**5. Factors influencing anaerobic co-digestion**

AD process is affected by several factors which led to a higher or lower biogas yield; these factors can be split into two main sources: operational conditions and substrates composition. Although both sources are widely related, they can be studied and controlled separately. The main operational conditions are temperature, pH, configuration of bioreactors, acclimation of inoculum, hydraulic retention time (HRT), organic load rate (OLR), and inoculum to substrate ratio (ISR) [1, 104]. The main factors related to substrates composition are the C/N ratio and macro and micronutrients [1, 5, 6, 104].

In this sense, AcoD is proposed as a feasible alternative in order to balance these factors and allow a better performance and a higher biogas production.

#### **5.1 Effect of initial conditions**

Most studies agree that the best operational temperatures for AD are under mesophilic (20–45°C) or thermophilic (>45°C) conditions [104]. Temperature affects, either directly or indirectly, the solubility of substrate compounds and the specific growth rate of the microorganisms involved, provoking a change in the HRT, the pH, and the methane yield [104]. Among the literature, the most common range is the mesophilic conditions due to its lower energy cost requirements and the similar methane yield when compared with higher temperature conditions [105]. However, temperature variations during AD performance have shown significant reductions in methane yield and the kinetics of the process [104, 106].

The effect of pH is mainly related to the optimum pH of the microorganism performance during AD. Based on that, a pH between 4.6 and 6.0 favored the hydrolysis, acidogenesis, and acetogenesis stages, while a pH between 6.0 and 8.0 favored the methanogenesis phase [1]. Literature shows that the most suitable pH range is between 6.5 and 7.5, where the methane production is most benefited [1, 104]. Furthermore, it has been reported that the initial pH of the substrate had a significant impact on methane yield, being the optimum value in the range of 7.0–7.5 for the co-digestion of swine manure and maize stalk [107]. However, pH is highly affected during the AD process, lowering its value if the buffer system is not strong enough due to the accumulation of volatile fatty acids (VFA) (e.g. acetic, propionic, butyric, and valeric acids) [104] or increasing it if ammonium nitrogen is accumulated (around 5.0 g NH3-N L−1) [33].

Temperature and pH have been linked to free ammonium nitrogen and ammonium ions equilibrium, showing that when one parameter is fixed there is a linear increase in methane yield when the other two independent variables increased up to a certain limit, after which the methane production decrease [104, 108]. A recent study showed optimal conditions for the mono-digestion of chicken manure of 34.0°C, 5.0 g NH3-N L−1, and pH 7.5 [108]. Moreover, in an earlier study, an increase of pH from 7 to 8 and above, enhanced the biogas production with similar methane proportion when temperature conditions increased from 37 to 55°C during the AD of buffalo manure [109].

As shown above, temperature and pH have a significant impact on AD performance and biogas production. Thus, co-digestion is presented as a suitable technique able to enhance the buffer system, the substrate pH, and free ammonium nitrogen values, allowing a higher methane yield and a more stable process [67, 77, 78, 99, 100]. Meneses-Reyes et al. showed that increasing the C/N ratio of the substrate by reducing the microalgae ratio provoked an increase in pH, however, the pH of digestate is similar among the different co-digestion ratios studied (7.33–7.51) [41]. AcoD of *Chlorella* sp*.* and food waste in batch mode (35°C) showed that methane yield was related to the initial pH of the substrates (7.3–8.7), being the optimum value 8.0 and reporting that the methane yield decreased almost linearly when pH differs from the optimum value, although these results were not conclusive as other variables were also different (e.g. VSfeeding from 8.0 to 9.2 g) [35]. However, a mixture of microalgae biomass with thermally treated wheat straw presented a pH of 12 with no significant effect on AcoD performance when compared with the AcoD of microalgae biomass and untreated wheat straw (pH: 6.82) [71]. Another study assessed the effect of temperature within the mesophilic range (25°C, 30°C, 35°C, 40°C) in biogas production, reporting a significant increase of biogas production (45%) when the temperature was increased up to 35°C, but a reduction of production or no significant improvement (depending on the C/N ratio of substrates) when the temperature was set up at 40°C [24]. This result is in accordance with other studies reporting a decrease of methane yield for the AcoD of microalgae with undigested sewage sludge in batch mode when increasing the temperature from mesophilic (37°C) to thermophilic (55°C) conditions, even though the pH was not affected (6.91–7.03) and the processes were stable [45]. AcoD of corn silage and *Nannochloropsis salina* in a semi-continuous mode (38°C; C/N: 21.2) do not present any stability deviation by the increasing OLR (2–4.7 g VS L−1 d−1), while the AcoD of two microalgae (*Scenedesmus* sp. and *Opuntia maxima*) in semi-continuous mode (37°C; C/N: 15.6) showed that pH was affected by OLR (2–6.67 g VS L−1 d−1) and ISR (6:8% VS basis), is the most stable conditions OLR: 2 g VS L−1 d−1 and ISR: 8% VS basis [60, 110]. Furthermore, a study assessing the effect of alkali, acid, and thermal pretreatment of *Oscillatoria tenuis*, before the AcoD with pig manure, reported that pH control affected the biogas production rather than physical or chemical pretreatments [18]. Similar results were reported during the AcoD of alkali pretreated microalgae consortium with swine wastewater, where the negative effect of ammonia inhibition at high pH (11) was stronger than the positive effect of the destruction of microalgae's cell walls [47].

