For more than a century, activated sludge in batch setups was used to treat sewage samples under continuous cycles of operation. Since then, the activated sludge process has rapidly expanded across the whole world, as industrialization and urbanization have entailed the need for the treatment of produced wastewaters. Nowadays, in the European Union (E.U.), the treatment of municipal wastewater complies with Directive 91/271/EEC, which, through the setting of rules regarding treatment efficiency and discharge, aims to protect the receiving water bodies from the vast amounts of wastewater produced.

The process involves the mixing and aeration of the activated sludge; the oxidation of organic carbon and inorganic nitrogen, wherein additional biomass is produced; the separation of the liquid and solid phases in order to control the concentration of the total suspended solids (TSS); the recirculation and retention of the biomass and possible disposal of the excess activated sludge [1]. During this step, the removal of phosphorus nutrients is also feasible, whereas the implementation of an anoxic stage results in the reduction and removal of the oxidized nitrogen compounds.

2. Agricultural By-Products and Wastewaters

The agriculture and food sectors globally face significant challenges in the 21st century. Food waste occupies an increasing section of waste treatment facilities and landfills. Today, remaining residues and losses throughout the food supply chain are drawing attention due to the vast waste of valuable resources, constituting a complex environmental problem. In low-income countries, food waste causes serious socio-economic consequences, while, on the other hand, consumer attitudes and the mass consumption of goods and products cause the production of huge amounts of household waste in middle and high-income countries ^[2][7]. Food waste can be classified into animal and agricultural origins. In the first, the main sources of waste are those from the dairy, meat, and fishery industries. In the latter, it is possible to classify a variety of residues according to the source, which may include cereals, roots and tubers, oil seeds and legumes, fruits, and vegetables. However, spoiled foodstuffs, along with heterogeneity, make them difficult to exploit, while a comprehensive characterization process is required in order to determine their composition. Nevertheless, both animal and agricultural wastes are often characterized by high organic matter ^[3][8]. As reported by Leite et al. ^[4][9], in the European Union alone in 2018, more than 21 million tons of waste were generated, which derived from the agriculture, fishery, and forestry sectors. Ravindran et al. ^[5][10] pointed out that one third of the food produced worldwide which is intended for human consumption is wasted every year, corresponding to losses of about 1.3 billion tons, while 40 to 50% (520–650 million tons) of global food waste per year derives from fruits, vegetables, and roots. In the E.U., food waste is estimated to reach 89 million tons per year, about half of which is generated during production, while the total production of agricultural residues (crop residues or parts of plants that are not consumed as food) amounts to 367 million tons per year, although some of these residues are commonly used at the farm level as bedding and fodder ^[6][11]. Recently, the Food and Agriculture Organization (FAO) in a report under the title “The Future of Food and Agriculture—Alternative Pathways to 2050” stated that the planet’s population is expected to increase to about 10 billion by 2050, with an expected increase in agricultural demand, in a scenario of moderate economic growth, of about 50% compared to 2013. Furthermore, an increase in income in low- and middle-income countries would hasten the dietary transition to a greater consumption of meat, fruits, and vegetables over cereals, necessitating subsequent production changes that are expected to put additional strain on natural resources. In fact, although investment in agriculture and available technological tools and innovations boost productivity, final output growth is less profound, as food losses account for a significant proportion of agricultural production; therefore, tackling the crucial parameters of food loss would in itself reduce the need to increase production, which is already hampered by the degradation of natural resources and the loss of biodiversity, as well as the cross-border spread of pests and diseases of plants and animals which are highly resistant to applied antimicrobials ^[7][12].

