The OLR variation can be derived from either variation in influent chemical oxygen demand (COD) or variation in flow rate with constant COD. An increase in OLR beyond the optimum level is followed by a decrease in the main process parameters such as COD removal, specific methane production. In addition, high amount of suspended solids "known as biomass wash-out" are observed in the effluent, indicating that the reactor suffered a process imbalance and that biomass accumulated in the reactor (Converti et al., 1993; Fezzani and BenCheikh, 2007; Rincon et al., 2008). This could be ascribed to an increase in the concentrations of the VFA with a consequent decrease in pH (Tiwari et al., 2006) or to escalated levels of inhibitory or toxic compounds such as phenols, lignin and others.

Therefore, there is a maximal operational value for this parameter. For instance, Rizzi and coworkers in the year of 2006 reported a decrease in COD removal and specific methane production when OLR was increased from 10 to 15 kg COD/m3-d. With the OLR increase to 20 kg COD/m3-d the biomass excess started to wash out, followed by deterioration of the reactor performance. In a different study, stable reactor performance was observed when the OLR increased from 1.5 to 9.2 kg COD/m3-d with the maximum methane production rate achieved for an OLR of 9.2 kg COD/m3-d. However, a significant decrease in the pH value (from 7.5 to 5.3) was observed when OLR was further raised to 11.0 kg COD/m3-d. In addition, the increase in the effluent COD with increased OLR was paralleled to a sharp increase in the effluent total volatile fatty acids (TVFA, g acetic acid/L) by about 400% (Rincon et al., 2008). This indicates that, at higher OLR the effluent total COD and mainly soluble COD is largely composed of the unused volatile acids produced in the reactor due to the inhibition of methanogenesis.

Methanobacteriaceae and Methanosaeta were found the main methanogens in a laboratory scale up-flow anaerobic digester treating olive mill wastewater (Rizzi et al., 2006). However, the authors also reported an interesting population shift by OLR variation. At lower OLR i. e. 6 kg COD/m3-d, hydrogenotrophic Methanobacterium predominated in the reactor but the number of cells/g sludge showed a 1000 fold decrease from 1011 to 108 when the OLR was increased to 10 kg COD/m3-d. In contrast, phylotypes belonging to the acetoclastic Methanosaeta were not affected by OLR variation and at 10 kg COD/m3-d, dominated in the biofilm (109 cells/g sludge) (Rizzi et al., 2006).

Olive oil wastewater is characterized by high levels of inhibitory compounds such as tannins, and lipids. As a result, increased OLR leads to higher concentration of these substances and a consequent inhibition of methanogenic cells. However, acetoclastic Methanosaeta due to its high affinity for acetate is capable of occupying the deepest and thus more protected niches in the granule or biofilm with low concentrations of substrate (acetate) (Gonzales-Gil et al., 2001). Phylotypes belonging to the genus Methanosaeta were also dominant independent of different OLR in other anaerobic digesters (Rincon et al., 2008).

In a different study was investigated the microbial ecology of granules in UASB reactor fed by synthetic wastewater under various OLR. The authors showed that the predominant microbial biomass was Methanosaeta. However, increasing the OLR led to a substantial increase of Methanosarcina in the granules (Kalyuzhnyi et al., 1996). The increase of Methanosarcina in the studied synthetic wastewater (toxin-free) due to increasing OLR is explained by the low affinity of these methanogens for acetate in comparison with Methanosaeta. Hence, by increasing OLR and consequent VFA concentration, Methanosarcina is favored.

As reviewed earlier, under mesophlic conditions Methanosaeta plays a significant role in making cores of sludge granules (Sekiguchi et al., 2001) and thus their ratio seems to control the speed of granulation (Rincon et al., 2008). Higher OLR, result in consequent higher concentration of substrates (i. e. acetate) in the reactor. Morvai and coworkers in 1990
investigate the influence of organic load ranging from 0.5-3.0 g/L on granular sludge development in an acetate-fed system. They argued that in the range of feed acetate levels examined, higher concentrations of acetate caused faster granulation of the sludge bed and, presumably of the microbial population, and resulted in better sludge structure and improved sludge settleability.

Low OLR has been reported to cause acute mass transfer limitation leading to disintegration of the larger granules (Ahn et al., 2002). The disintegration begins at the core of the granules due to substrate limitation with a consequent loss of granules strength and stability. However, this was not in agreement with the studies reported, which low OLR (<1.5 kg COD/m3-d) did not lead to disintegration of the granules in UASB reactors (Tiwari et al., 2005). This could be ascribed to the different experimental settings and wastewaters used in these studies. Teo and coworker (2000), treat a high iron bearing wastewater in a UASB reactor. Evidence shows that the presence of divalent and trivalent cations ions, such as Fe2+ and Fe3+, helps bind negatively charged cells together to form microbial nuclei that promote further granulation.

Tiwari et al. (2006) tried to enhance the granulation process by using natural ionic polymer additives. These may thus reduce the effect of low OLR (i. e. substrate limitation) on the granules and delayed the disintegration. Meanwhile was reported that COD removal rate, the COD specific removal rate (rs) and methane production rate were not suppressed by increasing OLR when treating wine wastewater and sewage mixture (Converti et al., 1990). That indicated that no inhibition factor related to the organic content of the effluent was present in both wine wastewater and sewage mixture studied.

This was further supported by the cell mass concentration varied very little with increasing the OLR. However as completely noticed by the authors, even at the absence of inhibitory compounds in the initial part, the removal rate increased with the OLR, following a first order kinetic. In the second part, instead the removal rate tended to a constant maximum value, following a zero order kinetic. Afterwards, the removal efficiencies as well as the methane production yield gradually decreased with increasing influent COD due to increasing the OLR, which evidently showed a substrate inhibition occurrence (Converti et al., 1990).

This supports the idea that even at the absence of the inhibitory compounds in the wastewater, increasing influent COD by the means of increasing OLR could lead to substrate inhibition and consequent reduced removal efficiencies. In other study is described the dependence of the removal rate on the OLR by an empirical equation similar to Monod’s model (Eq. 9) to compare the degradability of different effluents (Converti et al., 1990):

rs(max)OLR

(k+OLR)

where rs(max) (kg COD/kg of vss d) is the maximum value of rs, and k is a constant which physically is expressed in units of OLR, an increase of k indicates increased treatment ability of the studied effluent. The desired OLR is the function of the favorable effect of OLR on stimulating the growth of methanogens in the bioreactor by providing them with higher substrate concentrations, its reverse effect on elevating the concentration of inhibitory compounds and the buffering capacity of methanogenic community. In the other words, the maximal operational value of OLR is translated into the highest methane production
(indicating the highest conversion efficiency of the system) that the buffering capacity of methanogenic community is still capable of compensating for elevated concentrations of inhibitory compounds (Tabatabaei et al., 2011).