Evaluation of Parallel-Series Configurations of Two-Phase

sustainability

Article Evaluation of Parallel-Series Conﬁgurations of Two-Phase Partitioning Biotrickling Filtration and Biotrickling Filtration for Treating Styrene Gas-Phase Emissions

Pau San-Valero , Javier Álvarez-Hornos , Pablo Ferrero , Josep M. Penya-Roja, Paula Marzal and Carmen Gabaldón *

Research Group GI2AM, Department of Chemical Engineering, Universitat de València, Av. de la Universitat s/n, 46100 Burjassot, Spain; [email protected] (P.S.-V.); [email protected] (J.Á.-H.); [email protected] (P.F.); [email protected] (J.M.P.-R.); [email protected] (P.M.) * Correspondence: [email protected]

Received: 22 July 2020; Accepted: 18 August 2020; Published: 20 August 2020

Abstract: The removal of styrene from industrial representative gaseous emissions was studied using two reactors connected in series: a two-phase partitioning biotrickling filter (TPPB-BTF) and a conventional biotrickling filter (BTF). The system was operated under industrial conditions, which included steady and transient conditions and intermittent spraying. Silicone oil was used 1 in the TPPB-BTF with a quantity as low as 25 mL L− , promoting a faster start-up compared to the 3 1 BTF. By working at a styrene loading of 30 g m− h− , nearly complete removal efficiency (RE) was obtained. In addition, the removal was not adversely impacted by using non-steady emission patterns such as overnight shutdowns (97% RE) and oscillating concentrations (95% RE), demonstrating its viability for industrial applications. After 2 months from inoculation, two additional configurations (reverse series BTF + TPPB-BTF and parallel) were tested, showing the series configuration as the best approach to consistently achieve RE > 95%. After 51 days of operation, high throughput sequencing revealed a sharp decrease in the bacterial diversity. In both reactors, the microorganisms belonging to the Comamonadaceae family were predominant and other styrene degraders such as Pseudomonadaceae proliferated preferably in the first reactor.

Keywords: biological air treatment; biotrickling ﬁltration; silicone oil; styrene; two-phase partitioning bioreactor

1. Introduction The treatment of derivative air emissions of styrene became a priority given its detrimental effects on human health and the environment and its massive use, especially in the industries of coating (styrene resins) and of polystyrene polymers [1]. Apart from its known potential for photochemical ozone creation [2], the styrene classification was recently upgraded to Group 2A, “probably carcinogenic to humans” by the International Agency for Research on Cancer [1]. Therefore, there is a need to develop cost-effective technologies for the control of styrene emissions. The diluted gas-phase emissions of styrene encountered in such industrial activities [3] make the biotechnologies a suitable alternative. Moreover, bioprocesses have some advantages, such as lower energy impact and lower operational and energetic cost when compared to physical-chemical technologies. Among biotechnologies, biotrickling filtration (BTF) stands out—its total direct cost excluding capital recovery could be up to 3 times lower than a regenerative catalytic oxidizer when treating emissions from a fiber reinforce plastic industry [4].

Sustainability 2020, 12, 6740; doi:10.3390/su12176740 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 6740 2 of 14

For the three kinds of biotechnologies (biofilters, biotrickling filters and bioscrubbers), the mass transfer step has been identified as the controlling step of the process rather than the biodegradation, especially for poor solubility compounds such styrene. Therefore, different strategies have been developed to enhance the mass transfer [5–7]. In this regard, the addition of a non-aqueous phase (NAP) with affinity for styrene seems to be a promising way to improve the process efficiency [5,8]. In addition, other authors have also proven the benefits of introducing an NAP in the removal of o-xylene by BTF [9]. Silicone oil is an inert and non-toxic substance with a low price that appears between the most suitable NAPs due to the high solubility of styrene in it, which is 236 times higher than in water [10]. Previous studies showed the potential of two-phase partitioning bioreactors operated as BTF (TPPB-BTF) for treating styrene air emissions [11,12]. However, the enhanced performance of the TPPB-BTF is most notable at extreme conditions (high loads or low empty bed residence times), while at moderate conditions both configurations exhibited similar performances [12]. Furthermore, the additional cost of the NAP, the clogging problems derived from its use and the extra waste management associated with oil emulsions can compromise its benefits, making BTF more cost-effective than TPPB-BTF, especially at low styrene concentrations. Therefore, the use of both bioreactors in series (TPPB-BTF followed by BTF) could encourage the industrial application of the technology. This combination would take advantage of the TPPB-BTF, while the NAP quantity could be reduced by subsequently finalizing the treatment in a BTF. In addition, laboratory research should consider the influence of the characteristics of industrial gas-phase emissions on the process performance. Typically, the industrial gas-phase emissions of styrene randomly fluctuate with the 3 status of the manufacturing process in the range of 50–350 mg m− [4]. Moreover, industrial BTFs should be intermittently irrigated in order to reduce the energetic costs [13,14]. Another aspect of interest lies in the microbiology structure whose analysis would help to gain a better understanding of the process. Among the inoculation alternatives, TPBBs have been seeded with NAPs enriched with microorganisms previously adapted to the pollutant of interest [12]. However, the industrial BTFs are usually seeded with wastewater sludge without previous acclimation as a cost-effective strategy [4,15]. Therefore, the evaluation of TPPF-BTFs seeded with mixtures of industrial silicone-oil, water and wastewater sludge without prior acclimatization to styrene would be a further step for scaling up this technology. Recent high-throughput sequencing analysis identified Azoarcus and Pseudomonas as the dominant genera in BTFs treating styrene, showing that styrene loads modulated the community composition [16]. However, microbiological studies on the influence of the NAP are still scarce. This work aims to investigate the styrene removal efficiency of a TPPB-BTF plus a BTF operated in series under typical industrial conditions, which included steady and transient conditions, intermittent spraying and inoculation with activated sludge without previous acclimatization. Silicone oil was selected as the NAP for the TPPB-BTF and its influence on the microbial community was analyzed by high throughput sequencing after two months of operation of the TPPB-BTF + BTF configuration. In a second stage, the influence of the NAP on the long-term performance was assessed by comparing the TPPB-BTF + BTF scheme with the reverse one (BTF + TPPB-BTF) and with both bioreactors (TPPB-BTF and BTF) operating in parallel.

