Valorisation of industrial hemp (Cannabis sativa L.) residues and cheese whey into volatile fatty acids for single cell protein production.

The production of single cell protein (SCP) using lignocellulosic materials stands out as a promising route in the circular bioeconomy transition. However, multiple steps are necessary for lignocellulosics-to-SCP processes, involving chemical pretreatments and specific aerobic cultures. Whereas there are no studies that investigated the SCP production from lignocellulosics by using only biological processes and microbial biomass able to work both anaerobically and aerobically. In this view, the valorisation of industrial hemp (Cannabis sativa L.) biomass residues (HBRs), specifically hurds and a mix of leaves and inflorescences, combined with cheese whey (CW) was investigated through a semi-continuous acidogenic co-fermentation process (co-AF). The aim of this study was to maximise HBRs conversion into VFAs to be further used as carbon-rich substrates for SCP production. Different process conditions were tested by either removing CW or increasing the amount of HBRs in terms of VS (i.e., two and four times) to evaluate the performance of the co-AF process. Increasing HBRs resulted in a proportional increase in VFA production up to 3115 mg HAc L-1, with experimental production nearly 40% higher than theoretical predictions. The synergy between HBRs and CW was demonstrated, proving the latter as essential to improve the biodegradability of the former. The produced VFAs were subsequently tested as substrates for SCP synthesis in batch aerobic tests. A biomass concentration of 2.43 g TSS L-1 was achieved with a C/N ratio of 5.0 and a pH of 9.0 after two days of aerobic fermentation, reaching a protein content of 42% (g protein per g TSS). These results demonstrate the overall feasibility of the VFA-mediated HBR-to-SCP valorisation process.

Microbial protein production from lactose-rich effluents through food-grade mixed cultures: Effect of carbon to nitrogen ratio and dilution rate.

Overabundant agro-industrial side streams such as lactose-rich effluents from dairy activities offer multiple valorisation opportunities. In the present study, a food-grade mixed culture of bacteria and yeasts was tested under different operational conditions for the treatment and the valorisation of cheese whey permeate (CWP), the residue of whey protein recovery, into microbial protein (MP). Under continuous aerobic fermentation settings, the carbon-to-nitrogen (C/N) ratio showed little to no influence on the system performances and MP quality as compared to dilution rates (D), leading to a final protein content as high as 76%. Under high D values, instead, while biomass productivity increased, N-efficiency and protein content decreased. Unlike the bacterial community, the yeast one proved to be highly stable and less influenced by the increase of D. A preliminary estimate indicated that 2-11% of the future MP-based food production could be satisfied by only valorising lactose-rich dairy residues such as CWP.

Phenanthrene removal from a spent sediment washing solution in a continuous-flow stirred-tank reactor.

The issue of polycyclic aromatic hydrocarbons (PAHs) is widespread in marine sediments involving ecological systems and human health. Sediment washing (SW) has proven to be the most effective remediation approach for sediments polluted by PAHs, such as phenanthrene (PHE). However, SW still raises waste handling concerns due to a considerable amount of effluents generated downstream. In this context, the biological treatment of a PHE- and ethanol-containing spent SW solution can represent a highly efficient and environmentally-friendly strategy, but its knowledge is still scarce in scientific literature and no studies have so far been conducted in continuous mode. Therefore, a synthetic PHE-polluted SW solution was biologically treated in a 1 L aerated continuous-flow stirred-tank reactor for 129 days by evaluating the effect of different pH values, aeration flowrates and hydraulic retention times as operating parameters over five successive phases. A PHE removal efficiency of up to 75-94% was achieved by an acclimated PHE-degrading consortium mainly composed of Proteobacteria, Bacteroidota and Firmicutes phyla through biodegradation following the adsorption mechanism. PHE biodegradation, mainly occurring via the benzoate route due to the presence of PAH-related-degrading functional genes and a phthalate accumulation up to 46 mg/L, was also accompanied by a reduction of dissolved organic carbon and ammonia nitrogen above 99% in the treated SW solution.

How can we possibly resolve the planet's nitrogen dilemma?

