Removal of cyclohexane and ethanol from air in biotrickling filters inoculated with Candida albicans and Candida subhashii

Abstract

This paper presents investigations on the removal of cyclohexane and ethanol from air in polyurethane-packed biotrickling filters, inoculated with Candida albicans and Candida subhashii fungal species. Results on process performance together with flow cytometry analyses of the biofilm formed over packing elements are presented and discussed. The results indicate that the presence of ethanol enhances the removal efficiency of cyclohexane from air. This synergistic effect may be attributed to both co-metabolism of cyclohexane with ethanol as well as increased sorption efficiency of cyclohexane to mineral salt medium in the presence of ethanol. Maximum elimination capacities of 89 g m-3 h-1 and 36.7 g m-3 h-1 were noted for cyclohexane and ethanol, respectively, when a mixture of these compounds was treated in a biofilter inoculated with C. subhashii. Results of flow cytometry analyses after 100 days of biofiltration revealed that about 91% and 88% of cells in biofilm remained actively dividing, respectively for C. albicans and C. subhashii species, indicating their good condition and ability to utilize cyclohexane and ethanol as a carbon source. Removal of cyclohexane and ethanol from air in biotrickling fi lters inoculated with Candida albicans... 27 break the mass transfer barrier between gaseous and aqueous (i.e. biofilm) phases and the rate of their biodegradation is mainly governed by the rate of biodegradation within the biofilm. Contrary, for poorly water-soluble compounds, i.e. hydrophobic ones, the efficiency of biofiltration depends greatly on the mass transfer rate between the above mentioned phases, and thus the biofiltration efficiency is much lower than for hydrophilic compounds (Cheng et al. 2016; Gospodarek et al. 2019). Several measures may be applied to improve the biofiltration performance with respect to hydrophobic compounds, including the addition of surfactants, especially biosurfactants, application of selected microbial species, including fungi, reactor modification, selection of proper process conditions as well as co-treatment with hydrophilic compounds (Cheng et al. 2020; He et al. 2020; Miller et al. 2019; Miller et al. 2020; Rybarczyk et al. 2020, 2019b; Yang et al. 2018, 2010). In this paper, the possibility of using selected Candida fungi to simultaneously remove from air compounds with extremely different affinity to the aqueous phase was investigated. Hydrophobic cyclohexane and hydrophilic ethanol were used as model compounds. These compounds are found in post-processing gases from, e.g., paint, petroleum and food industries (Avalos Ramirez et al. 2007; Zhanga et al. 2018). Biotrickling filtration of air containing single cyclohexane or ethanol was previously investigated (Avalos Ramirez et al. 2007; Cox et al. 2001; Salamanca et al. 2017). In this paper, a mixture of cyclohexane and ethanol was subjected to biofiltration in two biotrickling filters, inoculated with Candida albicans and Candida subhashii, respectively. The composition of biofilms formed in two biotrickling filters was tested for purity of inhabiting fungi and compared between the process start-up and steady-state operation conditions using flow cytometry technique. To the best knowledge of authors of this paper, the above given fungi have not been used so far in biotrickling filters for air purification, and the search for new species of microorganisms capable of biodegradation of pollutants, especially of a hydrophobic nature, is an important trend in the environmental research. Materials and methods Investigations were performed on biotrickling filters made of plexi-glass columns of the following dimensions: 0.08 m in internal diameter and 0.68 m in height. Biofilters were packed with polyurethane foam discs (pore size PPI 10, Ultramare, Poland; dimensions of a single disc: 0.08 m in diameter, 0.01 m in height) up to the working volume of 2.5 dm3 each. Biofilters were fed with a gas mixture from the bottom, while the trickling liquid was supplied from the top of a bioreactor, by means of a peristaltic pump. Gaseous mixtures of air with cyclohexane and ethanol (POCH, Poland) were obtained by passing the purified and dried air via a porous sinter through vials containing liquid cyclohexane and ethanol. The gas flow rate was controlled and regulated using a precise mass flow controller (Vögtlin, Switzerland). Gas flow rate of 2.5 dm3 min-1 was used throughout the experiments, resulting in empty bed residence time (EBRT) equal to 1 min. Pressure drop across the packings of biotrickling filters was monitored using MPX5010dp sensors (NXP, the Netherlands) working in the range from 0 to 10 kPa. Maximum noted pressure did not exceed 2 kPa and no biomass overgrowth was observed throughout the experiments. Gaseous samples containing cyclohexane and ethanol were taken from inlet and outlet gas streams. Samples were collected in Tedlar bags and