**2. Monitorization of the anaerobic digestion process stability of organic solid waste during transitory states**

This section describes the control and operational parameters relevant for determining the stability and the microbial population adaptation of AD processes. These factors have been reported as fundamental in the literature for transitory states during the AD processes, when the feedstock type or composition changes.

#### **2.1 pH, alkalinity, and volatile fatty acids concentration**

Monitoring parameters such as pH, alkalinity, and VFAs concentration are fundamental indicators of the equilibrium and stability of the AD process [6, 18].

Microbial growth and activity strongly depend on the environmental pH [19–21]. According to literature, methane producers are most active at a neutral pH, i.e. between 6.5 and 8.5 [22, 23], while at lower pH (5.0–6.0), its activity decreases severely, being active only for the acids-producing microorganisms [21]. If pH is rapidly increased or decreased concerning the existing environmental conditions, the microbial activities of specific microbial species could be inhibited. Notably, the methanogenic archaea inhibition would affect the activity of anaerobic microorganisms and, subsequently, the whole AD process performance [24]. The rapid increase or decrease of pH values could mostly occur for substrates from different origins whose physicochemical characteristics are not similar [4]. Arhoun et al. [23] reported different pH buffering processes that, while remaining active, can hide possible instabilities. Still, when the buffering capacity is depleted in the long term, abrupt pH changes could cause severe problems to the digester operation. However, other parameters can be an early warning for pH buffer depletion. Among the most used are total (TA), partial (PA), or intermediate (IA) alkalinity.

Alkalinity could be defined as the ability of the AD liquor of the mixture to buffer the possible generation of acids produced during the biological process and, hence, mitigate potential pH changes [7]. The alkalinity in the AD liquor mixture is mainly provided by the non-protonated forms of VFAs and the carbonate system. If no other species interfere within the pH range of anaerobic digesters, a VFAs accumulation would be directly related to the breakdown of the bicarbonate buffering capacity [25]. The PA, IA, and TA measurements can evaluate the relative buffering substances concentrations. TA measures the combined effect of different buffer systems and is calculated as TA = PA + IA. PA corresponds to the buffer capacity of the carbonate system, just as ammonium/ammonia. In contrast, IA is the difference between TA and PA and corresponds to the buffer capacity of the non-protonated forms of VFAs. Alkalinity titrated down to 5.75 pH value is defined as a PA, whereas TA is titrated to 4.30 [20, 26]. Some authors have reported 2000–4000 mg of CaCO3 L−1 PA values as typical for properly performing digesters under mesophilic conditions and feeding organic solid waste [11, 27, 28]. However, in terms of stability, the evolution of parameters over time is more important than the actual concentration, acting as an early warning [23].

Several studies also reported alkalinity ratios as monitoring parameters used as early warning tools [23, 25, 29]. The process stability can be evaluated by the IA/PA ratio, which involves the acid concentration in the system (IA), and the buffer capacity provided by the carbonate species (PA). If the PA is insufficient to buffer the IA, the digester will be acidified, and the activity of microorganisms, especially methanogens, will be inhibited [29]. Therefore, to consider the process stable, the IA/PA ratio

must be kept below 0.4. Some authors also indicate IA/TA ratio as a parameter for monitoring the anaerobic digestion process. However, this has lower sensitivity than the IA/PA ratio [25].

The VFAs are produced during the anaerobic degradation of organic solid waste, and their evolution provides information about the performance of the different AD steps [7, 15, 30]. Especially, the substrates with high biodegradabilities, such as fruit, vegetable, or food waste, have a higher tendency to generate VFAs. The most common VFAs are acetic (C2), propionic (C3), butyric (C4), and valeric (C5) [8]. Acetic acid has been described as the least toxic fatty acid. On the contrary, propionic acid concentration has been associated with system failure, being even more inhibitory than butyric acid [18, 31]. The propionic acid accumulation is probably related to its conversion being the least thermodynamically favorable [6]. According to some studies, a propionic acid concentration in the range of 0.45–3.00 g COD L−1 (COD, chemical oxygen demand) has a high potential to inhibit the process. Obviously, inhibition will also depend on the substrate treated and the operating conditions [6, 32, 33]. As a result, propionic acid is usually presented as the main parameter to follow when analyzing the stability of AD [7].