Nitrogen is the most crucial element in the production of nutritious feeds and foods. The production of reactive nitrogen by means of fossil fuel has thus far been able to guarantee the protein supply for the world population. Yet, the production and massive use of fertilizer nitrogen constitute a major threat in terms of environmental health and sustainability. It is crucial to promote consumer acceptance and awareness towards proteins produced by highly effective microorganisms, and their potential to replace proteins obtained with poor nitrogen efficiencies from plants and animals. The fact that reactive fertilizer nitrogen, produced by the Haber Bosch process, consumes a significant amount of fossil fuel worldwide is of concern. Moreover, recently, the prices of fossil fuels have increased the cost of reactive nitrogen by a factor of 3 to 5 times, while international policies are fostering the transition towards a more sustainable agro-ecology by reducing mineral fertilizers inputs and increasing organic farming. The combination of these pressures and challenges opens opportunities to use the reactive nitrogen nutrient more carefully. Time has come to effectively recover used nitrogen from secondary resources and to upgrade it to a legal status of fertilizer. Organic nitrogen is a slow-release fertilizer, it has a factor of 2.5 or higher economic value per unit nitrogen as fertilizer and thus adequate technologies to produce it, for instance by implementing photobiological processes, are promising. Finally, it appears wise to start the integration in our overall feed and food supply chains of the exceptional potential of biological nitrogen fixation. Nitrogen produced by the nitrogenase enzyme, either in the soil or in novel biotechnology reactor systems, deserves to have a 'renaissance' in the context of planetary governance in general and the increasing number of people who desire to be fed in a sustainable way in particular.

Coupling of desorption of phenanthrene from marine sediments and biodegradation of the sediment washing solution in a novel biochar immobilized-cell reactor.

The recurrent dredging of marine sediments needs the use of ex-situ technologies such as sediment washing (SW) to effectively remove polycyclic aromatic hydrocarbons. Notwithstanding, the large volumes of generated spent SW effluents require adequate treatment by employing highly-efficient, inexpensive and environmentally-friendly solutions. This study proposes the phenanthrene (PHE) desorption from sediments using Tween® 80 (TW80) as extracting agent and the treatment of the resulting spent SW solution in a biochar (BC) immobilized-cell bioreactor. The SW process reached the highest PHE removal of about 91% using a surfactant solution containing 10,800 mg L-1 of TW80. The generated amount of spent PHE-polluted SW solution can be controlled by keeping a solid to liquid ratio of 1:4. A PHE degradation of up to 96% was subsequently achieved after 43 days of continuous reactor operation, aerobically treating the TW80 solution in the BC immobilized-cell bioreactor with a hydraulic retention time of 3.5 days. Brevundimonas, Chryseobacterium, Dysgonomonas, Nubsella, and both uncultured Weeksellaceae and Xanthobacteraceae genera were mainly involved in PHE biodegradation. A rough economic study showed a total cost of 342.60 € ton-1 of sediment, including the SW operations, TW80 and BC supply and the biological treatment of the SW solution.

Direct nitrogen stripping and upcycling from anaerobic digestate during conversion of cheese whey into single cell protein.

The environmental impact of the dairy industry is heavily influenced by the overproduction of nitrogen- and carbon-rich effluents. The present study proposes an innovative process to recover waste contaminated nitrogen from anaerobic digestate while treating excess cheese whey (CW) and producing high-quality, clean single cell protein (SCP). By relying on direct aeration stripping techniques, employing an airflow subsequently used in the aerobic cheese whey fermentation step, the investigated process was able to strip 41-80% of the total ammonium nitrogen (N-NH4+) from liquid digestate. The stripped ammonia gas (NH3) was completely recovered as N-NH4+ in the acidic CW, and further upcycled into SCP having a total protein content of 74.7% and a balanced amino acids profile. A preliminary techno-economic analysis revealed the potential to directly recover and upcycle nitrogen into SCP at costs (4.3-6.3 €·kgN-1) and energetic inputs (90-132 MJ·kgN-1) matching those of conventional feed and nitrogen management processes.

Valorisation of industrial hemp (Cannabis sativa L.) biomass residues through acidogenic fermentation and co-fermentation for volatile fatty acids production.

In line with the emerging circular bioeconomy paradigm, the present study investigated the valorisation of abundant hemp biomass residues (HBRs) such as hurds (HH) and a mix of leaves and inflorescences (Mix), and other organic wastes (i.e., cheese whey and grape pomace) through the volatile fatty acid (VFA) production in mono- and co-acidogenic fermentation. The highest VFA yields, measured as acetic acid (HAc) per unit of volatile solids (VS), were obtained with the untreated Mix in mono-fermentation (185 ± 57 mg HAc/g VS) and with the combination of Mix and CW in co-fermentation (651 ± 65 mg HAc/g VS), while the highest HAc percentage reached up to 94% of total VFAs. Finally, a preliminary techno-economic evaluation revealed that the mono-fermentation of alkali pretreated HH could lead to the highest revenues among HBRs, reaching up to 710-1810, 618-1577 and 766-3722 €/ha∙year for the production of HAc, single cell protein and polyhydroxybutyrates, respectively.