2. Materials and Methods

2.1. Materials All chemicals were purchased with analytical grade from VWR lnternational Eurolab. 1 Industrial-grade silicone oil with a kinematic viscosity of 50 cSt and a density of 0.96 g mL− at 25 ◦C was used (XIAMETER PMX-200; Univar, Spain). The stock of the mineral salt medium contained 1 1 (g L− ): 19.7 urea, 3.8 (NH4)2HPO4, 1.6 NaHCO3, 1.5 Na2SO4, 0.7 KCl, 4 yeast extract and 25 mL L− of a micronutrient solution containing (g L 1): 3.71 CaCl , 3.71 MgCl , 1.4 Fe (SO ) , 0.07 ZnSO 7H O, − 2 2 2 4 3 4· 2 Sustainability 2020, 12, 6740 3 of 14

Sustainability 2020, 12, x FOR PEER REVIEW 3 of 14 0.028 MnCl 4H O, 0.7 NiSO 6H O and 0.07 H BO . Activated sludge was collected from a municipal 2· 2 4· 2 3 3 2.2. Experimentalwastewater treatment set-up plant locally available.

2.2.Two Experimental bioreactors set-up of identical size were assembled in series, connecting the outlet gas from the first reactorTwo bioreactorsto the inlet of identicalgas of sizethe weresecond assembled reactor. in series,The first connecting reactor the operated outlet gas fromas athe two-phase first partitioningreactor to bioreactor the inlet gas (TPPB-BTF), of the second while reactor. the Thesecond first reactor reactor operatedoperated as as a a two-phase conventional partitioning biotrickling filterbioreactor (BTF) (Figure (TPPB-BTF), 1) (unless while theotherwise second reactor noted). operated The bioreactors as a conventional were biotricklingcylindrical filter methacrylate (BTF) columns(Figure (inner1) (unless diameter otherwise of 14.4 noted). cm) filled The bioreactorswith 20 L of were polypropylene cylindrical methacrylate rings (Flexiring columns®, Koch-Glitsch (inner B.V.B.A.,diameter Belgium) of 14.4 cm) of 25 filled mm with nominal 20 L of polypropylenediameter and rings a surface (Flexiring area® ,of Koch-Glitsch 207 m2 m− B.V.B.A.,3. The polypropylene Belgium) 2 3 ringsof had 25 mm a void nominal fraction diameter and andbulk a density surface areaof 92% of 207 and m 71m −kg. Them−3, polypropylenerespectively. ringsEach hadreactor a void had its 3 ownfraction trickling and tank bulk and density in order of 92% to and mimic 71 kg industrial m− , respectively. designs, Each the reactortanks’ volumes had its own were trickling set up tank to 10% v/v andrelative in order to the to mimicpacking industrial volume. designs, The air the stream tanks’, volumeswhose flow-rate were set up was to 10%adjusted v/v relative by a mass to the flow packing volume. The air stream, whose flow-rate was adjusted by a mass flow controller (Bronkhorst controller (Bronkhorst Hi-Tec, The Netherlands), was polluted with styrene by a syringe pump (New Hi-Tec, The Netherlands), was polluted with styrene by a syringe pump (New Era, NE 1000 model, Era, NE 1000 model, USA). The polluted air stream was introduced through the bottom of the column USA). The polluted air stream was introduced through the bottom of the column while the liquid whilestream the liquid flushed stream in counter flushed current in counter mode in current a closed mode loop configuration. in a closed loop The liquidconfiguration. velocity was The set liquid velocity was 1set to 3 m h−1 in both reactors. The pH of the TPPB-BTF was kept above 7.0 with a pH to 3 m h− in both reactors. The pH of the TPPB-BTF was kept above 7.0 with a pH control system controlconsisting system of consisting a pH probe, of a digitala pH probe, control a unit digital and acontrol LabQuest unit® data and acquisition a LabQuest card® data (Vernier, acquisition Oregon, card (Vernier,USA). Oregon, The pH controller USA). The was pH connected controller to a peristalticwas connected pump (Watson to a peristaltic Marlow, USA) pump which (Watson dosed Marlow, 1 M USA)NaOH which stock dosed solution. 1 M AsNaOH a cost-wise stock criterion,solution. the As percentage a cost-wise of siliconecriterion, oil wasthe percentage set to 2.5% v /ofv relative silicone oil was toset the to volume 2.5% v/v of therelative packing. to the Therefore, volume the of TPPB-BTF the packing. was seededTherefore, with athe mixture TPPB-BTF of silicone was oil seeded (0.5 L) with a mixtureand activated of silicone sludge oil (0.5 (0.5 L), L) while and theactivated BTF was slud seededge (0.5 with L), 0.5 while L of activated the BTF sludge. was seeded Both tanks with were 0.5 L of activatedcompleted sludge. up toBoth 2 L withtanks water were and completed mineral salt up medium.to 2 L with water and mineral salt medium.