3. Biotreatment of Agro-Industrial Wastewaters in Aerobic Bioreactor Systems

Aerobic biological treatment systems, including nitrification–denitrification plants, are commonly applied for the biotreatment of domestic wastewater and a range of agro-industrial wastewaters, due to their simplicity in operation, low cost of installation, high efficiency, and ability to biologically remove nitrogen through nitrification–denitrification. During the activated sludge process, organic matter is oxidized using air, mainly to carbon dioxide and water, and the microbial flocs formed are separated in a sedimentation tank ^[8][78]. Despite the fact that effluents of high organic content could be subjected to anaerobic digestion, the inability to biologically remove nitrogen in an efficient and simple manner and the high cost of installation often make the activated sludge process attractive for the biotreatment of certain agro-industrial effluents, especially those in which the COD concentration is low or moderate, or those that can be co-processed with municipal wastewater or washings. Even though the anaerobic treatment of agro-industrial wastewaters has the benefit of the production of biogas, this is balanced by the high HRT required, increasing the volume of the required digesters and resulting in specific space requirements, as well as the instability of the process, which provides no assurance of stable and satisfactory energy production ^[9][79]. Moreover, aerobic treatment enables the effective removal of nutrients, which is considered a strong benefit of the process, as high quality effluents are produced, capable of satisfying the stricter standards for disposal, which are not met in the case of anaerobic treatment systems ^[10][80]. Thus, there are several examples of using aerobic biological treatment systems for the depuration of agro-industrial effluents. For instance, activated sludge was immobilized on polyurethane particles in an aerobic bench-scale bioreactor for the treatment of winery wastewater under a maximum organic loading rate of 8.8 kg COD/m³·d and a hydraulic retention time of 0.8 d. Even at an OLR of 3 kg COD/m³·d, the ability of the aerobic immobilized cell bioreactor to remove COD was high, recording a COD removal efficiency of 87% ^[11][81]. Moreover, Roveroto et al. ^[12][82] treated brewery wastewater in a fixed-bed batch reactor, which operated under an intermittent aeration of 3 h aeration in 4 h cycle and a hydraulic retention time (HRT) of 0.83 d. The COD and BOD of the raw brewery wastewater ranged between 2 and 10 g/L and 1.2 to 3.6 g/L, respectively, while the total nitrogen reached up to 0.08 g/L. The highest removal efficiency, 92%, was recorded in the bioreactor when the influent COD was 2.7 g/L and the COD/N ratio was 107. Under these conditions, the nitrification efficiency was 88% and the total nitrogen (TN) removal was 85%. Antiloro et al. ^[13][40] investigated the biotreatment of citrus processing wastewater with a high organic content and essential oils concentration, i.e., between 20 and 30 g/L and 0.6 to 1.0 g/L, respectively, in an aerated lagoon system, reporting COD removal efficiencies from 59 to 97% and the establishment of a microbial community capable of coping with the increased concentration of essential oils. In addition, two aerobic granular sludge bench-scale SBRs operating under a sludge retention time (SRT) of 10 d and organic loading rates (OLRs) ranging from 3 to 15 kg COD/m³·d were used for the biotreatment of a citrus processing effluent of 5.5 g/L COD. At a neutral pH, the biosystem could remove COD by 90% regardless of the organic loading rate applied, although the reactor’s efficiency under acidic conditions declined to 75% when the OLR exceeded 7 kg COD/m³·d. Furthermore, Zema et al. ^[14][83] treated citrus processing wastewater of 5.0g/L COD and an essential oils concentration of 0.5 g/L under aerobic conditions in a full-scale treatment plant, reporting reasonable COD and essential oils removal efficiencies. Moore at al. ^[15][84] treated wastewater deriving from mixtures of fruits and vegetables in an aerobic pilot-scale ultrafiltration membrane bioreactor (MBR), for potential water reuse. Lettuce, beets, carrots, and cassava were processed to produce the first wastewater mixture, while potatoes, carrots, apples, onions, lettuce, beets, and bananas constituted the raw materials for the production of the second mixture of wastewater. The COD and total Kjeldahl nitrogen (TKN) content of the first mixture were 1.5 g/L and 0.01 g/L, respectively, whereas the respective concentrations in the second mixture were 7.1 g/L and 0.23 g/L. The HRT in the two experimental schemes examined varied from 24 to 52 h, whereas the OLR ranged from 0.82 to 2.7 kg COD/m³·d in the first and from 2.9 to 6.5 kg COD/m³·d in the second experimental setup. For both fruit- and vegetable-derived effluents treated in the MBR, high COD removal efficiencies of 97–98% were recorded, whereas the TKN removal efficiencies exceeded 91% for both wastewater mixtures. In this case, the activated sludge system, in combination with UV disinfection and the implementation of activated carbon for color removal, could produce an effluent of enhanced quality, which could be used in the agri-food sector. More than 10.5 million tons of coffee were exported by its producing countries in 2020 ^[16][85], a process that leads to the production of significant amounts of wastewater, since up to 45 kg of wastewater is generated during the pulping and washing of 1 kg of green coffee. Villa-Montoya et al. ^[17][3] treated coffee processing wastewater of a high organic content (COD of 7 to 15 g/L) and a TN concentration between 0.03 and 0.04 g/L in a sequencing batch reactor (SBR) under an OLR of 9 g COD/L.d, reporting that the intermittently aerated biological system achieved a COD removal efficiency of 92%. Coffee processing wastewater of a high COD concentration (17 g/L) was also treated in a constructed wetland system by Rossmann et al. ^[18][86], in order to achieve the efficient removal of nutrients and phenolic content. At an HRT of 11.8 d, the biosystem could remove total nitrogen (TN), total phosphorus, and total phenolic compounds by 69.1, 72.1, and 72.2%, respectively.

