Performance and microbial community evolution of toluene degradation using a fungi-based bio-trickling filter

Journal of Hazardous Materials 365 (2019) 642–649

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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat

Performance and microbial community evolution of toluene degradation using a fungi-based bio-trickling filter ⁎

Yun Zhanga, Jia Liua, , Yiwei Qina, Zhuhui Yanga, Jingyang Caoa, Yi Xingb, Jian Lia, a b

T

⁎

Key Laboratory of Beijing on Regional Air Pollution Control, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China School of Energy and Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Toluene Fungi Bio-trickling Microbial diversity

Fungi have their unique advantages in capturing and degrading hydrophobic VOCs. To study the performance of fungi-based bio-trickling filters (BTFs) with respect to the degradation of toluene, and the succession process of the fungal colony under different operating conditions, a three-layer BTF packed by dominant Fusarium oxysporum immobilized with ceramic particles were set up. The fungal BTF started quickly within 7 days and restarted less than 7 days after starvation; its average RE was higher than 92.5% when the toluene inlet loading rate (ILR) ranging from 7.0 to 100.9 g m−3 h−1 at steady state. Moreover, the maximum elimination capacity (EC) of 98.1 g m−3 h−1 was obtained at a toluene ILR of 100.3 g m−3 h−1. The microorganism analysis of time and space revealed that the dominant fungi Fusarium were replaced by Paramicrosporidium saccamoebae after a certain evolutionary period. The intermediate layer had more microbes and a more complex community than the other two layers, and was more suitable for the survival of the varieties of microbes.

1. Introduction Volatile organic compounds (VOCs), which are indispensable burgeoning atmospheric pollutants, need to be urgently governed because of their effects on the environment and on human life [1]. Toluene, a typical organic solvent, is widely used as an important raw material in

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the chemical industry. It is classified as a hydrophobic VOC, and does great harm to human health and the environment [2]. Therefore, efficient, cost-effective, and environment-friendly technologies are required for the treatment of volatile pollutants [3]. Traditional technologies such as thermal oxidation, incineration, photocatalytic oxidization, absorption, adsorption, and condensation

Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (J. Li).

https://doi.org/10.1016/j.jhazmat.2018.11.062 Received 19 March 2018; Received in revised form 14 November 2018; Accepted 15 November 2018 Available online 16 November 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.

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Next, 1.8% agar was added in the culture medium LA to manufacture medium LB, which was used to screen and reserve the fungi. All the media were autoclaved for 20 min at 121℃. However, the part of the composition that couldn’t be autoclaved was added to the media after irradiation. The acclimation and screening of toluene-degrading fungi proceeded in aerobic bottles. After 10 mL of the activated sludge was added into a bottle with 50 mL of the inorganic medium A, 0.0025 g streptomycin was added to the culture liquid (to inhibit bacterial growth), with a certain amount of toluene added to the medium. Then, the bottle was sealed (to prevent toluene from volatilizing), and the culture was shaken for 3 days at 120 rpm. The toluene concentration in the bottle was measured every few hours. Until the complete degradation of toluene, 10% of the inoculation liquid was transferred to fresh medium A, and then the cultivation of the fresh medium continued. The above processes were repeated three times. The final acclimated medium was coated on an LB plate after gradient dilution; then, a single colony was selected after constant-temperature incubation for 24 h. Subsequently, the single colony was streaked for separation and purification. Finally, effective toluene-degrading fungi were obtained.