Modelling the effect of SMP production and external carbon addition on S-driven autotrophic denitrification.

The aim of this study was to develop a mathematical model to assess the effect of soluble microbial products production and external carbon source addition on the performance of a sulfur-driven autotrophic denitrification (SdAD) process. During SdAD, the growth of autotrophic biomass (AUT) was accompanied by the proliferation of heterotrophic biomass mainly consisting of heterotrophic denitrifiers (HD) and sulfate-reducing bacteria (SRB), which are able to grow on both the SMP derived from the microbial activities and on an external carbon source. The process was supposed to occur in a sequencing batch reactor to investigate the effects of the COD injection on both heterotrophic species and to enhance the production and consumption of SMP. The mathematical model was built on mass balance considerations and consists of a system of nonlinear impulsive differential equations, which have been solved numerically. Different simulation scenarios have been investigated by varying the main operational parameters: cycle duration, day of COD injection and quantity of COD injected. For cycle durations of more than 15 days and a COD injection after the half-cycle duration, SdAD represents the prevailing process and the SRB represent the main heterotrophic family. For shorter cycle duration and COD injections earlier than the middle of the cycle, the same performance can be achieved increasing the quantity of COD added, which results in an increased activity of HD. In all the performed simulation even in the case of COD addition, AUT remain the prevailing microbial family in the reactor.

Phytoremediation of a pyrene-contaminated soil by Cannabis sativa L. at different initial pyrene concentrations.

This study proposes the phytoremediation of a pyrene (PYR)-contaminated soil by Cannabis sativa L. The experimental campaign was conducted along a 60 days period using three different initial PYR concentrations (i.e., 50, 100 and 150 mg kg TS-1 of soil) in 300 mL volume pots under greenhouse conditions (18-25 °C and 45-55% humidity). After 60 days of hemp growth and flourishing, the highest PYR removal reached approximately 95% in planted soil, 35% higher than in the unplanted control. PYR accumulation was observed in both roots and aerial parts of the plant, with a higher PYR uptake at increasing initial PYR concentrations in soil. The initial PYR concentration affected the growth and, thus, the phytoremediation potential of C. sativa L., which was the result of different removal mechanisms. Overall, the lowest initial PYR concentration was the one that resulted in the highest PYR removal. The interaction between the plant roots and microorganisms in rhizosphere was likely associated with PYR removal in this study. The highest DHO activity of 66.26 µg INTF g-1 TS-1 was observed in the soil spiked with 50 mg PYR·kg TS-1.

Biofilm carrier type affects biogenic sulfur-driven denitrification performance and microbial community dynamics in moving-bed biofilm reactors.

Autotrophic denitrification with biosulfur (ADBIOS) provides a sustainable technological solution for biological nitrogen removal from wastewater driven by biogenic S0, derived from biogas desulfurization. In this study, the effect of different biofilm carriers (conventional AnoxK™ 1 and Z-200 with a pre-defined maximum biofilm thickness) on ADBIOS performance and microbiomics was investigated in duplicate moving bed-biofilm reactors (MBBRs). The MBBRs were operated parallelly in continuous mode for 309 days, whilst gradually decreasing the hydraulic retention time (HRT) from 72 to 21 h, and biosulfur was either pumped in suspension (days 92-223) or supplied in powder form. Highest nitrate removal rates were approximately 225 (±11) mg/L·d and 180 (±7) mg NO3--N/L·d in the MBBRs operated with K1 and Z-200 carriers, respectively. Despite having the same protected surface area for biofilm development in each MBBR, the biomass attached onto the K1 carrier was 4.8-fold more than that on the Z-200 carrier, with part of the biogenic S0 kept in the biofilm. The microbial communities of K1 and Z-200 biofilms could also be considered similar at cDNA level in terms of abundance (R = 0.953 with p = 0.042). A relatively stable microbial community was formed on K1 carriers, while the active portion of the microbial community varied significantly over time in the MBBRs using Z-200 carriers.