Another commonly used parameter to monitor the stability of the anaerobic digestion process is the ratio of volatile fatty acids (VFAs) to total alkalinity (TA) (VFA/ TA or FOS/TAC ratio) [4, 34]. This parameter is related to the buffering capacity, represented by the total alkalinity, for a given effect of the VFA on the pH of the AD liquor mixture [23]. According to the literature, there are three critical VFA/TA ratio values. If VFA/TA ratio is lower than 0.40, the digester should be stable. When the ratio ranged from 0.40 to 0.80, the digester performance would present some signs of instability, while the VFA/TA ratio higher than 0.80 indicates significant instability in the digester [7, 11, 35].

#### **2.2 Specific energy loading rate (SELR)**

The specific energy loading rate (SELR) is, according to the literature, one of the parameters for evaluating the AD process stability, since it is useful for determining allowable organic loading rates. The SELR is defined as the quotient between the daily feed organic load (expressed in g of tCOD (L·d)−1) and the active biomass inside the digester (expressed in g VSS L−1) (VSS, volatile suspended solids) (Eq. (1)) and can be considered as an indicator of food to mass ratio (F/M) [19]. Thus, if the food mass in the feedstock exceeds the mass of decomposer microorganisms, it could cause a metabolic imbalance because of the acidification and inhibition of methanogenic microorganisms [36]. On the contrary, if the abundance of food available is insufficient, the metabolism of the microorganisms could be affected [37]:

$$\text{SELR} = \frac{Q \left[ t \text{COD} \right]\_{\text{inlet}}}{\left[ \text{VSS} \right] \cdot V\_{\text{working}}} \tag{1}$$

where *Q* is the inlet flow rate (L d−1), [*tCOD*] is feeding total COD concentration (g L−1), [*VSS*] is digestate volatile suspended solids concentrations (g L−1), and *V*working is the working volume of the digester (L).

According to Azevedo et al. [38], the limit value for SELR is 0.4 d−1. A higher value indicates a potential instability between the biomass of the microbial consortium and the loading of the feed mixture.

*Anaerobic Digestion of Organic Solid Waste: Challenges Derived from Changes in the Feedstock DOI: http://dx.doi.org/10.5772/intechopen.107121*

#### **2.3 Removal of organic matter (volatile solids and COD)**

Methane production should be stable if there is no accumulation of organic compounds inside the digester. A feeding of substrates with poor biodegradability could increase the content of volatile solids inside the reactor. Likewise, if the feed rate exceeds the rate of degradation by the microorganisms, organic compounds will accumulate inside the reactor, and methane production will be impacted. It is worth noting that the biodegradability capacity of a digester would depend on substrate characteristics and microbial degradation capacity. Therefore, this variable would be useful to evaluate the adaptation of an AD reactor to new substrates by comparing the biodegradability values in the digester with the expected for the added substrates. In that sense, biomethane potential tests can be a powerful tool to provide a reference framework for the biodegradability of the substrates [39]. The volatile solid removal is determined by Eq. (2) [23, 35]:

$$\text{VSS}\_{\text{removal}} = \frac{\text{VSS}\_{\text{ulet}} \cdot \text{VSS}\_{\text{digesante}}}{\text{VSS}\_{\text{ulet}}} \tag{2}$$

#### **2.4 Total ammonia nitrogen (TAN)**

Feedstocks with high content of proteins, i.e. with high content of nitrogen compounds, could induce high total ammonia nitrogen (TAN) in the AD process leading to biomass inhibition [6, 35]. A C/N ratio of 10–30 in the feedstock has been reported in the literature, which could avoid ammonium inhibition [20, 23]. Ammonia inhibition usually leads to a decrease in methane production rate and an increase in intermediate VFAs. Ammonia levels in the 200–1000 mg NH4-N L−1 have no adverse effect, while inhibition occurs between 1500 and 3000 mg NH4-N L−1, especially at higher pH values, and complete inhibition, at all pH values, above 3000 mg NH4- N L−1 [35].

