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Wastewater treatment plants (WWTP) are required to meet increasingly stringent effluent quality standards as well as to improve process sustainability in terms of energy efficiency, in line with the call for a paradigm shift towards the more sustainable concept of wastewater resource recovery facility (WRRF). In this context, optimised Instrumentation, Control and Automation (ICA) systems as well as process and plant-wide modelling have become key tools for optimising economic and environmental costs. The present work reports on the successfull integration of an innovative on-line continuous titrimeter and plant-wide modelling as a support tool for the optimisation of the nitritification unit in a large-scale industrial WWTP. The information gathered from the on-line titrimeter allowed for real-time estimation of the Ammonia Oxidation Rate of the activated sludge and, ultimately, of the nitrification residual capacity of the plant. Results on the titrimeter long-term operation and historical data modelling supported plant managing decisions, such as lowering the dissolved oxygen (DO) set-point of the oxidation unit, straightforwardly resulting in energy saving. Graphical abstract

Viable and metabolically active fungi in toxic mixed liquors, treating landfill leachates and municipal wastewaters, were identified by culture depending methods. A selective culture medium consisting of wastewater and agar (WA) restrained fungi that could be randomly present (94% of the 51 taxa retrieved on WA were sample-specific), overcoming the problem of fast growing fungi or mycoparasite fungi. Moreover, WA allowed the isolation of fungi with a possible role in the degradation of pollutants typically present in the two wastewaters. Phoma medicaginis var. medicaginis, Chaetomium globosum, and Geotrichum candidum were mainly found in municipal wastewater, whereas Pseudallescheria boydii, Scedosporium apiospermum, Aspergillus pseudodeflectus, and Scopulariopsis brevicaulis were typical of landfill leachate.

This study demonstrated that a microbial community dominated by fungi can be selected and maintained in the long-term under non-sterile conditions, in a pilot-scale packed-bed reactor fed with tannery wastewater. During the start-up phase, the reactor, filled with 0.6 m3 of polyurethane foam cubes, was inoculated with a pure culture of Aspergillus tubingensis and Quebracho tannin, a recalcitrant compound widely used by tannery industry, was used as sole carbon source in the feeding. During the start-up, fungi grew attached as biofilm in carriers that filled the packed-bed reactor. Subsequently, the reactor was tested for the removal of chemical oxygen demand (COD) from an exhaust tanning bath collected from tanneries. The entire experiment lasted 121 days and average removals of 29% and 23% of COD and dissolved organic carbon (DOC) from the tannins bath were achieved, respectively. The evolution of the microbial consortium (bacteria and fungi) was described through biomolecular analyses along the experiment and also developed as a function of the size of the support media.

In environmental biotechnology applications for wastewater treatment, bacterial-based bioprocesses are mostly implemented; on the contrary, the application of fungal-based bioprocesses, is still challenging under non-sterile conditions. In a previous laboratory-scale study, we showed that when specific tannins are used as the sole carbon source, fungi can play a key role in the microbial community, under non-sterile conditions and in the long term. In a previous study, an engineered ecosystem, based on fungal tannin biodegradation, was successfully tested in a laboratory-scale bioreactor under non-sterile conditions. In the present study, a kinetic and stoichiometric characterisation of the biomass developed therein was performed through the application of respirometric techniques applied to the biomass collected from the above-mentioned reactor. To this aim, a respirometric set-up was specifically adapted to obtain valuable information from tannin-degrading fungal biofilms. A mathematical model was also developed and applied to describe both the respirometric profiles and the experimental data collected from the laboratory-scale tests performed in the bioreactor. The microbial growth was described through a Monod-type kinetic equation as a first approach. Substrate inhibition, decay rate and tannin hydrolysis process were included to better describe the behaviour of immobilised biomass selected in the tannin-degrading bioreactor. The model was implemented in AQUASIM using the specific tool Biofilm Compartment to simulate the attached fungal biofilm. Biofilm features and transport parameters were either measured or assumed from the literature. Key kinetic and stoichiometric unknown parameters were successfully estimated, overcoming critical steps for scaling-up a novel fungal-based technology for tannins biodegradation.

