*4.2. Biological Methods*

Bioremediation is a technology that exploits the metabolic potential of microorganisms to purify contaminated sites. It can be performed in a non-sterile and open area containing numerous organisms. Among these, bacteria have a central role in the process thanks to their ability to degrade pollutants. In addition, fungi and other components (e.g., grazing protozoa) can also affect the process [61]. All these species require nutrients (i.e., carbon, nitrogen, phosphates, metal traces) to survive, so they break down organic compounds to attain them. Bioremediation can occur under aerobic or anaerobic conditions [62]. In the former case, the survival of microorganisms is mainly due to the consumption of atmospheric oxygen. In contrast, in the latter, microorganisms gain food by breaking down chemical compounds in the soil [27].

In recent decades, OMWW has been used in this way, acting as substrate for microorganisms' growth by providing nutritive substances. Some yeast species (i.e., *Candida tropicalis, Yarrowia lipolytica*) together with bacteria of the *Azotobacter vinelandii, Pseudomonas, Sphingomonas, Ralstonia* species have been proven to be helpful in the OMWW aerobic biodegradation and detoxification [63–65]. By way of example, the activity of a free-living N2-fixing bacterium, *Azotobacter vinelandii*, was investigated. In particular, OMWW was initially treated with calcium hydroxide to achieve the pH value of *ca.* 8–10 (stage I). Successively, it was mixed in a bioreactor in the presence of *Azotobacter vinelandii* (stage II). The process carried out according to the procedure reported by Arvanitoyannis et al. [27] resulted in an increasing level of nitrogen and its ammonium form throughout the whole remediation period. On the other hand, regarding phenols and sugar degradation, ca. 66–99% and 100% of phenols'abatement was observed after 3 and 7 days, respectively, whereas sugars were wholly degraded in only 3 days. Low phytotoxic features characterize the final product so it can be exploited as fertilizer [66].

In general, to reduce the high content of phenolic compounds in OMWW, water dilution represents a suitable strategy for a successful aerobic treatment. In fact, phenols are responsible for inhibition of microorganism growth [67,68]. Alternatively, OMWW could be mixed with additional waste and digested with the help of a solid substrate (i.e., straw, sesame bark, olive leaves, vineyard leaves, wood chips, animal manure) [69–71]. Then, when the phenol content of waste decreases, usually after 6–7 months, the final product can be exploited as fertilizer, giving profit [70,71]. Additionally, the composting stage could be coupled with physicochemical processes [72–74]. This last method requires high energy

demand and consequent high CO2 emissions. However, the energy demand can be reduced thanks to simultaneous methane production [75].

### *4.3. Physicochemical Treatments*

Among physicochemical treatments, dilution, evaporation, sedimentation, filtration, and centrifugation are commonly used to treat OMWW.

OMWW dilution is usually employed before biological treatments with the final aim to reduce its toxicity to microorganisms. On the other hand, evaporation and sedimentation result in a concentrated OMWW (*ca.* 70–75% more concentrated) thanks to both phase separation/dehydration and organic matter degradation [6,7]. In this context, solar distillation applied to OMWW can remove 80% COD in the distillate in 9 days, maintaining 25% water content [8].

Other strategies have also been investigated, mainly consisting of irreversible thermal treatments. This is the case with combustion and pyrolysis that require a reduced volume of waste and provide energy recovery. Still, unfortunately, they need expensive facilities, emit toxic substances into the atmosphere, and require an OMWW pre-concentration step [9,10].

Centrifugation and filtration increase the effluent pH and conductivity, removing the organic matter using phase separation and exclusion. Ordinarily, combining physical processes, coupled with coagulation/flocculation or adsorption techniques, gives rise to more efficient removal of organic matter. For example, it was found that when the sedimentation is followed by centrifugation and filtration, 21% and 15% decrease in COD and BOD, respectively, was observed, with the further 16% reduction in BOD due to the final filtration [11]. OMWW adsorption on activated clay causes an additional 71% COD reduction. However, a particular focus has to be put on the adsorption/desorption equilibrium since organic and phenolic features start to desorb after a precise contact time. The combination of treatment stages, i.e., settling, centrifugation, filtration, and adsorption on activated carbon, induce a maximum of 94% phenol abatement and 83% organic matter removal [12].

Regarding filtration, it is fundamental to point out that, besides the high efficiency of membranes, these processes require high operative pressures and energy consumption. However, proper membranes can be exploited to recover valuable by-products, such as phenols, which are mainly required for the pharmaceutical and chemical industry [13].

Lime treatment has been selected as a pre-treatment step for reducing OMWW polluting effect due to its inexpensiveness [76–79].

In this context, coagulation-flocculation is a very similar technology to lime treatment. Different coagulants (i.e., ferric chloride, polyelectrolytes) can be exploited [80]. On the other hand, electro-coagulation mainly consists of the suspension and precipitation of charged particles in the waste thanks to an applied voltage. Since this process is characterized by low cost and energy consumption, it is not so efficient in removing organic waste species.