FigureFigure 1. Schematic 1. Schematic of the of the experimental experimental set-up. set-up. Continuo Continuousus and and dashed dashed lines lines represent represent liquid liquid and gasand gas streams, respectively. streams, respectively.

2.3. Experimental Plan

2.3.1. TPPB-BTF + BTF Series Configuration: Influence of Spraying Frequency and Transient Loading of Styrene TPPB-BTF and BTF were operated in series simulating intermittent spraying of industrial BTFs. Experimental conditions are summarized in Table 1. The influence of the spraying frequency on the removal efficiency (RE) and elimination capacity (EC) of the system was evaluated at a constant and continuous styrene loading (phase A, days 0–44). Inlet concentration was fixed at 330 mg m−3 as a representative of styrene gas-phase emissions. The empty bed residence time (EBRT) was fixed at 20 s for each reactor, resulting in an EBRT of 40 s for the total system (TPPB-BTF + BTF). Therefore, the inlet load (IL) was of 60 g m−3 h−1 referring to the first reactor (TPPB-BTF) or 30 g m−3 h−1 referring to Sustainability 2020, 12, 6740 4 of 14

2.3. Experimental Plan

2.3.1. TPPB-BTF + BTF Series Configuration: Influence of Spraying Frequency and Transient Loading of Styrene TPPB-BTF and BTF were operated in series simulating intermittent spraying of industrial BTFs. Experimental conditions are summarized in Table1. The influence of the spraying frequency on the removal efficiency (RE) and elimination capacity (EC) of the system was evaluated at a constant and 3 continuous styrene loading (phase A, days 0–44). Inlet concentration was fixed at 330 mg m− as a representative of styrene gas-phase emissions. The empty bed residence time (EBRT) was fixed at 20 s for each reactor, resulting in an EBRT of 40 s for the total system (TPPB-BTF + BTF). Therefore, the inlet 3 1 3 1 load (IL) was of 60 g m− h− referring to the first reactor (TPPB-BTF) or 30 g m− h− referring to the whole system. The bioreactors were operated without water purge during the first two weeks to avoid losses of silicone oil and biomass. Afterwards, each water tank was daily purged at a rate of 5% v/v, replacing the purged volume with water and without silicone oil. Nutrient addition was fixed to 40 mL 1 1 d− for the first reactor (TPPB-BTF) and 15 mL d− for the second reactor (BTF).

Table 1. Summary of the spraying frequencies and the styrene loading modes tested.

Phase Days Spraying Styrene Loading A-I (Start-up) 0–3 Continuous A-II 4–9 15 min on/45 min off Continuous A-III 10–14 15 min on/105 min off Inlet conc. = 330 mg m 3 A-IV 15–21 15 min on/45 min off − A-V 21–44 3 min on/9 min off 1 Discontinuous: 16 h d− B-I 45–51 3 min on/9 min off 3 Inlet conc. = 330 mg m− Discontinuous: 16 h d 1 B-II 52–62 3 min on/9 min off − Oscillating inlet conc. 1 Discontinuous: 16 h d− B-III 63–65 3 min on/9 min off 3 Inlet conc. = 330 mg m−

From phase B in Table1, the best spraying frequency (3 min spraying on /9 min spraying off) was 1 selected and styrene feeding was reduced to 16 h d− to simulate typical night closures at industry (phase B-I). Fresh air without styrene was supplied at the same flow rate during the other 8 h of the day. In phase B-II, the effect of discontinuous and oscillating inlet concentrations of styrene ranging 3 3 1 from 200 to 600 mg m− was tested. The EBRT and the average IL were kept constant at 60 g m− h− and 20 s (data referred to the first reactor). From day 63 onwards, the experimental conditions of phase B-I were restored to evaluate the impact that the oscillating concentrations had on the resilience of the system.