The information available on the microbial communities responsible for pollutant degradation is increasingly accessible. Its use to optimize process design and operation is an important challenge in the field of effluent treatment research. Therefore, a prototype of a moving bed biotrickling filter (MBBTF) reactor was designed and, for the first time, operated at full-scale for the removal of sulfides desorbing from tannery industrial wastewater. The bacterial community operating in this innovative reactor was studied, and its composition and response to different operating conditions were characterized. A stable biomass, dominated by sulfur-oxidizing bacteria of the genus Acidithiobacillus was selected from inside the MBBTF reactor, and temperature, pH and bed rotation were shown to be the main factors driving the community structure. Moreover, data from different approaches indicated an uneven spatial distribution of biofilm inside the studied reactor, due to the combined effect of fluid dynamics and substrate gradients within the bed volume. Despite the high removal efficiency achieved by this innovative prototype (80% on average), the data suggested that the result could be improved by adopting solutions for a more stable and even biofilm distribution. It was shown that short frequent bed rotations, rather than long scattered rotations, ensured biomass stability. Furthermore, diversifying biofilm support media as a function of expected local pollutant concentrations should be considered. Data obtained from the bacterial community can therefore provide indications for possible further improvement of MBBTF reactor design and performance.

Tannins are polyphenolic compounds produced by plants and they are used in industrial vegetable tanning of leather. Tannins represent one of the low biodegradability substances in tannery wastewaters with high recalcitrant soluble chemical oxygen demand, furthermore high concentration of tannins can inhibit biological treatment. In the present study, four novel rotating submerged packed bed reactors were inoculated with a selected fungal strain to reach a biological degradation of tannins in non-sterile conditions. The selected fungal strain, Aspergillus tubingensis MUT 990, was immobilised in polyurethane foam cubes carriers and inserted inside a submerged rotating cage reactors. The reactors were feed with a solution composed of four tannins: Quebracho (Schinopsis spp.), Wattle (Mimosa spp.), Chestnut (Castanea spp.) and Tara (Caesalpinia spp.). Four reactors with a volume of 4 L each were used, the co-substrate was pure malt extract, the hydraulic retention time was 24 h and the pH setpoint was 5.5. The reactors configuration was chosen to allow the study of the effect of rotation and the co-substrate addition on tannins removal. The experiment lasted two months and it was achieved 80% of chemical oxygen demand and up to 90% dissolved organic carbon removal, furthermore it was detected an important tannase activity.

Tannins are polyphenolic compounds produced by plants that are used in the vegetable tanning of leather at industrial scale. Quebracho tannin and Tara tannin are intensively used by the tanning industry and are two of the most recalcitrant compounds that can be found in tannery wastewaters. In this study two reactors fed with Quebracho tannin and Tara tannin, respectively, were inoculated with polyurethane foam cubes colonized with a fungal strain biofilm of Aspergillus tubingensis MUT 990. A stable biofilm was maintained in the reactor fed with Quebracho tannin during 180 days of operation. Instead, biofilm got detached from the foam cubes during the start-up of the reactor fed with Tara tannin and a bacterial-based suspended culture was developed and preserved along the operational period (226 days). Soluble chemical oxygen demand removals up to 53% and 90% and maximum elimination capacities of 9.1 g sCOD m−3 h−1 and 37.9 g sCOD m−3 h−1 of Quebracho and Tara tannins, respectively, were achieved in the reactors without the addition of co-substrates. Next generation sequencing analysis for bacteria and fungi showed that a fungal consortium was developed in the reactor fed with Quebracho tannin while fungi were outcompeted by bacteria in the reactor fed with Tara tannin. Furthermore, Quebracho and Tara tannins were successfully co-treated in a single reactor where both fungi and bacteria were preserved.

Fungi are known to be present in the activated sludge of wastewater treatment plants (WWTP). Their study should be at the base of an overall vision of the plant effectiveness and of effluents sanitary impact. Moreover, it could be fundamental for the implementation of successful bioaugmentation strategies aimed at the removal of recalcitrant or toxic compounds. This is one of the first studies on the cultivable autochthonous mycoflora present in the mixed liquors of two WWTP treating either vegetable or chromium tannery effluents. All samples showed a risk associated with potential pathogens or toxigenic species and high ecotoxicity (Lepidium sativum and Raphidocelis subcapitata were the most sensitive organisms). Diverse fungal populations developed, depending on the origin of the samples (63% of the 102 identified taxa were sample-specific). The use of a fungistatic was determinant for the isolation and, thus, for the identification of sample-specific species with a lower growth rate. The incubation temperature also affected the mycoflora composition, even though at lower extent. A selective medium, consisting of agarised wastewater, allowed isolating fungi with a biodegradation potential. Pseudallescheria boydii/Scedosporium apiospermum species complex was ubiquitously dominant, indicating a possible role in the degradation of pollutants in both WWTP. Other species, i.e. Trichoderma spp., Trematosphaeria grisea, Geotrichum candidum, Lichtheimia corymbifera, Acremonium furcatum, Penicillium simplicissimum, Penicillium dangeardii, Fusarium solani, Scopulariopsis brevicaulis potentially could be involved in the degradation of specific pollutants of vegetable or chromium tannery wastewaters. However, several of these fungi are potential pathogens and their application, for an in situ treatment, must be carefully evaluated.