Phenanthrene biodegradation in a fed-batch reactor treating a spent sediment washing solution: Techno-economic implications for the recovery of ethanol as extracting agent.

The continuous dredging of sediments contaminated by polycyclic aromatic hydrocarbons such as phenanthrene (PHE) has required the employment of high-efficiency technologies, including sediment washing (SW). However, the large amount of generated spent SW effluents requires the development of effective, eco-friendly and cost-saving approaches, which can tackle the waste formation in favor of the recovery of chemicals. This study proposes the treatment of a spent SW solution containing ethanol (EtOH) as extracting agent, by testing different initial PHE concentrations (i.e. 20-140 mg L-1) within six consecutive cycles in a fed-batch bioreactor under aerobic conditions. The biological process achieved a PHE removal of 63-91% after the enrichment of PHE-degrading bacteria and the proper supplementation of nutrients, and was mainly affected by the initial PHE concentration value and the excessive decrease of pH and dissolved oxygen. Achromobacter, Sphingobacterium and Dysgonomonas genera were mainly involved in PHE degradation, which followed a first-order kinetic model (R2 = 0.652-0.928) with a degradation rate and half-life time of 0.127-1.177 d-1 and 0.589-2.912 d, respectively. A techno-economic assessment revealed that a virtuous operation of SW, EtOH recovery and biodegradation of the SW solution can allow the recovery of up to 1.35 tons of EtOH per ton of remediated sediment and the decrease of the overall costs by 50%.

Phytoremediation of pyrene-contaminated soils: A critical review of the key factors affecting the fate of pyrene.

Soil contamination by pyrene has increased over the years due to human-related activities, urgently demanding for remediation approaches to ensure human and environment safety. Within this frame, phytoremediation has been successfully applied over the years due to its green and cost-effectiveness features. The scope of this review includes the main phytoremediation mechanisms correlated with the removal of pyrene from contaminated soils and sediments to highlight the impact of different parameters and the supplement of additives on the efficiency of the treatment. Soil organic matter (SOM), plant species, aging time, environmental parameters (pH, soil oxygenation, and temperature) and bioavailability are among the main parameters affecting pyrene removal through phytoremediation. Phytoextraction only accounts for a small part of the entire phytoremediation process, but the addition of surfactants and chelating agents in planted soils could increase pyrene accumulation in plant tissues by 20% as a consequence of the increased pyrene bioavailability. Rhizodegradation is the main phytoremediation mechanism involved due to the activity of bacteria capable of degrading pyrene in the root area. Inoculated-planted soil treatments have the potential to decrease pyrene accumulation in shoots and roots by approximately 30 and 40%, respectively, further stimulating the proliferation of pyrene-degrading bacteria in the rhizosphere. Plant-fungi symbiotic association results in an enhanced accumulation of pyrene in shoots and roots of plants as well as a higher biodegradation. Finally, pyrene removal from soil can be improved in the presence of amendments, such as natural non-ionic surfactants, biochar, and bacterial mixtures.

Enhanced lignocellulosic component removal and biomethane potential from chestnut shell by a combined hydrothermal-alkaline pretreatment.

This study proposes new perspectives for the management and biorefinery of wastes deriving from the agri-food sector such as chestnut shell (CS), which was here used as an organic feedstock for biomethane production through anaerobic digestion (AD). 1-5% alkaline (i.e. NaOH and KOH), hydrothermal (i.e. at 100 °C) and combined hydrothermal-alkaline pretreatments were employed to enhance the CS biodegradability prior to biochemical methane potential (BMP) tests conducted under mesophilic conditions. The hydrothermally-pretreated CS with 3% NaOH achieved the highest biomethane yield of 253 (±9) mL CH4·g VS-1 coupled to a volatile solid reduction of 48%. The hydrothermal-alkaline pretreatment positively affected both delignification and hemicellulose polymerization, promoting an approximately 2.4-fold higher substrate biodegradability compared to the untreated CS, which only reached a CH4 production of 104 (±5) mL CH4·g VS-1. AD proceeded via volatile fatty acid accumulation, subsequently followed by methane production that was effectively simulated via the modified Gompertz kinetic having a R2 of 0.974-0.999. Among the physical-chemical parameters characterizing the CS, the soluble chemical oxygen demand (sCOD) was highly correlated with the BMP showing a Pearson coefficient of 0.952. The cumulative biomethane yield, the sCOD and the cellulose, hemicellulose and lignin amount of CS were also processed through the least square method, obtaining a useful regression equation to predict the BMP. The economic assessment indicated that the hydrothermal-alkaline pretreatment is a cost-effective method to improve the BMP of CS, also for future full-scale applications.