4. Biomass Valorization of Aerobic Biosystems Treating Agro-Industrial Wastewaters

Microorganisms are an important source of enzymes, as they grow rapidly in a short period of time. In addition, a wide variety of agro-industrial residues and wastes can be used as substrate, thus reducing overall production costs and the use of natural resources while value-added products are produced. Enzymes of microbial origin can find a variety of applications in industry, such as in the production of food and beverages, as well as in the manufacture of chemicals and pharmaceuticals. The properties and activities of an enzyme are considered to be directly dependent on the strain that is capable of inducing them, while their effectiveness in biotechnological applications is being constantly and increasingly evaluated. Therefore, there is a strong scientific interest and a wide scientific field in the search for new strains capable of producing high-activity enzymes at a reduced cost with potential uses in industry ^[19][87]. Moreover, aerobic bioreactor systems treating agro-industrial wastewater can be considered as microbial cell factories producing a wide range of industrial enzymes, such as cellullases, xylanases, glycosidases, lipases, and proteases. For instance, Zerva et al. ^[20][4] assessed the hydrolytic potential of an immobilized cell bioreactor treating caper wastewater at an elevated salinity (3.12 to 101 g/L). The non-halotolerant microbiota of the immobilized cells at a salinity of up to 20 g/L were able to highly hydrolyse celluloses, hemicelluloses, starch, fats, and proteins. Increased endo-1,4-β-xylanase activity above 1785 U/g protein was recorded throughout the experimental period. Endo-1,4-β-D-glucanase activity of 250 U/g protein was also reported, even though it was highly affected by the elevated salinity. Regarding polygalacturonase, its activity exceeded 533 U/g protein and further increased to 959 U/g protein under the highest salinity. Furthermore, β-1,4-D-glucosidase activity was above 510 U/g protein, while the increase in the organic loading rate and low salinity resulted in the elevation of α-1,4-D-glucosidase activity up to 905 U/g protein. Initial lipase activity was above 352 U/g protein but was affected by a salinity concentration of 1% w/v and decreased to 130 U/g protein. Moreover, Zerva et al. ^[21][5] treated pepper processing wastewater in an aerobic immobilized cell bioreactor and monitored the hydrolytic potential of bacteria isolated from the immobilized biomass of the biosystem, reporting a high endo-1,4-β-xylanase activity of 107,000, 72,000 and 70,000 U/g protein for three bacterial isolates belonging to the genera Nocardia and Gordonia. Bacterial isolates related to Aquincola, Microbacterium, Planococcus, Sphigopyxis, and Xanthobacter were also found to exert endo-1,4-β-xylanase activity from 29,700 to 37,400 U/mg protein. In addition, several white-rot fungi can be used for the biotreatment of various agro-industrial enzymes and produce ligninolytic enzymes. For instance, a Phanerochaete chrysosporium strain was immobilized by Sharari et al. ^[22][88] on polyurethane foam for the treatment of bagasse wastewater and the simultaneous production of ligninolytic enzymes, reporting peroxidase activity of 260 U/L and laccase activity of 131 U/L, whereas xylanase activity of 74 U/L was also detected. Moreover, Mafakher et al. ^[23][89] isolated lipase-and citric acid-producing yeasts from agro-industrial wastewater treatment plants. Among the 300 yeast isolates examined, 6 exhibited a high lipase activity, which were identified as Yarrowia lypolitica isolates.