2.3.2. Alternative Parallel-Series Conﬁgurations The order of the reactors was reversed on day 72, and thus the polluted air stream was introduced to the conventional BTF and its outlet air stream was subsequently fed to the TPPB-BTF. 1 Operational conditions of phase B-III were used (discontinuous styrene feeding during 16 h d− , 3 inlet concentration of 300 mg m− ). Moreover, the same oscillating styrene pattern as that of phase B-II was applied for several days to evaluate the individual response of both reactors in the reverse conﬁguration. To compare the TPPB-BTF and BTF performances after a long term of operation, the experiment 3 was ended by setting up both reactors in parallel. The air stream polluted with 330 mg m− of 3 styrene was split into the two reactors. The IL of the whole system was kept constant at 30 g m− Sustainability 2020, 12, 6740 5 of 14

1 1 h− , while the single EBRT to each reactor was doubled (40 s). Styrene feeding was set to 16 h d− . Fluctuating transient conditions (those of phase B-II) were also tested in this conﬁguration.

2.4. Microbial Community Analysis Biomass samples from both bioreactors were taken at day 51 from the bottom and top ports which were located at 20 and 105 cm from the inlet gas. The inoculum sample was also analyzed. DNA was extracted using the PowerSoil DNA isolation kit (Mobio Laboratories, Qiagen, USA). DNA extraction was checked by electrophoresis in an agarose gel. The V4 hyper variable region of the 16S rRNA gene was amplified using the primers 515F (50-GTG CCA GCM GCC GCG GTA A-30) and 806R (50-GGA CTA CHV GGG TWT CTA AT-30). Afterwards, the amplicons were sequenced using the MiSeq system (Illumina, San Diego, USA). The sequences were screened and trimmed by using the Quantitative Insights Into Microbial Ecology (QIIME) software with a sequence length of 200 nt and a mean quality score cut-off of 25 nt. Alpha diversity (within the sample) was assessed by the Shannon diversity index and richness estimator (Chao1), which were calculated by normalizing the lowest number of the sequences for all five samples. Beta diversity (similarity between samples) was assessed using principal coordinates analysis (PCoA) based on weighted UniFrac distances.

2.5. Analytical methods The styrene concentration was monitored by a total hydrocarbon analyzer (Nira Mercury 901, + 3- Spirax Sarco, Spain). Conductivity, pH (WTW, pH/Cond 340i, Germany) and NH4 and PO4 concentrations (Merck MQuant® test strips: 110024 and 110428, respectively) were checked daily. Pressure drop was checked periodically (MP101 model, KIMO, Spain).

3. Results and Discussion

3.1. TPPB-BTF + BTF Series Configuration: Influence of Spraying Frequency The time course of the inlet and outlet styrene concentration of both reactors (TPPB-BTF and BTF) is shown in Figure2a. After inoculation, both reactors were irrigated for 24 h per day for promoting the attachment of the biomass to the media and the silicone oil in the TPPB-BTF (phase A-I). The use of silicone oil enhanced the start-up of styrene biotrickling filtration. As it can be observed, TPPB-BTF had a faster start-up than BTF. At day 3, RE of the TPPB-BTF (reactor 1) was 26.3% (outlet concentration = 3 3 267 mg m− ) while the BTF (reactor 2) reached only 8.6% (outlet concentration = 244 mg m− ). This was accompanied by a faster development of biomass (visual inspection) in the TPPB-BTF than in the BTF. From day 4 onwards, the system was intermittently irrigated for 15 min every hour (15 min spraying on/45 min spraying off, phase A-II). Under these conditions, the decrease in the outlet concentration of both reactors continued following the same tendency as during the start-up: higher improvement in RE for the TPPB-BTF than for the BTF. After ~one week of operation under this trickling frequency (day 9), the TPPB-BTF and the BTF achieved styrene outlet concentrations of 3 133 and 57 mg m− respectively, thus corresponding to 60% RE for the TPPB-BTF (TPPB-BTF EC = 35 g 3 1 3 1 m− h− ) and a RE of 83% for the whole system (total EC = 24 g m− h− ). The approach for using both bioreactors connected in series (TPPB-BTF plus conventional BTF) allowed a faster start-up of the process with significant savings in the use of silicon oil. The reduction in the spraying frequency to 15 min every 2 h (phase A-III, days 10–14) caused a worsening of the RE of the TPPB-BTF by decreasing it to 37%, negatively affecting the global 3 3 1 performance of the system (total outlet concentration = 166 mg m− , total EC = 16 g m− h− ). The stronger impact on the TPPB-BTF when compared to the BTF was due to its higher nutrient demand, indicating that enlarging the non-frequency periods could limit the biodegradation rate in biotrickling filtration. In fact, when the spraying frequency was increased to 15 min every 1 h (phase A-IV, days 15–20) the RE of the TPPB-BTF improved to 62 3% (EC = 35 2 g m 3 h 1), ± ± − − while the combined RE of the TPPB-BTF + BTF was 81 3% (total EC = 23 1 g m 3 h 1). From day ± ± − − Sustainability 2020, 12, x FOR PEER REVIEW 5 of 14 lowest number of the sequences for all five samples. Beta diversity (similarity between samples) was assessed using principal coordinates analysis (PCoA) based on weighted UniFrac distances.

