detailed description of the THP treatment followed by ultrafiltration (UF) for the recovery of protein from DS has been described elsewhere [21]. We have utilized a series of instrumental characterizations of the recovered protein hydrolysates (PHs): sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy, and amino acid analysis (AAA). Then, we evaluated the efficacy of PHs as an antioxidant by the in-vitro oxygen radical absorbance capacity (ORAC) measurements. All methods were described in our previous work [21].