can be used for indoor air pollution control [4–9]. They are defective because of their considerably large energy requirements and by-products. In contrast, biological technologies are methods which use natural capabilities of microorganisms to remove low-concentration and large-intake VOCs. They are considered as alternative techniques and have the advantages of high efficiency, lack of byproducts, and low investment and operating costs [10]. Among these biological technologies, bio-trickling filtration has been proven to be efficient in eliminating different types of odorous compounds and VOCs. Because of its relatively small footprint and the cost-effective ability to convert contaminants into harmless end-products under optimal conditions, it has attracted considerable attention [11]. Thus far, the research on bio-trickling filtering has mainly concentrated on five aspects; the treatment of different target pollutants [12], selection of various fillers [13], optimization of the reactor and process conditions [14], microbial strains and biodegradation performance [15], and the establishment and analysis of the dynamics model [16], while studies on the structure of microbial populations in the reactor and their action mechanisms have rarely been reported. Most of the earlier bio-trickling filter (BTF) studies have focused on the original bacterium resources such as activated sludge and bacteria [17,18]. However, BTF studies on the use of immobilized fungi as the major original strain for toluene removal are scant [2]. Compared to biological technologies focused on bacteria, filamentous fungi have become a growing field of research on VOC biotechnologies. The mycelium produced by fungi can easily catch the hydrophobic pollutants in the gas phase. The fungi have the ability to tolerate low pH and adapt to a dry environment, and can degrade hydrophobic pollutants under a broad range of process conditions [19,20]. In recent studies, the main fungi for separating and degrading benzene and toluene have been Scedosporium apiospermum [20], Paecilomyces variotii [21], Aspergillus niger [22], Cladophialophora [23,24], Exophiala oligosperma [25], and Phanerochaete chrysosporium [26]. However, the evolution of fungal communities in a BTF has not been reported in these articles. Currently, more interest has been concentrated on the mysterious relationship of the biodiversity–ecosystem function [27]. The studies mainly include the change in the microbial flora inoculated with a single bacterium [27]; a comparative study to determine the relationship between the microbial community and the degradation performance of BTFs with fungi, bacteria and fungi-bacteria membrane [19]; the gene encoding of a toluene-degrading enzyme, the community structure of bacteria [28]; etc. Among them, although the RE and EC of toluene-degrading BTF immobilized with specific strains have been studied intensively, few studies have completely examined the toluene RE, process robustness, fungi diversity, and the abundance of functional strains in the upper, intermediate, and bottom layers of a BTF inoculated with a specific fungal strain. In this study, the performance of the fungi-based BTF on the degradation of toluene, and the succession process of the fungal colony under different operating conditions were evaluated. High-throughput sequencing technology with real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) were used to investigate the abundance of the functional strains and to trace Fusarium oxysporum. It was selected to form the biofilm in the BTF fungal community to describe the variations of the performance parameters in the different layers of the BTF. Particularly noteworthy was the exploration of the fungal mechanism for toluene purification.

2.2. Preliminary identification of fungi The selected fungi were preliminarily identified by an optical electron microscope (Olympus BX51) using the insert culture method; the living fungi were observed, and diverse morphological features of the strains were identified. Then, the fungal sample was taken for sequencing. 2.3. BTF setup A three-layer BTF was set up in laboratory scale as shown in Fig. 1. The BTF was made of polymethyl methacrylate and had a diameter of 120 mm; the thickness of the tower was 5 mm. Ceramic pellets with a diameter of 4–5 mm, stacking density of 0.35 g cm−3, and a specific surface area of 0.99 m2 g−1 was used as the packing material in the BTF. To observe the changes in the microbial diversity at different tower heights, the BTF was divided into three sections: top layer (T), intermediate layer (I), and under layer (U). The height of each of these layers was 260 mm, and the height of the packing material was T = I = U = 150 mm. Further, each section was equipped with a sampling port at the intermediate of the packing layer. Therefore, the volume of the total packing material was 4.27 × 10−3 m3. An air compressor was used for the negative pressure operation in the experiment. Further, toluene vapor was generated and it flowed through the BTF [19]. To provide a counter-current operation mode, purified gas was discharged through the top of the reactor, while the nutrient liquid was supplied from the top of the BTF. The flow rate of the recirculation liquid was controlled by the time relay and an electromagnetism valve. Moreover, a liquid whose composition was the same as that of culture A was sprayed regularly onto the packing by using a centrifugal water pump from a recirculation tank to supply nutrients and moisture to the microorganisms. 2.4. Inoculation and operation To ensure that the biofilm strains are in adequate contact with the biomass initially attached to the packing bed, an immobilization method with three steps was used to start the BTF [29]. First, the fungi strains liquid were poured into an aeration flask with ceramic pellets and the necessary nutrients for microorganisms. The aeration time was 2 h. Second, the ceramics attached with microorganisms were transferred from the aeration flask into another flask containing higher concentrations of nutrient elements. Lastly, the biological ceramic pellets were poured into the bio-trickling filter, with the recycle liquid

2. Materials and methods 2.1. Microorganism domestication and cultivation medium The origins of used strains were two types of activated sludge from a sewage treatment plant. Three different media were used in this experiment. The compositions of the nutrition solutions A and LA are shown in Table 1. The final pH was adjusted to 5.6 by using HCl (1M). 643