2.5. Analytical methods The styrene concentration was monitored by a total hydrocarbon analyzer (Nira Mercury 901, Spirax Sarco, Spain). Conductivity, pH (WTW, pH/Cond 340i, Germany) and NH4+ and PO43- concentrations (Merck MQuant® test strips: 110024 and 110428, respectively) were checked daily. Pressure drop was checked periodically (MP101 model, KIMO, Spain).

3. Results and Discussion

3.1. TPPB-BTF + BTF Series Configuration: Influence of Spraying Frequency The time course of the inlet and outlet styrene concentration of both reactors (TPPB-BTF and BTF) is shown in Figure 2a. After inoculation, both reactors were irrigated for 24 h per day for promoting the attachment of the biomass to the media and the silicone oil in the TPPB-BTF (phase A- I). The use of silicone oil enhanced the start-up of styrene biotrickling filtration. As it can be observed, TPPB-BTF had a faster start-up than BTF. At day 3, RE of the TPPB-BTF (reactor 1) was 26.3% (outlet −3 concentrationSustainability 2020 = 267, 12, 6740mg m ) while the BTF (reactor 2) reached only 8.6% (outlet concentration6 of= 14244 mg m−3). This was accompanied by a faster development of biomass (visual inspection) in the TPPB- BTF than in the BTF. 21From onwards day (phase4 onwards, A-V), the the system spraying was strategy intermittent was modifiedly irrigated by for irrigating 15 minutes each every reactor hour for 3(15 min min sprayingevery 12 on/45 min. Themin systemspraying was off, evaluated phase forA-II). long-term Under performancethese conditions, by keeping the decrease these conditions in the outlet for ~3 weeks. The RE of the TPPB-BTF remained at similar values, 63 8% (TPPB-BTF EC = 34 4 g concentration of both reactors continued following the same tendency± as during the start-up:± higher m 3 h 1), indicating that this reactor already achieved its maximum removal capacity working at an improvement− − in RE for the TPPB-BTF than for the BTF. After ~one week of operation under this IL of 60 g m 3 h 1 and 20 s of EBRT. Increasing the spraying frequency had a positive effect on the trickling frequency− − (day 9), the TPPB-BTF and the BTF achieved styrene outlet concentrations of 133 BTF, enhancing the RE of the TPPB-BTF + BTF series configuration up to 91 4% (total EC = 24 1 g −3 and 573 mg1 m respectively, thus corresponding to 60% RE for the TPPB-BTF± (TPPB-BTF EC± = 35 g m− h− ). This was accompanied by a higher development of biofilm in the BTF (visual observation), m−3 h−1) and a RE of 83% for the whole system (total EC = 24 g m−3 h−1). The approach for using both showing that a more frequent nutrient/water addition is advisable to promote biomass growth in bioreactorsthe gas-phase connected styrene-degrading in series (TPPB-BTF bioreactors, plus especially conventional at low BTF) inlet allowed concentrations. a faster Furthermore,start-up of the process with significant savings in the use of silicon oil. 1 low pressure drops were achieved in both bioreactors (8 Pa m− ).

Figure 2. Evolution of the styrene concentration in the series configuration two-phase partitioning Figurebiotrickling 2. Evolution filter (TPPB-BTF) of the styrene+ a conventionalconcentration biotrickling in the series filter configuration (BTF). (a) Influence two-phase of the partitioning spraying 1 biotricklingfrequency filter for continuous (TPPB-BTF) loading + a conventional experiments, biotrickling (b) discontinuous filter loading(BTF). ( experimenta) Influence (16 of hthe d− spraying). frequency for continuous loading experiments, (b) discontinuous loading experiment (16 h d−1). Although there are no literature data on a combined TPPB-BTF + BTF system for styrene removal, several studies can be found regarding TPPB-BTF and BTF alone. Previous authors pointed out that a BTF required a minimum EBRT of 30 s to achieve high and stable removal. For example, San-Valero et al. [12] reported a decrease in the RE from 92% to 66% of a BTF treating a styrene 3 concentration of 184 mg C m− when EBRT was decreased from 30 to 20 s (ILs varying from 22 to 33 g C 3 1 m− h− ). Similarly, Rene et al. [11] also observed a decrease in the RE for an EBRT of 20 s, although their 3 1 ILs were much higher than that of this study (211 g m− h− ). In this regard, the addition of a NAP such as silicone oil allowed the reduction in the EBRT without compromising the performance [11,12], although the main disadvantage derived from the greater amount of silicone oil required. Herein, we demonstrated that the silicone oil usage can be significantly reduced (up to three times) by dividing the required packing volume in two reactors connected in series and operating only the first reactor as a TPPB. By using this strategy, the complexity of the system increased but the silicone oil usage was reduced to the minimum value reported in the literature, facilitating the industrial application. Only a 3 1 single dose of silicone oil of 0.83 m per kg h− of styrene emitted is required to achieve REs >90%. Sustainability 2020, 12, 6740 7 of 14

