Geographic Information System (GIS) is a powerful instrument that can be used for the spatial representation of waste and by-product flows at various levels, allowing to improve municipal solid waste (MSW) management. The mapping obtained can be advantageously targeted to build a regional network of technological, economic, social and environmental linkages and to boost circular economy practices. In this work, the data on MSW produced in the Lodzkie region, Poland, during 2021 were used to generate a geolocalized database and an interactive web map, using ArcGIS software. The geodatabase and the map visualization were organized in three layers of information with increasing detail to foster a map-driven symbiosis between waste suppliers and waste recipients, paving the way for a more circular regional economy.

Heterotrophic microalgae Chlorella protothecoides, either as whole (H) or pre-treated by ultrasound method (HPU), were anaerobically digested to provide information on the light effect on the simultaneous production of biogas/methane and pigments, under conditions of continuous absence or presence of light. The simultaneous production of red pigments and biogas/methane was achieved by using continuous lighting, while under complete darkness only methane was obtained as anaerobic digestion's product but with higher amounts; values of 36/68 (H and HPU) against of 115/150 mL methane (Hc and HPUc) were registered. Ultrasonic pre-treatment doubled the methane yield from 120 to 278 and 382 to 615 L CH4/kg VSIN (H vs HPU and Hc vs HPUc, respectively), confirming its positive effect on anaerobic digestion process. From the results, the permanent supply of light to the digestion units is necessary for the simultaneous production of biogas/methane, digestate flow and pigments.

A methodology was developed to assess the allocation of different types of endogenous waste biomass to eight technologies for producing electricity, heat, biogas and advanced biofuels. It was based on the identification of key physicochemical parameters for each conversion process and the definition of limit values for each parameter, applied to two different matrices of waste biomass. This enabled the creation of one Admissibility Grid with target values per type of waste biomass and conversion technology, applicable to a decision process in the routing to energy production. The construction of the grid was based on the evaluation of 24 types of waste biomass, corresponding to 48 sets of samples tested, for which a detailed physicochemical characterization and an admissibility assessment were made. The samples were collected from Municipal Solid Waste treatment facilities, sewage sludges, agro-industrial companies, poultry farms, and pulp and paper industries. The conversion technologies and energy products considered were (trans)esterification to fatty acid methyl esters, anaerobic digestion to methane, fermentation to bioethanol, dark fermentation to biohydrogen, combustion to electricity and heat, gasification to syngas, and pyrolysis and hydrothermal liquefaction to bio-oils. The validation of the Admissibility Grid was based on the determination of conversion rates and product yields over 23 case studies that were selected according to the best combinations of waste biomass type versus technological solution and energy product.

Microalga Chlorella protothecoides materials were assessed as substrates for anaerobic digestion (AD) aiming at the simultaneous production of biogas/methane and pigments: whole autotrophic (AA) and heterotrophic algae (H); extracted heterotrophic microalgae from lipid production (HExt); and pretreated heterotrophic microalgae through enzymatic (HPEnz), autoclave (HPA), and ultrasound (HPU) processes. AA was more suitable for AD than H, as it was more efficiently converted into methane (279 vs. 180 L CH4/kg VSin). In comparison, the pretreatment of heterotrophic microalgae had a positive effect on AD, with registered methane yield increases from 263 to 290 L CH4/kg VSin (HPU, HPA, HExt). Reddish pigmentation developed in H and HPU units due to the presence of purple non-sulfur bacteria (PNSB). This phenomenon and the changes in microbiota structure during AD were confirmed by metagenomic analysis. At the end of the process, the relative abundance of Clostridiales and Bacillales increased, enhancing the hydrolysis of compounds in acetate. Consistently, Methanosaeta became the comparatively dominant methanogen, meaning that methane was produced through the acetoclastic methanogenesis pathway. The obtained results indicate for AD biorefinery feasibility—regarding the simultaneous production of biogas/methane—a digestate flow and pigments (bacteriochlorophyll a and carotenoids).

Wastes generated during the cultivation of marine microalga Crypthecodinium cohnii and after the lipid extraction process, were energetically valorized into biogas production through anaerobic digestion (AD). The tested wastes were extracted microalgae (Ae) with hexane (AeH) using supercritical extraction methods (AeS) and the supernatant obtained after culture medium centrifugation (M). The digestion of the algae biomass in the admixture with the supernatant medium (AeH+M+I and AeS+M+I) provided a higher methane content and a higher methane yield (582 and 440 L CH4/kg VS) than the substrates Ae and M, individually digested (155 and 96 L CH4/kg VS, respectively). Flow cytometry monitoring processes during AD indicated that the yield of the accumulated biogas was influenced by the operating conditions. The mixture of AeH+M+I was the only assay with a proportion of cells with less damaged membranes after AD, providing the highest methane yield and productivity (582 L CH4/kg VS and 31 L CH4/kg VS.d, respectively) and the highest energetic potential of 5.8 KWh/kg VS of all the substrates. From the results, AD integration to lipid production by C. cohnii to recover energy from the generated wastes enhanced the sustainability of the entire process and promoted the practice of zero waste.

