Respirometric Methods Applied in the Characterisation of a Fungal Biofilm

Introduction. Respirometry is a technique based on the measurement of oxygen consumption rate usually applied with suspended biomass. To apply respirometry for biofilms characterization it is possible to detach the biomass; however, this approach changes the behaviour of the biofilm due to modifications in substrate diffusion and microbial communities stratification. Respirometry can be also applied directly to a biofilm without any removal from the support media and has been used (heterogenous respirometry) to characterise the process kinetics in a biotrickling filters for gas treatment (Bonilla-Blancas et al., 2015). Respirometry has rarely been applied to fungal biomasses: the majority of studies were focus on homogeneous respirometry in sterile conditions, on sterile solid matrices, on soil matrices or with olive mill wastewater treatment. However, respirometry on submerged fungal and bacterial biofilm in non-sterile conditions and undisturbed has never been applied. Fungi, thanks to their enzymatic systems, are one of the main players in the biosphere in the recycling of organic matter. Recently, fungi have gained attention for their potential use in the remediation of wastes and wastewaters. Nevertheless, a combined ecosystem of fungi and bacteria is interesting due to its potential synergies: fungi could reduce the general toxicity of the effluent and break the chemical bonds of recalcitrant compounds, while bacteria could bring these compounds to a final mineralisation. The main operational concerns are related to bacterial contamination and to the robustness of the bioprocess in the long-term, since fungi are easily outcompeted by bacteria. Currently, a bioreactor able to maintain the stable growth and performance of fungi under sterile and non-sterile conditions is still a challenging task. This work report on the use of respirometry as a tool for the monitoring and modelling of a reactor inoculated with fungi and able to partially, but stably and continuously remove a target recalcitrant compound in non-sterile conditions. Tannins are polyphenolic compounds produced by plants that are used in the vegetable tanning of leather at an industrial scale and represent one of the lowbiodegradability substances in tannery wastewaters. In the present work, the respirometry was applied to assess the biological activity and the diffusions processes of this fungal biofilm. Since fungal biomass is poorly characterised with modelling and respirometry, dedicated experiments were designed and validated along with a respirometric procedure. Material and Methods. The selected fungal strain was immobilised in polyurethane foam (PUF) cubes carriers and inoculated in a novel rotating, submerged, packed bed reactor fed with Quebracho tannin (QT) for 180 days. In the setup developed, the treatment reactor consisted of a 5 L vessel (4 L of effective volume and 1 L of headspace), equipped with pH stabilisation at 5.8 ± 0.2, achieved by dosing 1 M solutions of NaOH and HCl. Air was injected through a stone diffuser and manually controlled with a rotameter (100 NL h−1). The air flow allowed for complete mixing inside the reactor. Inside the vessel, a submerged plastic cylindrical cage was inserted containing 100 immobilised PUF cubes. Dissolved Oxygen (DO) and temperature were measured with a galvanic DO sensor (Oxi 340i with CellOx 325, WTW, Germany) and pH electrodes (53 33, Crison, Spain), respectively. The reactor was located in a room with temperature control. A computer and a SCADA system developed in-house were used for data acquisition and pH regulation. The respirometer used was controlled by a computer with dedicated control software in Visual Basic. The 0.3 L vessels had a stainless steel cover and a stainless steel air diffuser. The respirometer was jacketed with temperature control via the recirculation of a thermostatic water batch (Polystat24, Fisher Scientific, Spain). The air flow was regulated at 10 N mL min-1 with a mass flow controller (TecFluid, USA), and mixing was guaranteed with a magnetic stirrer. Temperature and pH probes (SenTix82, WTW, Germany) and a DO probe (CellOx 325, WTW, Germany) were connected to a control station (Inolab Multi 740, WTW, Germany) and the computer for data monitoring. The pH set point was maintained by dispensed burette and the dosage of diluted solutions of NaOH and HCl (Multi-Burette 2-SD, Crison Instruments, Spain). The biodegradable sCOD fractions (sbCOD) of medium solution containing 10 g L-1 of QT filtered at 0.45 μm were estimated with a respirometer and the conventional procedure for COD fractioning was adopted, as described elsewhere The same pH set point (pH 5.8 ± 0.2) and temperature set point (25°C) of fungal biofilm reactor were used. Results and Conclusions. The use of respirometric tests with immobilised biomass is a new technique without a defined conventional procedure, especially when fungi are considered. Pure fungal pellets and immobilised biomass sampled from the treatment reactor were used to test and define the respirometric procedure. Since the dry mass was assessed with 3 PUF cubes, the respirometric test procedure (using a 0.3 L vessel) required the removal of 10 + 3 PUF cubes sampled from the treatment reactor. The PUF cubes were placed in sterile water for 24 hours in endogenous conditions before the test water was replaced. The PUF cubes were fixed in the middle of the vessel, far from the surface and the bottom of the vessel to avoid direct contact with the final part of the probe and to minimise fluctuations. The resulting profile was accepted as repeatable after multiple pulses. Before each pulse, the endogenous conditions and Kla were evaluated with an air cut (Mora et al., 2016). The concentrations tested were within the range of 47 mg L-1 to 476 mg L-1 of sCOD for QT. These tests were performed also to verify the tannins biodegradation capacity of selected fungal strain versus QT. These tests were repeated until reaching an equal response after each pulse: approximately 3-4 pulses were needed. The QT pulses were chosen, as shown in Table 1, in order to obtain a similar substrate to biomass (S/X) ratio among the respirometer and the different condition tested in the treatment reactor. In Figure 1 four pulses of QT are represented, with an increasing concentration of COD, tested in the respirometer filled with the immobilised biomass sampled from treatment reactor. This work report on the use of respirometry as a tool for the monitoring and modelling and a procedure to apply respirometry of a fungal and bacterial biofilm in non-sterile conditions and undisturbed has been developed and respirometric protocol was defined. Respirometry respirometry was applied to assess the biological activity and the diffusions processes of this fungal biofilm. The obtained Yield factor was 0.45 ± 0.01 mg COD mg COD-1 and the results of the respirometric tests on biomass samples was coupled to the continuous operation performance of treatment reactor for kinetics parameters estimation. The modelling was based on Monod kinetics with diffusion and substrate inhibition, the model was designed with Aquasim software. Few studies in the literature have performed a stoichiometric and kinetic characterisation of fungal biomass applied in environmental biotechnologies related to wastewater treatment in non-sterile conditions. Respirometer could be used as a modelling tool for the fungal bioprocess. Acknowledgements. The project was funded by Italian MIUR (FIR RBFR13V3CH) and Regione Toscana (POR FESR 2014-2020, Lightan) and the MANUNET project FUNCELL MNET17/ENER-1143.