3.2. TPPB-BTF + BTF Series Conﬁguration: Inﬂuence of Transient Styrene Loading

1 Styrene feeding was reduced to 16 h d− to evaluate the impact of discontinuous emissions typically found at industrial sites on the RE (phase B-I). The spraying pattern from which the highest RE was achieved (3 min spraying on/9 min spraying off) was used. The results are shown in Figure2b. Both reactors maintained the same performance as under continuous emissions (phase A-V, Figure2a). The RE of the TPPB-BTF was 63 6% (EC = 32 4 g m 3 h 1) with a combined RE of the series ± ± − − configuration of 90 3% (total EC = 22 1 g m 3 h 1). The slight difference in EC with respect to ± ± − − the previous phase is due to a ~10% decrease in the inlet styrene concentration associated to inherent variations of the experiment. Therefore, the robustness of this configuration for its application to factories with night closures was corroborated. Emissions from industrial facilities are usually characterized by fluctuating conditions of loading associated with variations on the manufacturing processes which could hinder the performance of the process. In this study, fluctuating and discontinuous styrene feeding was tested over ~2 weeks (days 52–66, phase B-II) by applying two identical 8h cycles with styrene inlet concentrations ranging 3 3 1 from 200 to 575 mg m− . The average IL during the feeding period was 60 g m− h− referred to the SustainabilityTPPB-BTF 2020, (first12, x FOR reactor) PEER and REVIEW the EBRT of each reactor was 20 s (total EBRT = 40 s). The transient7 of 14 response for the first 8 h after styrene feeding resumption from night shutdown for a representative reactorsday isfollowed shown in the Figure inlet3. Asconcentration it can be observed, pattern. the Despite outlet concentration the outlet forpeaks both occurring reactors followed when inlet styrenethe rose inlet up concentration to 575 mg m pattern.−3, the rest Despite of the the time, outlet low peaks styrene occurring concentrations when inlet were styrene emitted rose up from to the 3 BTF (reactor575 mg m 2).− The, the average rest of the outlet time, emission low styrene of th concentrationse system (TPPB-BTF were emitted + BTF) from during the BTF the (reactor 8 h cycle 2). was The average outlet emission of the system (TPPB-BTF + BTF) during the 8 h cycle was 33 mg m 3. 33 mg m−3. Although this value was slightly higher when compared to the previous stage (phase− B-I, Although this value was slightly higher when compared to the previous stage (phase B-I, 27 mg m 3), 27 mg m−3), it can be concluded that the two bioreactors operating in series is a robust configuration− it can be concluded that the two bioreactors operating in series is a robust configuration for the efficient for themanagement efficient management of industrial emissionof industrial fluctuations. emission In fluctuations. fact, although In the fact, performance although ofthe the performance TPPB-BTF of the TPPB-BTF was slightly lower (RE = 60%,3 EC~371 g m−3 h−1) than that of phase B-I, the global RE was slightly lower (RE = 60%, EC~37 g m− h− ) than that of phase B-I, the global RE increased to 95% increaseddue to to the 95% progressive due to the improvement progressive of improvement the BTF performance of the (reactorBTF performance 2). (reactor 2).

FigureFigure 3. Response 3. Response to inlet to inlet styrene styrene concentration concentration peaks ofof the the series series conﬁguration configuration TPPB-BTF TPPB-BTF+BTF+BTF under discontinuous loading (16 h d 1). A representative day of phase B-II is displayed. under discontinuous loading (16 h d−1−). A representative day of phase B-II is displayed. 3 1 On day 64, a constant inlet concentration of 330 mg m− for 16 h d− was restored for a week toOn evaluate day 64, the a constant resilience inlet of the concentration system after the of transitory330 mg m loading−3 for 16 conditions h d−1 was (phase restored B-III). for The a week RE to evaluateof the the TPPB-BTF resilience was of 66 the 2%system (EC =after38 the3 g tran m 3sitoryh 1), slightly loading higher conditions than the (phase RE obtained B-III). onThe the RE of ± ± − − the TPPB-BTF was 66 ± 2% (EC = 38 ± 3 g m−3 h−1), slightly higher than the RE obtained on the previous days to starting the oscillating emissions (days 45–51). Interestingly, the total system achieved nearly complete removal (97 ± 1%; total EC = 27 ± 2 g m−3 h−1). This fact was associated with the clear improvement of the performance of BTF (reactor 2) during the disturbance period, corroborating the slower progress of the BTF on developing enough degrading biofilm. This phenomenon was also observed in pilot BTFs, where improvements in removal overtime despite styrene load increase were achieved [4,15]. These results show that although the use of silicone oil has a clear impact on the performance of biotrickling filtration during the first weeks after inoculation, its long-term effect remains unclear.