#### **2.5 Biogas production and composition**

Biogas production is a crucial measure of the AD process status. If at a given OLR, there is a decrease in biogas production or biogas production rate that does not correspond to the degradation of the fed load, it could be considered a warning sign that the process is not working at its optimum [20]. According to the literature, the production of biogas or methane can be expressed as gas production rate (GPR), specific gas production (SGP), specific methane production (SMP), or specific methane yield (SMY), among others [6, 38, 40]. The GPR is expressed as the biogas volume generated per day to the reactor volume. The SGP and SMP/SMY are the biogas or methane volume generated by the mass of volatile solids feeding [6]. Generally, specific parameters are used to compare the stability of anaerobic digestion processes developed at different OLR values.

The organic matter degradation by microorganisms produces different types of gasses contained in the biogas. The biogas composition is mainly methane (45–85%), carbon dioxide (15–45%), and other gases such as hydrogen sulfide, ammonia, and nitrogen. The accurate proportion of gasses in the biogas depends on the process conditions and the feedstock [20].

A change in the microbial community could generate a different biogas composition. If there is an accumulation of hydrogen in the process, the hydrogenotrophic

methanogenic microorganisms could be inhibited. On the contrary, if acetic acid accumulates, the acetoclastic methanogenic microorganisms could be inhibited. In both situations, methane production could be affected [18]. Also, the different compositions of the feedstock can directly affect biogas generation. Alibardi & Cossu [41] found that a higher proportion of carbohydrates in the substrate results in a more significant biogas generation. Not so when the substrate is mainly composed of lipids or proteins.

#### **2.6 Microbial population adaptation**

Commonly, the AD process involves several stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis [20, 21]. First, hydrolytic microorganisms are responsible for breaking down complex organic matter, such as proteins and carbohydrates, into simpler compounds. In the acidogenesis stage, the simpler compounds are biodegraded by acidogenic into VFAs, alcohols, H2, and CO2. Then, acetogenic microorganisms transform them into acetic acid, H2, and CO2. Finally, methanogens convert these products into CH4 and CO2 [42] following two pathways. One is carried out by acetoclastic methanogens, which can convert acetate into CH4 and CO2; the other is performed by hydrogenotrophic methanogens, which convert H2/CO2 to CH4 [43]. These relationships play a crucial role in the anaerobic process leading to a balance between populations. For example, hydrogenotrophic methanogens are responsible for maintaining a low partial pressure of H2 (<10 Pa), which is necessary for the functioning of the intermediate trophic group [44]. Therefore, the AD process stages efficiency is closely associated with the abundance and activities of specific anaerobic microbial communities. Many studies have reported that the diversity and abundance of microbial communities are closely associated with the digestion conditions, such as OLR, pH, temperature, HRT, and types of digestion substrates [2, 21], and an active anaerobic microbial communities imbalance could reduce the efficiency of the AD process [21, 45].

In anaerobic digesters, the stability of the microbial population and the relationships between groups (i.e. acetate utilizing methanogens/hydrogen utilizing methanogens ratio, and sulfate-reducing bacteria/methanogens ratio) are widely used parameters to establish the stability of the digesters. However, there is a lack of studies reporting the effect of feedstock's type and composition on community structure and microbial activity changes. Zahedi et al. [42] have observed that although the number of microorganisms is essential in many microbial ecology studies of anaerobic digestion, operating with actual and changing wastes under realistic circumstances, MSW could be not a key parameter to control the process. It was concluded that stability and good microbial community dynamics (flexibility to adapt in response to changes in environments, particularly to changes in the substrate and operating conditions) are essential factors for the stable performance of the reactors [42].

So, although some researchers have documented that feedstock composition and OLR may influence bacterial and archaeal communities, there is a lack of consensus on the impact assessment that drastic changes in the type and composition of feedstock might have on community structure changes, bacterial density increases, and microbial diversity. Furthermore, nowadays, the complexity, cost, and high expertise required for this kind of analysis advocate for using the microbial analyses as a supplementary tool for gaining deep knowledge of the reactors, but not for the routine monitoring of the AD process.