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Table 1 Compositions of inorganic nutrition. Code

Nutrition solution

Composition (g L−1)

A

Nitrogen source Phosphate buffer

2 0.47 0.45 0.50 0.01 0.001 0.001 0.001 0.001 0.05 5.0 15.0 5.0

Magnesium salt Calcium salt Microelement

LA

Bacterial inhibitor Carbon sources, phosphate and vitamins Nitrogen sources and vitamins Mineral salt

Application Ammonium tartrate Na2HPO4 KH2PO4 MgSO4·7H2O Anhydrous CaCl2 MnSO4·H2O FeSO4 ZnSO4·7H2O CuSO4·5H2O Chloramphenicol Beef extract Peptone NaCl

This medium was used in the selection and cultivation of toluene degrading fungi.

This medium was applied for the enrichment incubation of fungi.

The ceramsite was observed using scanning electron microscopy (SEM) (Hitachi S-4300, Japan). Microbial morphology was analyzed by using an electron microscope. The pressure drop of the BTF was measured daily by using a glass U-tube piezometer. The pH value of the recirculation solution was determined daily by using an acidometer (pHs-3C). 2.6. DNA isolation, PCR, and DGGE The ceramic pellet samples were collected from the BTF at days 10, 39, 70, and 100 of regular operation for the DGGE analysis. Moreover, the samples were gathered from the BTF sections T, I, and U on each of the above-mentioned days; then, the samples were named T10, I10, U10, T39, I39, U39, T70, I70, U70, T100, I100, and U100, respectively, with the number denoting the day on which the ceramic samples were collected. To explore the evolution of microbial communities in the biomass attached on the ceramsite, genetic sequencing analyses were performed. DGGE fingerprint experiments of the DNA extracted from the biofilm samples of the fungal BTF were analyzed, and then the gel consistent with the six leading bands in the DGGE fingerprint device was excised and sequenced. The total DNA of microbes on ceramsite was extracted using an MP kit (USA); then, the 18S rDNA genes in the DNA samples were amplified using two universal primers GC-FR1 containing a CG clamp (5′-CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGAICCATTCAATCGGTAIT-3′) and FF390 (5′-CGATAACGAACGAGACCT-3′). The volume of the amplification reaction mixture was 25 μL, which contained 2.5 μL of the 10× ExTaq buffer, 2 μL of dNTP (2.5 mM), 0.25 μL of ExTaq Polymerase (5 U μL−1), 1 μL of GC-FR1 (10 mM), 1 μL of FF390 (10 mM), 50 ng of template DNA, and ddH2O. The reaction conditions for 35 cycles were as follows: 94 °C for 5 min, 94 °C for 3 min, and renaturation 30 s at 50 °C, followed by a 1-min extension at 72 °C. Finally, we performed elongation for 10 min at 72 °C. For the DGGE analysis, 10 μL of the PCR product obtained from each sample was separated using a Gel-Doc2000 DGGE System (Bio-Rad, USA). The gel was run in a 1 × TAE buffer at 150 V and 60 °C for 7 h. The DGGE bands were cut to recycle the objective band by using a polygel DNA extraction kit (Omega, USA). And following that, the sequence determination was performed. As reported by Montebello et al. [30], the sequence was divided into appropriate taxonomic levels on the basis of the sequence identity percentage.

Fig. 1. Schematic diagram of the bio-trickling filter (1.thermostatic water bath; 2.toulene solution; 3.air mixed bottle; 4.rotameter; 5.sampling port; 6.biotrickling filter; 7.recirculation tank; 8.Immersible pump; 9.solution upflow tube; 10.overflow tube; 11.collecting tank; 12.downflow tube; 13.electromagnetism valve; 14.air condensate bottle; 15.air compressor). Table 2 Experimental conditions for the bio-trickling filter. Stage

Time(day)

Toluene concentrations (mg m−3)

Empty bed resident time (s)

Inlet loading rate (g m−3 h−1)

Start up Stable Starvation Re-start Stable

1–7 8–18 19–27 28–34 35–62 63–93 94–117

80–750 750–1000 – 150–300 100–1500 100–1000 100–700

96 77 – 77 55 32 43

3–35 30–50 5–15 10–100 10–105 8–55

(containing lower concentration nutrients) intermittent trickling for 2 h. Table 2 summarizes the range of the selected operating parameters for five different stages. 2.5. Analytical methods The toluene concentration was measured using an Agilent 6890 A gas chromatograph (GC) equipped with a flame ionization detector (FID), and an Agilent 19091J-413 capillary column (30 m × 320 μm × 0.25 μm). The conditions for the detection were as follows: column temperature of 60℃, detector temperature of 300℃, injection port temperature of 100℃, and N2 as the carrier gas.