concentrations of the above given volatile organic compounds were determined using gas chromatography technique using a DB-WAX column (30 m × 0.53 mm × 1 μm; Agilent Technologies, USA) and flame ionization detector (Varian CP-3800, VarianAnalytical Instruments, USA). Nitrogen was used as a carrier gas. The parameters of the analytical program were as follows: oven temperature: 100°C; FID detector temperature: 200°C, carrier gas flow rate: 3 cm3 min-1; split ratio: 10. During the start-up period (first 7–10 days of biofiltration process), packing elements of biofilters were trickled with a Buffered Peptone Water medium (Merck, Germany). Then, mineral salt medium (MSM) was introduced. MSM contained the following salts dissolved in 1 dm3 of distilled water: Na2HPO4·2H2O (7.39 g), KH2PO4 (3 g), NaCl (0.5 g) and NH4Cl (1 g) (POCH, Poland). Trickling liquid solutions were autoclaved before introducing to biofilters (Prestige Medical, England) and the MSM solution was exchanged once a week throughout the whole reported time period. Trickling liquid was sprayed over the packing elements with a frequency of 0.5 minutes per each hour, with a volumetric flow rate of 0.2 dm3 min-1. Prior to the biofiltration start-up, packing elements made of polyurethane foam discs were inoculated with Candida albicans (biotrickling filter “A”) and Candida subhashii (biotrickling filter “B”). Immobilization of fungi on polyurethane discs was realized using sterile beakers (each 1 dm3 in volume) in which 600 cm3 of Sabouraud medium (BTL, Poland) containing 10% (v/v) of selected fungi species inoculums was placed. These beakers were agitated in an orbital shaker (100 rpm, 24°C). After 24 hours of shaking, half of the medium volume was replaced with a fresh MSM solution. After the next 24 hours of shaking, the whole volume of the medium was replaced by a fresh portion of MSM solution. Then, a similar procedure was repeated, but MSM solution was replaced with Sabouraud medium in two steps as described above. The inoculation procedure lasted for 120 hours. Each day of inoculation, samples of media were taken and optical density measurements at wavelength of 595 nm using Thermo Scientific Multiskan FC spectrophotometer (Thermo Fisher Scientific, Finland) were routinely performed. Two series of experiments were performed. In the first series (I), the performance of two biotrickling filters A and B was studied. Both biotrickling filters were initially fed with cyclohexane only, and at the 38th day of the process, ethanol was introduced into the gas stream. Additionally, flow cytometry analyses aiming at the determination of the general condition of microorganism populations inhabiting packing elements of biofilters were performed. In the second series of experiments (II), one biotrickling filter inoculated with C. subhashii was investigated. Selection of these fungi species out of two investigated was done due to pathogenic characteristics of C. albicans as well as due to expected high performance of C. subhashii in the biotrickling filtration of hydrophobic volatile organic compounds. In this series, the biofilter was fed with a mixture of cyclohexane and ethanol from the process initiation. This approach was intended in order to study the effect of ethanol addition on the 28 P. Rybarczyk, M. Marycz, B. Szulczyński, A. Brillowska-Dąbrowska, A. Rybarczyk, J. Gębicki performance of cyclohexane biofiltration, in comparison to experiments in series I. In series II, the liquid phase (MSM) was also investigated in terms of variations of pH as well as the concentrations of treated compounds during the biofiltration process. The experimental staining technique with methylene blue (Sigma Aldrich, USA) was used to assess the formation of biofilm, containing the tested fungi, on the polyurethane foam elements. Photos of immobilized fungi were taken using transmitting light optical microscope with a 10× working distance lens (LAB 40 Series Optical Microscope, OPTA-TECH, Poland). For cytometric analyses, single polyurethane discs were taken from each of the working biofilters. Each disc was placed in a beaker and suspended in 40 cm3 of 0.01 M phosphate buffered saline solution (PBS, pH = 7.6) and shaken (4 times of 15 s shaking) in an ultrasonic bath (Bandelin Sonorex, Germany). After each shaking step, a beaker with a disk was placed in an water-ice bath for 15 s. The cell suspension was filtered using a 400-mesh nylon net to remove impurities. The precipitates were washed twice with PBS solution and suspended in 35 cm3 of PBS solution after the centrifugation (6000 rpm, 6 min, 4°C; Eppendorf Centrifuge 5418R, Germany). A cell count was determined using a flow cytometry technique (Merck Millipore Guava easyCyte 8, Germany). A suspension volume containing 1 million of fungi cells was used in further investigations. For the determination of microbial population condition using flow cytometry, 100 μL of AAB buffer (Annexin V Binding Buffer, BD Biosciences, Pharmingen, USA), 0.5 μL of FITC Annexin V (Annexin V fluorescein conjuga