Sludge generated in three Portuguese food processing companies - chestnut, dairy and fruit-cereal – were anaerobically digested, over 77-day experimental time, under batch mode and mesophilic conditions of temperature, to assess the anaerobic digestion process applicability to the energetic valorisation of these organic material surpluses. The biogas production of 106-144 mL (STP conditions) and the methane content of 61-72% indicate that all tested sludge reserve an energetic potential through anaerobic process. Comparatively, the sludge digestion from the fruit-cereal processing industry provided the largest volume of biogas, while the milk-yogurt reached the highest methane yield amount (209 L CH4 kg-1 VSinfluent). Anaerobic digestion is an alternative process that, according to the obtained results, can advantageously contribute to solving the problem of excess sludge in the food industry by converting it into a digestate for agricultural purposes and an energy carrier vector (biogas/methane).

Olive oil and pig productions are important industries in Portugal that generate large volumes of wastewater with high organic load and toxicity, raising environmental concerns. The principal objective of this study is to energetically valorize these organic effluents-piggery effluent and olive mill wastewater-through the anaerobic digestion to the biogas/methane production, by means of the effluent complementarity concept. Several mixtures of piggery effluent were tested, with an increasing percentage of olive mill wastewater. The best performance was obtained for samples of piggery effluent alone and in admixture with 30% of OMW, which provided the same volume of biogas (0.8 L, 70% CH4), 63/75% COD removal, and 434/489 L CH4/kg SVin, respectively. The validation of the process was assessed by molecular evaluation through Next Generation Sequencing (NGS) of the 16S rRNA gene. The structure of the microbial communities for both samples, throughout the anaerobic process, was characterized by the predominance of bacterial populations belonging to the phylum Firmicutes, mainly Clostridiales, with Bacteroidetes being the subdominant populations. Archaea populations belonging to the genus Methanosarcina became predominant throughout anaerobic digestion, confirming the formation of methane mainly from acetate, in line with the greatest removal of volatile fatty acids (VFAs) in these samples.

A hybrid anaerobic reactor was operated under the complementary effluents concept to reduce the unbalanced/inhibitory capacity of the provided piggery effluent. Brewery wastewater was chosen to complement piggery effluent (60:40% v/v, respectively). The HRT reduction from 6.7 to 3.0 days allowed the testing of an organic load increase from 4.5 to 10.0 g COD/L·d, which resulted in the almost doubling of biogas production. Biogas volumes (1.2 and 2.1 L/L·d, respectively) associated with its quality (>77% CH4) revealed that the hybrid anaerobic reactor responded positively to the operational changes and that piggery effluent can be advantageously digested using the brewery wastewater as the complementary effluent. The unit bottom and the packing bed were the main functional sections recognized in the hybrid. At the beginning of anaerobic digestion, bacterial populations belonged mostly to Bacteroidales (33%) and Clostridiales (35%). The process stability and the biogas quality at 3-d HRT were related to a change in the structure composition, since Flavobacteriales (18%), Bacillales (7%), Pseudomonadales (11%) and members of the Alcaligenaceae family (5%) also integrated the microbial communities. An evident change had also occurred in archaeal populations at this phase. Methanosaeta became the dominant genus (95%), confirming that acetoclastic methanogenesis was the main way for methane production.

A hybrid anaerobic reactor (HAR) operated to digest brewery wastewater, complemented with piggery effluent (60% and 40% v/v, respectively), under three hydraulic retention times: HRT: 5.7, 3.0 and 1.0 d. Along the first phase, the biogas of 0.9 LL−1d−1 evolved to 1.2 LL−1d−1 with a methane content of 77–78%. The HRT reduction to 3 days promoted the production until 2.1 LL−1 d−1, with methane proportions of 79.5%. The operation with an even lower HRT (1 d) allowed to obtain a higher biogas (2.9 LL−1d−1, 79.5% CH4). HAR was successfully applied to the mixture digestion, even with a HRT as low as 1 day. Phenolic compounds with antioxidant capacity in effluent adds value, besides the supply of biogas/methane.