3. Results and discussion 3.1. Evaluation of isolated strains The morphology of the isolated strain after the separation was as 644

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Fig. 2. Morphology of the isolated strain under an electron microscope ((a) spores, (b) hyphae). Table 3 Sequence alignment results of PCR products. Identifer

Most similar strain

Logon number

Similarity degree/%

Original fungus

Fusarium oxysporum

JF807402

99

shown in Fig. 2. The results showed that the hyphae and the spores of the screened strains could be clearly observed under the electron microscope, which could be initially considered a fungus. To further confirm the category of the selected strains, the strain fluid was analyzed by PCR sequencing. The genomic DNA of the extraction liquid was amplified by PCR, and the sequence alignment result was Fusarium oxysporum (Table 3). Note that the fungi were dominant in the BTF with some other strains also present. 3.2. Performance analysis 3.2.1. Start-up stage At the start-up stage of the BTF, toluene domestication was adopted from a low concentration to a high concentration. The air input at this stage was 0.16 m3 h−1, and the inlet concentration of toluene was between 100 mg m-3 and 1000 mg m-3. Empty bed resident time (EBRT) was 96 s, and the room temperature was 19℃–31℃. On days 1–2, the RE of the reactor was higher than that on the third day; the efficiency then declined substantially, which was attributed to the toluene adsorption ability of the packing material in the tower [31]. With an increase in the inlet toluene concentration and the operation days, the fungi in the BTF began to gradually play a leading role, and the purification efficiency of the vertical fungal BTF gradually increased and tended to be stable. The reactor’s RE increased from 42.23% on the first day to 90.07% on the seventh day; on days 8–10, EBRT decreased at 77 s, the inlet concentration was maintained at approximately 700 mg m-3, and the RE was approximately 90%. On days 11–18, the inlet concentration swung back and forth at 750–1000 mg m-3 with the EBRT continuously maintained at 77 s, while the purification efficiency remained consistent at approximately 90%. Even when the inlet air concentration fluctuated continuously, the BTF maintained a high treatment effect, which indicated that the trickling filter had strong robustness. Certainly, the temperature, which increased from 22℃ to 31℃, had a good effect during this period (Fig. 3). This indicated that the BTF immobilized with fungi could achieve success in 7 days, which was shorter than the period reported in many studies on immobilization by active sludge [18].

Fig. 3. Evolution of the RE (green triangle), inlet concentration (red diamond), outlet concentration (blue circular) and temperature (hollow circular) in the bio-trickling filter (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

period of time [2]. In this case, the microbial community of the tower varies in terms of the degree of starvation and the growth of the fungal community is inhibited, even in a dormant state, which causes a recovery phase after the restart operation. The recovery time of the BTF directly reflects its performance. Most of the studies have analyzed the re-acclimation performance after the starvation following a steady stage [1], while because of the long-term stable operation of the BTF, its stability improved, and it easily recovered after a short period of stagnation. Therefore, it is more meaningful to study the recovery performance of the BTF after the start-up stage. In this experiment, the recovery performance of the BTF was studied after a 9-day holding period; during this period, neither the nutrient solution nor the target pollutant was provided to the microorganisms. During the restart period, the air intake was 0.20 m3 h−1 (EBRT was 77 s), and the inlet toluene concentration was approximately 150–300 mg m−3. After starvation, the RE of toluene on the first day was more than 80%; then, it fluctuated to more than 95% and ultimately, achieved stability. Results revealed that the recovery performance of the BTF after the start-up stage was steady, and the restart time was less than 7 days. This could be attributed to the higher appetency for hydrophobic VOCs, the stronger resistance to dry fungi, and the existence of aerial mycelia, which could promote the adsorption and capture of organics in the fungal biomass [19].