The addition of biochar as a sustainable strategy for the remediation of PAH-contaminated sediments.

The contamination of sediments by polycyclic aromatic hydrocarbons (PAHs) has been widely spread for years due to human activities, imposing the research and development of effective remediation technologies for achieving efficient treatment and reuse of sediments. In this context, the amendment of biochar in PAH-contaminated sediments has been lately proposed as an innovative and sustainable technology. This review provides detailed information about the mechanisms and impacts associated with the supplementation of biochar to sediments polluted by PAHs. The properties of biochar employed in these applications have been thoroughly examined. Sorption onto biochar is the main mechanism involved in PAH removal from sediments. Sorption efficiency can be significantly improved even in the presence of a low remediation time (i.e. 30 d) when a multi-PAH system is used and biochar is provided with a high dosage (i.e. by 5% in a mass ratio with the sediment) and a specific surface area of approximately 360 m2 g-1. The use of biochar results in a decrease (i.e. up to 20%) of the PAH degradation during bioaugmentation and phytoremediation of sediments, as a consequence of the reduction of PAH bioavailability and an increase of water and nutrient retention. In contrast, PAH degradation has been reported to increase up to 54% when nitrate is used as electron acceptor in low-temperature biochar-amended sediments. Finally, biochar is effective in co-application with Fe2+ for the persulfate degradation of PAHs (i.e. up to 80%), mainly when a high catalyst dose and an acidic pH are used.

Effect of nickel on the comparative performance of inverse fluidized bed and continuously stirred tank reactors for biogenic sulphur-driven autotrophic denitrification.

The comparative performance of an inverse fluidized bed reactor (IFBR) having high density polyethylene beads as carrier materials for biofilm formation and a continuous stirred tank reactor (CSTR), both maintaining autotrophic denitrification using biogenic sulphur (ADBIOS) in the absence and presence of nickel (Ni2+), was studied. The reactors were compared in terms of NO3--N and NO2--N removal and SO42--S production throughout the study. A simulated wastewater with an inlet NO3--N concentration of 225 mg/L and a decreasing concentration of biogenic sulphur (bio-S) from 1.5 to 0.375 g/L was used. Both reactors were operated at a hydraulic retention time (HRT) of 48 h for 140 days and at an HRT of 42 h for the following 68 days. A more efficient ADBIOS was observed in the CSTR than IFBR throughout the study due to a better mixing of the feed wastewater in the bulk liquid and a higher availability of bio-S to the suspended cells. The NO3--N removal efficiency in the IFBR decreased by approximately 41% when the feed bio-S was reduced to 0.375 g/L, while it remained unaffected in the CSTR. Conversely, the presence of Ni2+ did not significantly affect NO3--N removal in both reactors even at a feed Ni2+ concentration of 120 mg/L. The highest NO3--N removal rates achieved were 86 and 108 mg NO3--N/(L·day) in the IFBR and CSTR, respectively, in the presence of 120 mg/L of feed Ni2+ at an HRT of 42 h. Batch studies conducted with acclimatized biomass showed that the continuous-flow operation mode in both reactors played a major role in helping the autotrophic denitrifiers to tolerate Ni2+ toxicity.

Comparing performances, costs and energy balance of ex situ remediation processes for PAH-contaminated marine sediments.

This study proposes a comparison of different ex situ technologies aimed at the removal of polycyclic aromatic hydrocarbons from marine sediments in terms of performances, costs and energy balance. In accordance with the principles of water-energy nexus, anaerobic bioremediation, soil washing and thermal desorption were investigated under low liquid phase and temperature conditions using phenanthrene (PHE) as model compound. After 42 days of anaerobic bioremediation, the highest PHE biodegradation of 68 and 64% was observed under denitrifying and methanogenic conditions, respectively, accompanied by N2 and CH4 production and volatile fatty acid accumulation. During soil washing, more than 97% of PHE was removed after 60 min using a solid-to-liquid ratio of 1:3. Along the same treatment time, low-temperature thermal desorption (LTTD) allowed a PHE removal of 88% at 200 °C. The economic analysis indicated that LTTD resulted in a higher cost (i.e. 1782 € m-3) than bioremediation and soil washing (228 and 371 € m-3, respectively). The energy balance also suggested that bioremediation and soil washing are more sustainable technologies as a lower required energy (i.e. 16 and 14 kWh m-3, respectively) than LTTD (i.e. 417 kWh m-3) is needed.