Olive mill wastewaters (OMW) are effluents originated from olive oil extraction. As an oil-rich residue, OMW is a potential source of lipase-producing microorganisms and a complex medium potentially suitable for lipase production. The aim of the present study was to isolate yeasts with the ability to produce extracellular lipases from OMW. Thirty-two yeast isolates were obtained and screening for esterase/lipase activity using rapid plate detection methods allowed the selection of five isolates. Subsequently, extracellular lipolytic activity was determined in shake-flasks, and the best activity was found in the isolate JT5 (0.85 U/mL). This isolate was identified as Magnusiomyces capitatus by DNA sequencing. Growth and lypolytic activities by M. capitatus JT5 were assessed in undiluted OMW, and optimization of lipase production was achieved by a positive interaction of two factors (oxygen availability and nitrogen concentration). The highest lipase activity (1.4 U/mL) was obtained at NH4Cl concentration of 2.8 g/L and kLa of 0.65 min−1. The growth of M. capitatus JT5 in a stirred tank bioreactor, using undiluted OMW, allowed the improvement of lipase production (up to 3.96 U/mL) by increasing olive oil concentration in the medium, under the selected conditions of nitrogen concentration and oxygen availability. This study highlighted the isolate M. capitatus JT5 as a lipase-producing microorganism that is able to grow in undiluted OMW under controlled conditions. Results obtained in shake-flasks have been reproduced satisfactorily in the stirred tank bioreactor.

The anaerobic digestion of a peculiar piggery effluent (PE), with a high organic content (93 g/L), was carry out using olive mill wastewater (OMW) as complementary substrate. From the different tested conditions – [100%PE], [70%PE+30%OMW], [50%PE+50%OMW], [20%PE+80%OMW] – units containing only PE and the lowest proportion of OMW in the mixture (30% OMW), provided the highest biogas volume of about 780 mL (70% CH4). Comparatively, identical quantities of each substrate ([50%PE+50%OMW]) generates some gas (320 mL, 60% CH4), understood as the result of an adaptation process by the microbial consortium, while the [20%PE+80%OMW] condition provided even less gas volume (120 mL, 6% CH4), probably due to the antimicrobial capacity of the phenolic compounds in OMW, confirming the negative influence of using so high OMW proportion.

Brewery wastewater (BWW) was digested anaerobically in mesophilic conditions and batch mode. The presence of a reddish pigmentation associated to the better removal capacity of the BWW digestion (64%), indicates that the treatment and energetic valorisation of an organic effluent can occur at the same time and inside the same unit as the production of a photosynthetic pigment. The reddish pigmentation found in this experiment was mainly attributed to bacteriochlorophyll a, and to carotenoids pigments of the spirilloxantin series (characteristics of purple non-sulfur bacteria). Microbial identification through Next-generation sequencing of 16S rRNA genes showed the presence of bacterial genus Rhodobacter in the inoculum and in anaerobic digestion of BWW.

Olive oil extraction is one of the ancient agricultural industries all over the Mediterranean area and even today it is of fundamental economic importance for many industries found over the whole Mediterranean. However, this industry generates large amounts of olive mill wastewater (OMW) and due to its physicochemical characteristics it causes severe environmental concerns and management problems in the Mediterranean area, which is facing water scarcity. Technologies to reuse this wastewater will have a high impact at the economic and environmental level. The work presented aims to improve the use of jet-loop reactors technology for the aerobic biotreatment of OMW. A jet-loop reactor (100 L) coupled with an ultrafiltration (UF) membrane (MBR) system (JACTO.MBR_100 L) were tested for the influence of hydraulic parameters on OMW degradation and scale-up to 1,000 L. Chemical oxygen demand and total phenols (TP) decreased notably (up to 85% and 80% removal efficiency, respectively) after the biological treatment. The treated OMW (UF permeate) was evaluated as a source for irrigation and its impact on the soil and plant growth and their quality parameters.

Chlorella vulgaris microalgae biomass was cultivated in brewery secondary effluents and used as a recalcitrant effluent to be valorised energetically by anaerobic digestion process. All previous techniques applied for cellular disruption – autoclave, freeze/heating, ultrasound, microwave - provided either high absorption values and release of reducing sugars in the medium or membrane cells damage, compared to the untreated sample, indicating that the pre-treatment action was effective. The highest methane production was attained by the autoclave and untreated microalgae assays (samples with less permeabilized cells) while the lowest was provide by the microwaves biomasses pre-treatment: 163-178 mL versus 67 mL CH4. COD removal of 27-29 % and 16 % and TS removal of 28-32 % and 17 % were obtained, respectively. The corresponding methane yield achieved values of 0.04 and 0.030 L g-1 COD and 0.205-0.235 L g-1 TS related to concentrations determined in the influent.