3.2.2. BTF response to VOC starvation In an actual industrial production process, because the process conditions fluctuate, the form of the emission of the exhaust gas is usually intermittent and the BTF is in a state of stagnation for a certain 645

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3.3. Elimination capacity To find the optimum technological conditions for the treatment of toluene with BTF, the relationships between ILR and EC at different EBRTs are displayed in Fig. 4. Obviously, the maximum toluene EC of 98.09 g m−3 h-1 was achieved with the maximum ILR of 100.32 g m−3 h-1 at the EBRT of 55 s, which was higher than that of the bacterialfungal system [25]. Moreover, the BTF could treat a wide concentration range of toluene vapor with a high RE at the EBRT of 55 s and the reactor exhibited a very stable RE on days 35–65, while RE of more than 90% could be retained in the case of diverse fungi, even at toluene ILR of around 100 g m−3 h-1. In particular, when ILR between 40 and 50 g m−3 h-1 of EBRT was 43 s, the EC spots in the red block diagram significantly deviated from the 100% line and the room temperature was observed to be approximately 40℃ for several days, as shown in Fig. 3. As the optimal microbial life temperature was approximately 29℃, the continuous high temperature affected the normal life activities of microorganisms, which led to the low RE and EC. This indicated that the high temperature had a certain inhibitory effect on the living state of the microorganisms, which affected the ability of the microbial degradation of toluene. Note that the pH of the nutrient solution fluctuated between 5.6 and 8.0, and the total air pressure drop of the reactor was maintained within 5–200 Pa.

Fig. 4. Effect of the inlet loading rate on elimination capacity at different empty bed resident times.

3.2.3. Potential of fungal BTF for toluene removal In many industrial production processes, although the inlet waste gas flow is continuous, the pollutant concentration is uncertain. In order to further study the effects of different inlet mass concentrations on the RE of toluene and test the potential of fungal BTF for the removal of toluene, a four-stage experiment was performed at an inlet toluene concentration of 100–900 mg m−3, EBRT of 32 s, and room temperature of 31℃–41℃. This showed that (Fig. 3) in the range of the toluene intake concentration required by microbes, when the concentration of toluene suddenly increased, the RE declined at first, but because of adequate nutrition, the biofilm on the packing surface grew rapidly and reproduced at the maximum removal capacity to remove more of the toluene. Thus, the tower recovered from a higher level of degradation in the short term. When the inlet toluene concentrations exceeded the range that the BTF could withstand, the RE decreased gradually when the inlet toluene concentration increased suddenly, and finally remained constant at a certain level. The elimination capacity (EC) was still relatively high (Fig. 4); therefore, the actual toluene elimination quantity was still maintained at a certain level. This subsequently reduced the inlet mass concentration and the BTF recovered to a higher efficiency within a short period of time. These phenomena illustrated that stable-stage BTF could well resist the impact load (from 10.5 g m−3 h-1 to 103.4 g m−3 h-1) and showed a considerable purification effect for a certain concentration range of toluene (from 93.3 mg m−3 to 920.6 mg m-3). The response to the fluctuation in the inlet concentrations depended on the complex microbial structure (Fig. 7) in the BTF [32].

3.4. Microbial community structure analysis 3.4.1. Analysis of species diversity of inoculation fungi To determine the species of the microorganism in the membrane fungi, the inoculum strains were analyzed using a high-throughput sequencing technique to known phyla and genera. Fig. 5 shows that Ascomycota was the predominant fungal phyla group in the membrane strains. Fusarium was the dominant strain and accounted for 86.21% of the entire fungus community. The second was Myrothecium, which accounted for 2.45%, and the occupancy of the other strains was extremely low. This showed that the majority of the strains at the genus level were Fusarium, which could effectively degrade toluene. A comparison of the virgin pellets before the inoculation (Fig. 6(a)) with the biological ceramsite pellets after the inoculation (Fig. 6(b)) revealed that the latter were covered with a mature biofilm. Filamentous structures clearly formed on the ceramsite, which indicated that the predominant microorganisms were mycelia fungi; these fungi grew well in the considered BTF [19]. 3.4.2. Analysis of DGGE fingerprint The Shannon–Wiener indices obtained showed that the diversity and the evenness of the microbial population in the tower changed throughout the experiment under the toluene atmosphere. The diversity index varied from 0.89 (biomass attached in the under layer) to a high

Fig. 5. Microbial diversity of membrane strains at the (a) phylum and (b) genus. 646

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Fig. 6. Microorganism scanning electron microscopy photographs of the ceramics ((a) before inoculation, (b) after inoculation).