3.3. Alternative Series and Parallel Configurations In order to elucidate the long-term influence of the use of silicone oil during the start-up of BTF treating styrene, two alternative configurations were evaluated after more than two months of operation of the TPPB-BTF + BTF series configuration. Each configuration was tested over a week by using the same styrene feeding than in phase B-III (inlet concentration = 330 mg m−3 during 16 h d−1). Firstly, a series configuration reversing the order of the two bioreactors was used, so that the EBRT was maintained to 20 s for each reactor. The BTF was moved to the first position while the second reactor was the TPPB-BTF (BTF + TPPB-BTF series configuration). The BTF reached 74 ± 5% RE (EC = 40 ± 2 g m−3 h−1) with a total RE of the two reactors in series of 98 ± 1% (total EC = 26 ±1 g m−3 h−1). By comparing the efficiency of the first reactor in the two series configuration, the original and the reverse ones, the BTF showed a slightly higher RE than the TPPB-BTF. Indeed, both configurations achieved a nearly complete removal of styrene, with outlet concentrations <6 mg m−3, demonstrating Sustainability 2020, 12, 6740 8 of 14 previous days to starting the oscillating emissions (days 45–51). Interestingly, the total system achieved nearly complete removal (97 1%; total EC = 27 2 g m 3 h 1). This fact was associated with the clear ± ± − − improvement of the performance of BTF (reactor 2) during the disturbance period, corroborating the slower progress of the BTF on developing enough degrading biofilm. This phenomenon was also observed in pilot BTFs, where improvements in removal overtime despite styrene load increase were achieved [4,15]. These results show that although the use of silicone oil has a clear impact on the performance of biotrickling filtration during the first weeks after inoculation, its long-term effect remains unclear.

3.3. Alternative Series and Parallel Configurations In order to elucidate the long-term influence of the use of silicone oil during the start-up of BTF treating styrene, two alternative configurations were evaluated after more than two months of operation of the TPPB-BTF + BTF series configuration. Each configuration was tested over a week 3 by using the same styrene feeding than in phase B-III (inlet concentration = 330 mg m− during 16 h 1 d− ). Firstly, a series configuration reversing the order of the two bioreactors was used, so that the EBRT was maintained to 20 s for each reactor. The BTF was moved to the first position while the second reactor was the TPPB-BTF (BTF + TPPB-BTF series configuration). The BTF reached 74 5% ± RE (EC = 40 2 g m 3 h 1) with a total RE of the two reactors in series of 98 1% (total EC = ± − − ± 26 1 g m 3 h 1). By comparing the efficiency of the first reactor in the two series configuration, ± − − the original and the reverse ones, the BTF showed a slightly higher RE than the TPPB-BTF. Indeed, both configurations achieved a nearly complete removal of styrene, with outlet concentrations <6 mg 3 m− , demonstrating that biotrickling filtration is a very competitive alternative to thermal oxidizers for the control of styrene air emissions. These results also reveal that after more than two months of start-up, there were no differences between styrene degradation regarding the presence or absence of a NAP. These results are of high interest for the industrial scalability of the process, as the use of silicone oil increases the operational cost while showing a positive impact on performance only in the short-term. In the next experiment, both bioreactors were set-up in parallel configuration by splitting the polluted air stream into two parts. Consequently, the EBRT increased by two-fold (40 s each one) 3 1 and the IL applied to each reactor was ~30 g m− h− . Similar REs were obtained for both reactors (88% 1% for the TPPB-BTF and 91% 1% for the BTF) with an average outlet styrene concentration ± ± of 30.0 8.1 mg m 3. The results corroborate that after more than 2 months after silicone oil addition, ± − its effect on the process performance could be considered negligible. Despite the greater operational cost due to greater pressure drop, the series configuration is the preferred option versus the parallel one when tight and very low emissions limits are required. Both configurations (reverse serial and parallel modes) were tested under transient styrene loading (Figure4). The reverse series configuration (BTF + TPPB-BTF, Figure4a) showed a similar outlet emission pattern to that obtained with the TPPB-BTF + BTF series configuration (Figure3). A slightly lower emission was achieved (average outlet emission during 8 h cycle: 18 mg m3) due to the progressive improvement of the BTF overtime. Therefore, the silicone oil does not provide an advantage for buffering peaks two months after its application. It could be hypothesized that the progressive growth and thickening of the biofilm results in outer layers with similar physical and chemical properties than those of a conventional biofilm. For the parallel configuration, both reactors showed 3 3 similar results (TPPB-BTF: 54 g m− , BTF: 40 mg m ), being worse than the series configurations. In any case, BTF performed better than the TPPB-BTF in any of the tested configurations to handle fluctuating emissions. These results indicate that the BTF can achieve similar or even better removals than the TPPB-BTF if enough time is given for the biofilm development. Furthermore, it can be concluded than the series configuration offers a clear safety margin for achieving very low emissions when compared to the parallel configuration. The removal obtained herein using the series configuration of two reactors Sustainability 2020, 12, x FOR PEER REVIEW 8 of 14 that biotrickling filtration is a very competitive alternative to thermal oxidizers for the control of styrene air emissions. These results also reveal that after more than two months of start-up, there were no differences between styrene degradation regarding the presence or absence of a NAP. These results are of high interest for the industrial scalability of the process, as the use of silicone oil increases the operational cost while showing a positive impact on performance only in the short- term. In the next experiment, both bioreactors were set-up in parallel configuration by splitting the polluted air stream into two parts. Consequently, the EBRT increased by two-fold (40 s each one) and the IL applied to each reactor was ~30 g m−3 h−1. Similar REs were obtained for both reactors (88% ± 1% for the TPPB-BTF and 91% ± 1% for the BTF) with an average outlet styrene concentration of 30.0 ± 8.1 mg m−3. The results corroborate that after more than 2 months after silicone oil addition, its effect on the process performance could be considered negligible. Despite the greater operational cost due to greater pressure drop, the series configuration is the preferred option versus the parallel one when tight and very low emissions limits are required. Both configurations (reverse serial and parallel modes) were tested under transient styrene loading (Figure 4). The reverse series configuration (BTF + TPPB-BTF, Figure 4a) showed a similar outlet emission pattern to that obtained with the TPPB-BTF + BTF series configuration (Figure 3). A slightly lower emission was achieved (average outlet emission during 8 h cycle: 18 mg m3) due to the progressive improvement of the BTF overtime. Therefore, the silicone oil does not provide an advantage for buffering peaks two months after its application. It could be hypothesized that the progressive growth and thickening of the biofilm results in outer layers with similar physical and chemical properties than those of a conventional biofilm. For the parallel configuration, both reactors showed similar results (TPPB-BTF: 54 g m−3, BTF: 40 mg m3), being worse than the series configurations. In any case, BTF performed better than the TPPB-BTF in any of the tested configurations to handle fluctuating emissions. These results indicate that the BTF can achieve similar or even better removals than the TPPB-BTF if enough time is given for the biofilm development. Furthermore,Sustainability 2020 it, 12 can, 6740 be concluded than the series configuration offers a clear safety margin9 offor 14 achieving very low emissions when compared to the parallel configuration. The removal obtained herein using the series configuration of two reactors (RE >98% for stationary emissions and >95% for oscillating(RE >98% forones) stationary is the best emissions reported and for> treating95% for 30 oscillating g m−3 h−1 ones) of styrene is the at best an reportedEBRT as forlow treating as 40 s. 30 g 3 1 m− h− of styrene at an EBRT as low as 40 s.