High rate continuous biohydrogen production by hyperthermophilic Thermotoga neapolitana.

This study focused on continuous-flow hydrogen production by Thermotoga neapolitana at a hydraulic retention time (HRT) decreasing from 24 to 5 h. At each HRT reduction, the hydrogen yield (HY) immediately dropped, but recovered during prolonged cultivation at constant HRT. The final HY in each operating period decreased from 3.4 (±0.1) to 2.0 (±0.0) mol H2/mol glucose when reducing the HRT from 24 to 7 h. Simultaneously, the hydrogen production rate (HPR) and the liquid phase hydrogen concentration (H2aq) increased from 82 (±1) to 192 (±4) mL/L/h and from 9.1 (±0.3) to 15.6 (±0.7) mL/L, respectively. Additionally, the effluent glucose concentration increased from 2.1 (±0.1) to above 10 mM. Recirculating H2-rich biogas prevented the supersaturation of H2aq reaching a value of 9.3 (±0.7) mL/L, resulting in complete glucose consumption and the highest HPR of 277 mL/L/h at an HRT of 5 h.

Assessing practical identifiability during calibration and cross-validation of a structured model for high-solids anaerobic digestion.

High-solids anaerobic digestion (HS-AD) of the organic fraction of municipal solid waste (OFMSW) is operated at a total solid (TS) content ≥ 10% to enhance the waste treatment economy, though it might be associated to free ammonia (NH3) inhibition. This study aimed to calibrate and cross-validate a HS-AD model for homogenized reactors in order to assess the effects of high NH3 levels in HS-AD of OFMSW, but also to evaluate the suitability of the reversible non-competitive inhibition function to reproduce the effect of NH3 on the main acetogenic and methanogenic populations. The practical identifiability of structural/biochemical parameters (i.e. 35) and initial conditions (i.e. 32) was evaluated using batch experiments at different TS and/or inoculum-to-substrate ratios. Variance-based global sensitivity analysis and approximate Bayesian computation were used for parameter optimization. The experimental data in this study permitted to estimate up to 8 biochemical parameters, whereas the rest of parameters and biomass contents were poorly identifiable. The study also showed the relatively high levels of NH3 (i.e. up to 2.3 g N/L) and ionic strength (i.e. up to 0.9 M) when increasing TS in HS-AD of OFMSW. However, the NH3 non-competitive function was unable to capture the acetogenic/methanogenic inhibition. Therefore, the calibration emphasized the need for target-oriented experimental data to enhance the practical identifiability and the predictive capabilities of structured HS-AD models, but also the need for further testing the NH3 inhibition function used in these simulations.

Mineral characterization of the biogenic Fe(III)(hydr)oxides produced during Fe(II)-driven denitrification with Cu, Ni and Zn.

The recovery of iron and other heavy metals by the formation of Fe(III) (hydr)oxides is an important application of microbially-driven processes. The mineral characterization of the precipitates formed during Fe(II)-mediated autotrophic denitrification with and without the addition of Cu, Ni, and Zn by four different microbial cultures was investigated by X-ray fluorescence (XRF), Raman spectroscopy, scanning electron microscopy equipped with energy dispersive X-Ray analyzer (SEM-EDX), Fourier transform infrared spectroscopy (FTIR) and X-ray Powder Diffraction (XRD) analyses. Fe(II)-mediated autotrophic denitrification resulted in the formation of a mixture of Fe(III) (hydr)oxides composed of amorphous phase, poorly crystalline (ferrihydrite) and crystalline phases (hematite, akaganeite and maghemite). The use of a Thiobacillus-dominated mixed culture enhanced the formation of akaganeite, while activated sludge enrichment and the two pure cultures of T. denitrificans and Pseudogulbenkiania strain 2002 mainly resulted in the formation of maghemite. The addition of Cu, Ni and Zn led to similar Fe(III) (hydr)oxides precipitates, probably due to the low metal concentrations. However, supplementing Ni and Zn slightly stimulated the formation of maghemite. A thermal post-treatment performed at 650 °C enhanced the crystallinity of the precipitates and favored the formation of hematite and some other crystalline forms of Fe associated with P, Na and Ca.