The sum of the numbers of bands of samples T, I, and U on days 10, 39, 70, and 100 was 12, 20, 22, and 28, respectively. These results indicated that the species diversity in the reactor gradually became abundant by the operation time. This phenomenon is related to the unpasteurized nutrient solution in the reservoir and the packing material in the reactor. Further, in the environment where toluene was the sole carbon source, the other strains could survive and grow [27]. The largest number of general fungal populations in the BTF (bands 23, 24, and 28, Fig. 7) occurred towards the end of the experiment, illustrating that the origin of these strains was in the nutrient solution or in the packing material. To understand the mechanism of the microbial degradation of

value of 2.25 corresponding to the biomass attached onto the intermediate layer. This indicated significant differences in the microbial diversity between the different layers during the different running times. Fig. 7 shows the DGGE image of the BTF samples on days 10, 39, 70, and 100 of the experimental period from the top, intermediate, and under layer, respectively. The bands numbers changed from T10 to T100, I10 to I100, and U10 to U100, showing that with an increase in the number of days, the number of bands in each of the layers of the reactor increased gradually. Note that the number of bands of T70 was 4, which was less than that of T39; this might be attributed to the extraction, as their abundance was lower than the detection limit [27].

Fig. 7. Denaturing gradient gel electrophoresis images of the bio-trickling filter samples on days 10, 39, 70, and 100 of the experimental period. 647

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Table 4 Sequences alignment analysis results. Band no.

Closest relative

Accession no.

Similarity (%)

Most similar strain

Band7 Band9 Band12 Band23 Band24 Band28

Paramicrosporidium saccamoebae uncultured cercozoan uncultured cercozoan uncultured fungus Cystofilobasidium lari-marini uncultured fungus

JQ796369 AY605185 AY605185 HM486985 LECSSRD5 JX268080

93 98 98 98 90 92

Fungi Rhizaria Rhizaria Fungi Fungi Fungi

operating conditions in a BTF. In contrast, bands 23, 24, and 28 were not found on day 10 but appeared in most of the other samples collected during different operation periods. These phenomena may be attributed to the changed concentration of toluene, gas flow rate, and environmental temperature, resulting in the stimulation of heterotrophic microbial communities and the accumulation of metabolites during the operation process. Moreover, it was interesting that many bands were not observed during one operation period but reappeared during another period. This suggested that the microbes were very sensitive to specific conditions.

toluene, the change in the microbial community in space was analyzed, including the differences in the number of bands between the upper, intermediate, and the lower layers at the same sampling time. As can be seen from the DGGE fingerprint, the number of bands and the Shannon–Wiener index in the intermediate layer was the highest at any sampling time compared with that in the upper and lower layers, while the difference between the upper and the lower layers was not obvious. This indicates that the species diversity and the number of microbes in the intermediate layer were the richest, which can be correlated to the adequate nutrition for microbial growth in this layer. The reactor was divided into three sections, with toluene as the sole carbon source traveling through the reactor from the bottom to the top layer, and the carbon content decreased gradually in steps. The nutrient solution was sprayed from the top to the bottom, and the upper layer obtained the most nutrients, followed by the intermediate layer, and then the lower layer. As a result, although the nutrient solution was sufficient in the top layer, the carbon source was insufficient, which was exactly opposite to that in the under layer. Therefore, factors that were detrimental to the growth of a large number of microbes in these two layers existed. As the carbon source and the nutrient content were moderate, there were rich species and a large number of microbes in the intermediate layer. This suggested that in the counter-current flow-type vertical BTF, both the nutrient solution and the carbon source were important for the growth of the microorganisms. Furthermore, the number of bands in T100, I100, and U100 was 7, 11, and 10, respectively, implying that the species diversity of the intermediate and the lower layers was more plentiful than that of the top layer at the end of the stable operation. It further illustrated that the microbial growth was related to the carbon source, which was abundant at the lower end of the reactor in the counter-current flow-type tower. The Shannon–Wiener diversity indices are marked in the bottom part of the gel.