Conventional wastewater treatment technologies are ineffective for remediation of old LandFill Leachate (LFL), and innovative approaches to achieve satisfactory removal of this recalcitrant fraction are needed. This study focused on old LFL treatment with a selected fungal strain, Bjerkandera adusta MUT 2295, through batch and continuous tests, using packed-bed bioreactors under non-sterile conditions. To optimize the process performance, diverse types of co-substrates were used, including milled cellulose from beverage cups waste material. Extracellular enzyme production was assayed, in batch tests, as a function of a) cellulose concentration, b) leachate initial Chemical Oxygen Demand (COD) and Soluble COD (sCOD), and c) co-substrate type. Bioreactors were dosed with an initial start-up of glucose (Rg) or cellulose (Rc). An additional glucose dosage was provided in both reactors, leading to significant performance increases. The highest COD and sCOD removals were i) 63% and 53% in Rg and ii) 54% and 51% in Rc.

In Moving Bed Biofilm Reactors (MBBR) the biofilm grows protected within small plastic carriers (known as biofilm carriers), which are designed with high internal surface area. The biological wastewater treatment process consists of adding biofilm carriers in aerated or anoxic tanks to support biofilm attachment and growth. Some of the authors conceived, designed and realised an improved carrier (new shape and material) for fungal biofilm development. The improved biofilm carriers were tested in laboratory conditions and good results were obtained. This new biofilm carrier will be used to treat cellulosic (tannery and papermill) wastewaters and is grafted with cellulose fibers for the self-sustainability of the fungal biomass. This will facilitate substrate adhesion, due to biochemical capabilities of the selected strains, which involve secretion of extracellular enzymes, that can break down substrate by combined action of several degradative processes, such as demethylation, oxidative cleavage of the propane side chain, cleavage of ether bonds between monomers etc.

Recalcitrant compounds limit the efficiency of conventional biological processes for wastewater treatment, representing one of the major issues in the field. This study focused on the treatment of three effluents with White-Rot-Fungus (WRF) Bjerkandera adusta MUT 2295 in batch tests, with biomass cultivated in attached form on polyurethane foam cubes (PUFs) to test its efficiency in the removal of the target effluents’ recalcitrant fraction. Treatment efficiency of B. adusta was evaluated on landfill leachate (Canada) and two solutions containing synthetic recalcitrant compounds, which were prepared with tannic and humic acid. Chemical Oxygen Demand (COD) and color removal, the production of manganese peroxidases, and the consumption of a co-substrate (glucose) were monitored during the experiment. Biological Oxygen Demand (BOD5) and fungal dry weight were measured at the beginning and at the end of the experiment. After co-substrate addition, effluent COD was 2300 ± 85, 2545 ± 84, and 2580 ± 95 (mg/L) in raw leachate and tannic and humic acids, respectively. COD removal of 48%, 61%, and 48% was obtained in raw leachate and in the synthetic effluents containing tannic and humic acids, respectively. Color removal of 49%, 25%, and 42% was detected in raw leachate and in tannic and humic acid solutions, respectively. COD and color removals were associated with the increase of fungal dry weight, which was observed in all the trials. These results encourage the use of the selected fungal strain to remove tannic acid, while further investigations are required to optimize leachate and humic acid bioremediation.

Hydrogen sulfide (H2S) is one of the major tannery wastewater pollutants. Up today, chemical scrubbing is the most established technology for H2S removal in wastewater treatment plants. However, this procedure increases both the operating costs and the carbon footprint of the treatment. On the other hand, biological treatment is an emerging and sustainable technology for air pollution control. Our study focuses on the biological sulfur-oxidizing potential of autochthonous sludge of a plant treating tannery wastewater, located in Santa Croce (Tuscany, Italy). We propose a multidisciplinary approach to investigate two aspects: first, the composition of the microbial community both in the biological sludge and in the primary sludge and second, the sulfur-oxidizing potential of these native matrices. In order to do that, biological and primary sludge were used as inoculum in a reactor fed with sulfides. Then, traditional cultivation techniques were combined with several molecular approaches for analyzing the microbial community structure with a special focus on sulfur-oxidizing bacteria (SOB). Our study demonstrated that the use of different techniques was fundamental in order to detect the largest number of sulfur-oxidizing bacterial components; in particular, the detection of less represented components was guaranteed only by the performed multidisciplinary approach. Our study demonstrated the optimal performances of the reactor in selecting a sulfur-oxidizing biomass from autochthonous matrices. In addition, the importance of the primary sludge as inoculum for sulfur-oxidizing reactors was proved.