Olive mill wastewater (OMW) is a major waste stream resulting from numerous operations that occur during the production stages of olive oil. The resulting effluent contains various organic and inorganic contaminants and its environmental impact can be notable. The present work aims at investigating the efficiency of (i) jet-loop reactor with ultrafiltration (UF) membrane system (Jacto.MBR), (ii) solar photo-Fenton oxidation after coagulation/flocculation pre-treatment and (iii) integrated membrane filtration processes (i.e. UF/nanofiltration (NF)) used for the treatment of OMW. According to the results, the efficiency of the biological treatment was high, equal to 90% COD and 80% total phenolic compounds (TPh) removal. A COD removal higher than 94% was achieved by applying the solar photo-Fenton oxidation process as post-treatment of coagulation/flocculation of OMW, while the phenolic fraction was completely eliminated. The combined UF/NF process resulted in very high conductivity and COD removal, up to 90% and 95%, respectively, while TPh were concentrated in the NF concentrate stream (i.e. 93% concentration). Quite important is the fact that the NF concentrate, a valuable and polyphenol rich stream, can be further valorized in various industries (e.g. food, pharmaceutical, etc.). The above treatment processes were found also to be able to reduce the initial OMW phytotoxicity at greenhouse experiments; with the effluent stream of solar photo-Fenton process to be the least phytotoxic compared to the other treated effluents. A SWOT (Strength, Weakness, Opportunities, Threats) analysis was performed, in order to determine both the strengths of each technology, as well as the possible obstacles that need to overcome for achieving the desired levels of treatment. Finally, an economic evaluation of the tested technologies was performed in an effort to measure the applicability and viability of these systems at real scale; highlighting that the cost cannot be regarded as a 'cut off criterion', since the most cost-effective option in not always the optimum one.

Report for Deliverable 5: Activity 7 of MEDOLICO Project - Mediterranean Cooperation in the Treatment and Valorisation of Olive Mill Wastewater, EU Programme ENPI-CBCMED I-B/2.1/090

1 Laboratorio Nacional de Energia e Geologia, I.P. (LNEG), Unidade de Bioenergia, Estrada do Paco do Lumiar, 1649038 Lisboa, Portugal ana.eusebio@lneg.pt; isabel.paula@lneg.pt 2 Centro de Biodiversidade, Genomica Integrativa e Funcional, Faculdade de Ciencias, Universidade de Lisboa, 1749016 Lisboa, Portugal sichaves@fc.ul.pt; rptenreiro@fc.ul.pt Institute of Agro-environmental and Forest Biology (IBAF), National Research Council of Italy via P. Castellino, 111 80131 Naples Italy francesco.lacara@ibaf.cnr.it; alessandra.morana@ibaf.cnr.it; elena.ionata@ibaf.cnr.it

MEDUSA WATER, EU Programme INCO-MED ICA3-CT-1999-00010; OLEICO, EU Programme LIFE04/ENV/IT/000409; MOTIVE, Portuguese FCT Programme POCI/AMB/56616/2004; LIFEN-Energia, Portuguese FCT Programme PTDC/ENR/69755/2006; MEDOLICO, EU Programme ENPI-CBCMED I-B/2.1/090

The major parcel of the degradation occurring along wastewater biotreatments is performed either by the native microbiota or by added microbial inocula. The main aim of this study was to apply two fingerprinting methods, temperature gradient gel electrophoresis (TGGE) and length heterogeneity-PCR (LH-PCR) analysis of 16S rRNA gene fragments, in order to assess the microbiota structure and dynamics during mixed olive oil and winery wastewaters aerobic biotreatment performed in a jet-loop reactor (JLR). Sequence homology analysis showed the presence of bacterial genera Gluconacetobacter, Klebsiella, Lactobacillus, Novosphingobium, Pseudomonas, Prevotella, Ralstonia, Sphingobium and Sphingomonas affiliated with five main phylogenetic groups: alpha-, beta- and gamma-Proteobacteria, Firmicutes and Bacteroidetes. LH-PCR analysis distinguished eight predominant DNA fragments correlated with the samples showing highest performance (COD removal rates of 67 up to 75%). Cluster analysis of both TGGE and LH-PCR fingerprinting profiles established five main clusters, with similarity coefficients higher than 79% (TGGE) and 62% (LH-PCR), and related with hydraulic retention time, indicating that this was the main factor responsible for the shifts in the microbiota structure. Canonical correspondence analysis revealed that changes observed on temperature and O(2) level were also responsible for shifts in microbiota composition. Community level metabolic profile analysis was used to test metabolic activities in samples. Integrated data revealed that the microbiota structure corresponds to bacterial groups with high degradative potential and good suitability for this type of effluents biotreatments.