4. Conclusions In general, the fungal BTF could quickly start within 7 days, tolerate highly transient shock loadings, and had high elimination capacity and RE along with high robustness. Moreover, during a long experimental period of 117 days, no clogging phenomenon was observed in the fungal BTF. The results of PCR-DGGE revealed that the species richness of the tower gradually increased with an increase in the runtime, and the largest species richness was observed in the intermediate layer. The dominant strains in the tower changed alternately with the operating conditions, and the non-dominant strains were various, which enhanced the tower’s robustness. Acknowledgements This work was supported by the Science and Technology Project of Beijing Municipal Education Commission (KM201810005034). References [1] P. Balasubramanian, L. Philip, S. Murty Bhallamudi, Biotrickling filtration of complex pharmaceutical VOC emissions along with chloroform, Bioresour. Technol. 114 (2012) 149–159. [2] E.R. Rene, B.T. Mohammad, M.C. Veiga, C. Kennes, Biodegradation of BTEX in a fungal biofilter: influence of operational parameters, effect of shock-loads and substrate stratification, Bioresour. Technol. 116 (2012) 204–213. [3] J. Repeckiene, J. Svediene, A. Paskevicius, R. Tekoriene, V. Raudoniene, E. Gudeliūnaite, P. Baltrenas, A. Misevicius, Succession of microorganisms in a plate-type air treatment biofilter during filtration of various volatile compounds, Environ. Technol. 36 (2015) 881. [4] Y. Liao, L. Jia, R. Chen, O. Gu, M. Sakurai, H. Kameyama, L. Zhou, H. Ma, Y. Guo, Charcoal-supported catalyst with enhanced thermal-stability for the catalytic combustion of volatile organic compounds, Appl. Catal. A-Gen. 522 (2016) 32–39. [5] R. Dziembaj, M. Molenda, M.M. Zaitz, L. Chmielarz, K. Furczon, Correlation of electrical properties of nanometric copper-doped ceria materials (Ce1−xCuxO2−δ) with their catalytic activity in incineration of VOCs, Solid State Ion. 251 (2013) 18–22. [6] V. Hequet, C. Raillard, O. Debono, F. Thevenet, N. Locoge, L.L. Coq, Photocatalytic oxidation of VOCs at ppb level using a closed-loop reactor: the mixture effect, Appl. Catal. B- Environ. 226 (2018) 473–486. [7] W. Wang, X. Ma, S. Grimes, H. Cai, M. Zhang, Study on the absorbability, regeneration characteristics and thermal stability of ionic liquids for VOCs removal, Chem. Eng. J. 328 (2017) 353–359. [8] A.H. Mamaghani, F. Haghighat, C. Lee, Gas phase adsorption of volatile organic compounds onto titanium dioxide photocatalysts, Chem. Eng. J. 337 (2018) 60–73. [9] B. Belaissaoui, Y.L. Moullec, E. Favre, Energy efficiency of a hybrid membrane/ condensation process for VOC (Volatile Organic Compounds) recovery from air: a generic approach, Energy 95 (2016) 291–302. [10] N. Xue, Q. Wang, J. Wang, J. Wang, X. Sun, Odorous composting gas abatement and microbial community diversity in a biotrickling filter, Int. Biodeter. Biodegr. 82 (2013) 73–80.

3.4.3. Analysis of microflora evolution The sequence of six dominant bands in DGGE gel rendered two different phyla, as shown in Table 4. Bands 7, 23, 24, and 28 belonged to fungi; the functional characteristics of bands 23 and 28 could not be inferred because neither of them matched with the known organisms listed in the NCBI BLAST nucleotide sequence database. Further, bands 7 and 24 were respectively Paramicrosporidium saccamoebae and Cystofilobasidium lari-marini. Bands 9 and 12 were Rhizaria, which were identified as close relatives (98%) of uncultured cercozoan. Obviously, the dominant strains were no longer Fusarium oxysporum after ten days of tower operation; they were replaced by others, demonstrating that during the initial stage of the tower operation, the species experienced ‘the survival of the fittest’ and eventually reached equilibrium. Although Fusarium oxysporum dominated the degradation of toluene in the shake flasks, they were not dominant in the tower. Band 7, which widely exists in the BTF, was the dominant strain in the fungal communities throughout the entire operation time. This suggested that the microbial community changed with operating conditions and was essential in maintaining high VOC REs and steadiness of the BTF throughout the runtime [33]. Band 9 was observed on day 10; however, it disappeared during the anaphase operation period, which indicated that cercozoan was not suitable for toluene degradation under different 648

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