*2.2. Feeding Phases*

The reactor was filled with digestate coming from an anaerobic digestion industrial plant treating corn silage and triticale. The digestate was used as received from the industrial plant. Then, a start-up phase was performed. During this phase, the temperature was increased by 2 ◦C daily until a constant value of 39 ◦C (mesophilic conditions) was reached.

Subsequently, the daily feeding rate of admixtures (Q, [m3substrate·d−1]) was increased during the first phase to reach an adaptation of the bacterial consortium to the specific substrate. After the start-up phase, the hemp to digestate proportion (hereinafter: percentage of new hemp straw in the admixture C, [% wt/wt]), Q, and digestate recirculation ratio (R, adimensional) were changed during the experimental period.

C and R values were chosen keeping in account the fluid-dynamics behavior of the admixtures. An increase of the hemp straw amount, indeed, could make the pumping of the admixtures itself difficult.

Two reference values of C (C1: 3% wt/wt; C2: 5% wt/wt) were set to evaluate the process. These conditions were tested by considering the presence/absence of specific bioenhancers. Treatments were randomly applied during the entire experiment.

Concerning the ranges of the organic loading rate (OLR, [kgVS·(m3reactor·d)−1]), hydraulic retention time (HRT, [d], dependent on Q), R, and C, different regimes were defined.

The abovementioned variables were analyzed along with the specific gas production (SGP, [m3biogas or methane·kgVS−1]) and the gas production rate (GPR, [m3biogas or methane·(m3reactor·d)−1]).

A commercial enzymatic preparation (Micropan Biogas ®from Eurovix, IT) was applied to reduce the current supply of hemp straw residues and to maximize biogas production at the same time. It is made of microbial enzymes containing cellulase, lipase, xylanase, active principles of *Fucus Laminariae*, algae *Lithothamnium calcareum*, natural nutrients/grow factors, selected yeast, mineral biological catalysts rich in oligo elements, and selected microorganisms (facultative anaerobic bacteria, such as: *Bacillus subtilis*, *Bacillus macerans*; strictly anaerobic bacteria genus *Methanobacterium*). The specific gravity was 0.8 <sup>t</sup>/m3.

The daily intake was introduced into the reactor by dissolving the powder into water (1:4 wt/v).

The dosage was divided into two parts, depending on the fiber composition of lignocellulosic feedstock (Table 4), C, and Q. The daily intake was defined by multiplying the percentage of cellulose and hemicellulose of hemp residues (see Table 4) with the hemp mass in Q and a coefficient of 0.05, as suggested by the producers. Thus, 20 g per day were obtained.

The first enzymatic inoculum of the reactor sludge was calculated by dividing the working volume of the reactor by Q, and by multiplying this value with the daily intake (500 g of bioenhancers were introduced into the reactor).

#### *2.3. Management of the Reactor and Process Stability*

Both the process stability and reactor features were controlled and managed by using a set of parameters.

Management of the reactor: As regards the reactor, the following were considered:


$$HRT = \frac{V}{Q'} \tag{1}$$

where V is the total digester volume [m3reactor] and Q is the daily feeding rate [m3substrate·d−1];

• OLR ([kgVS·(m3reactor·d)−1]), calculated as:

$$OLR = \frac{V \cdot S}{Q},\tag{2}$$

where S is the VS concentration on a wet basis in the feeding admixtures [%wtw.b.].

Management of process stability: The process stability was monitored by considering the below listed parameters:


Performance parameters: The two main performance parameters considered in the anaerobic digestion trials carried out on hemp straw residues are:

• SGP [Nm3biogas or methane·kgVS−1], calculated as:

$$SGP = \frac{G}{Q \cdot S} \tag{3}$$

where Q and S were already described, G is the daily production of biogas/methane [m3biogas or methane·d−1];

• GPR ([Nm3biogas or methane·(m3reactor·d)−1]), calculated as the daily production of biogas/methane per m<sup>3</sup> of sludge accumulated in the reactor.

The abovementioned parameters are related to:

• C (percentage of new hemp in the admixture, [% wt/wt]), calculated as:

$$\mathcal{C} = \frac{\text{Mass}\_{\text{hemp}}}{\text{Mass}\_{\text{hemp}} + \text{Mass}\_{\text{digestate}} + \text{Mass}\_{\text{water}}} \cdot 100,\tag{4}$$

where: Masshemp, Massdigestate, and Masswater are the mass of the hemp, digestate, and water composing the admixtures;

• R (digestate recirculation ratio, adimentional), following Equation (5):

$$R = \frac{\sum\_{i} \text{Mass}\_{digesrate}}{\sum\_{i} \text{Mass}\_{hcmp}},\tag{5}$$

where <sup>Σ</sup>iMasshemp and <sup>Σ</sup>iMassdigestate [g·<sup>10</sup>3] are the cumulative amounts of new hemp or digestate composing hemp-digestate admixtures in a specific time window.