5. Microbial Communities’ Structure in Aerobic Biosystems Treating Agro-Industrial Wastewaters

The recent development and application of high-throughput sequencing techniques have led to a better understanding of microbial communities’ structure and functions in bioengineering systems. In the last decade, the implementation of molecular methods, such as next generation sequencing techniques, has permitted the elucidation of the microbial ecology and biotechnological potential of certain aerobic bioreactor systems treating agro-industrial wastewaters. In that direction, by implementing high-throughput sequencing techniques, Fang et al. ^[24][90] stated the dominance of Zoogloea in the activated sludge of an SBR treating rice winery wastewater under an OLR of 2.4 g COD/L.d. Apart from the presence of Zoogloea species, Rhodobacter and Rubrivax were also detected in high abundances. The dominance of Zoogloea spp. in the activated sludge of this aerobic bioreactor system can find a biotechnological application potential, since this genus is considered an important PHA accumulating microorganism ^[25][91]. Bacteria of the genus Amaricoccus, Zoogloea, and Azoarcus were also identified in winery wastewater using FISH, whereas Amaricoccus species dominated the constructed clone library ^[26][92]. Moreover, in meat processing wastewater treated in an SBR, the activated sludge microbial community was dominated by the class Alphaproteobacteria, which are frequently identified in similar samples ^[27][93], where Amaricoccus spp. covered 11% of the microbial diversity in the SBR. Furthermore, the biotreatment of dairy wastewater in a full-scale aerobic SBR under an OLR of 2.5 kg COD/m³·d revealed the predominance of the genera Proteiniphilum, Byssovorax, Acidobacterium, and Zoogloea, which covered 35.9, 14.5, 10.1, and 8.3% of the total relative abundance ^[28][94], despite the fact that Proteiniphilum and Byssovorax bacteria are rarely reported as microbial constituents of activated sludge. The same researcheuthors also reported that Thiothrix and Leptothrix spp. were the main filamentous bacteria of the activated sludge system, with the presence of Thauera being involved with the formation of granular structures and the cohesion of the activated sludge due to the release of extracellular polymeric substances (EPS). Other major inhabitants of activated sludge systems treating agricultural wastewater are members of the genera Bacillus, Pseudomonas, Thauera, Xanthomonas, Spingobacterium, and Comamonas, such as in aerobic biosystems treating olive mill ^[29][95], winery ^[30][96], and dairy ^[31][97] wastewaters. In addition, Pires et al. ^[32][98] isolated bacterial and fungal strains from a coffee processing wastewater treatment plant. The bacterial isolates were mainly members of the phyla Proteobacteria, e.g., Acetobacter, Serratia, and Enterobacter spp.; Actinobacteria, e.g., Corynebacterium and Arthrobacter; and Bacteroidetes, e.g., Chrysobacterium. Regarding the fungal community structure, the majority of isolates were identified as yeasts of the order Sacharomycetales, such as Wickerhamomyces, Torulaspora, Kazachstania, Saturnispora, Meyerozyma, Hanseniaspora, and Pichia spp., which have been often detected in municipal wastewater treatment plants and in other biosystems treating agro-industrial wastewater, e.g., palm oil effluent ^[33][34][99,100]. Pires et al. ^[32][98] also detected filamentous fungi, such as Alternaria alternata and Fusarium oxysporum. Petruccioli et al. ^[35][101] also isolated yeasts from the activated sludge of an aerobic jet loop reactor treating winery wastewater, identifying microbiota such as Saccharomyces, Candida, and Trichosporum, reporting a link between the presence of Saccharomyces and the formation of biofilm.