### *Anaerobic Co-Digestion of Microalgae and Industrial Wastes: A Critical and Bibliometric Review DOI: http://dx.doi.org/10.5772/intechopen.104378*

pH is also being widely used as a control parameter able to indicate the stability of the reactors since is strongly linked to VFA accumulation [40, 43]. AcoD of *Arthrospira platensis* with several carbon-rich co-substrates proved to be stable at low OLR (1 g VS L−1 d−1) in a semi-continuous mode but unstable at higher OLR (2–5 g VS L−1 d−1) due to VFA accumulation and pH dropped, this study is in accordance with the AcoD of *Chlorella* sp. and glycerol that presented a stable pH range (6.6–7.32) when the HRT was above 5 days, at which point volatile fatty acids (VFA) accumulation inhibit the biogas production [24, 33]. However, AcoD of naturally grown microalgae consortium with WAS in a semi-continuous mode (37°C) showed that when the HRT was increased from 1 to 3 to 4–6, pH was reduced (from 7.51 to 7.04), although VFA accumulation was not observed and the system remained stable with no difference in methane production except at HRT of 6 were slightly dropped [46].

Another important factor is alkalinity, which helps to prevent large changes in pH due to the accumulation of volatile fatty acids, or the generation of ammonia due to protein hydrolysis. Alkalinity provides the necessary buffering capacity to counteract possible changes in pH, produced by the balance between carbonate and bicarbonate. The ideal alkalinity values for AD would be between 2000 and 4000 mg CaCO3 L−1 [99, 100]. A study assessed the relation between pH and alkalinity, where the initial pH was fixed at 7.0 while the initial alkalinity changed (70–3200 mg CaCO3 L−1), however, pH remained around neutral values (6.9–7.2) during the AcoD process suggesting that initial alkalinity has no impact avoiding an ammonium concentration from nitrogen-rich substrates as microalgae [30].

The C/N ratio is another factor that influences the AD process [29]. A good substrate C/N ratio can range between 20 and 30, with an optimal value of 25 [102]. With a C/N ratio below 20, there is an imbalance between C and N in the reactor, which ultimately releases a large amount of NH3, which usually happens with the degradation of microalgae [102]. The high concentration of NH3 in the digester affects the growth and metabolism of microorganisms and produces an accumulation of volatile fatty acids, which results in a decrease in biogas yield. This factor can also be supplied by choosing a good co-substrate and optimizing this C/N ratio [94].

The ISR is another key parameter that influences AD and methane production [13]. To find the maximum methane potential, a proper balance between the substrate and the microorganisms is necessary so that limitations and inhibitions do not occur due to the loading of the substrate. For biochemical methane potential (BMP) assays, a ISR ≥ 2 is suggested as the default value [111].

### **5.2 Effect of pretreatments**

As a common way to improve the methane yield of AcoD of microalgae, several studies have reported the effect of pretreatments on microalgae biomass. **Table 3** shows some of these pretreatments and the effect on AcoD calculated as the increase of biomethane production in percentage over the non-pretreated AcoD.

It has been reported that although some pretreatments can improve the methane yield of microalgae as a sole substrate, these have a negative effect during the AcoD, mainly due to the high organic matter consumption or the inefficacy of pretreatments breaking down the cell wall [47, 86, 97].

From **Table 3** it can be seen that low thermal pretreatments (60–55°C; 1−2d) have none or very little effect on AcoD [19, 86], however, when the temperature increased to 120°C, the biomethane production improve greatly (up to 43%) [18, 49]. Other successfully tested pretreatments are ultrasonication, hot water, and a


#### **Table 3.**

*Effect of pretreatment on AcoD.*

combination of thermal and alkaline pretreatments [57, 71]. Nevertheless, based on the higher improvement on biomethane production, thermal pretreatment at 120°C is the most effective process, where time and pressure would be the variables to analyze [49, 57].