1 Figure 4. Response to inlet styrene concentration peaks under discontinuous loading (16 h d− ). −1 Figure(a) Series 4. configurationResponse to inlet BTF styrene+ TPPB-BTF, concentration (b) parallel peaks configuration under discontinuous of TPPB-BTF loading and BTF. (16 h d ). (a) Series configuration BTF + TPPB-BTF, (b) parallel configuration of TPPB-BTF and BTF. 3.4. Microbial Community Analysis 3.4. Microbial Community Analysis Pyrosequencing yielded a range from 29,125 to 63,295 raw reads for the inoculum (sample 0, day 0) and for the four biofilm sections (samples 1 to 4, day 51) of the series configuration TPPB-BTF + BTF (Table2). Therefore, samples 1 to 4 corresponded to biomass exposed to progressive decreases in 3 styrene concentration. Inlet and outlet styrene concentrations were (day 51): 302 and 91 mg m− for 3 TPPB-BTF, and 91 and 30 mg m− for BTF. From the lowest number of normalized sequences for the five samples (29,125), between 1565 and 565 operational taxonomic units (OTUs) were detected, the biggest number corresponding to the inoculum. The richness estimator (Chao1) and Shannon diversity index are summarized in Table2. Both parameters achieved much lower values in the samples from the two reactors (samples 1 to 4) than from the inoculum (sample 0), indicating a sharp shift in the bacterial diversity after 51 days of operation. Therefore, a specialization of the microbial community occurred, probably associated with the change in the carbon source from a complex wastewater to styrene only. In addition, the lowest value was obtained for the biomass exposed to the highest styrene concentration 3 (sample 1 nearest to inlet gas to the system containing 302 mg m− of styrene). Moreover, the highest value corresponded to the biomass exposed to the lowest styrene concentration (sample 4 nearest 3 to the outlet gas from the system with 30 mg m− of styrene). Therefore, this study corroborates that styrene concentration is a critical factor in decreasing the microbial diversity of BTFs, as was previously reported [16]. To evaluate the similarity between samples, beta diversity analysis was carried out through PCoA analysis based on weighted Unifrac distances (Figure5). PCoA revealed that the samples from each reactor clustered together, indicating that the recirculation of the trickling water contributes to the homogenization, to some extent, of the microbial population along the reactor. Despite being exposed to similar styrene concentration, samples 2 and 3 showed a greater dissimilarity between themselves than with the other sample of its own reactor due to the use of independent water tanks. Sustainability 2020, 12, 6740 10 of 14

Table 2. Summary of processed sequences from pyrosequencing including richness estimator and diversity index from the inoculum and each biomass sample of the series conﬁguration TPPB-BTF + BTF.

Sample Sampling Day OTUs 1 Chao1 Shannon Index 0 Inoculum 0 1565 1655 (1575–1734) 2 8.25 (8.20–8.31) 2 1 TPPB-BTF—Inlet 3 51 545 566 (506–627) 5.83 (5.81–5.85) 2 TPPB-BTF—Outlet 4 51 616 736 (667–805) 5.94 (5.92–5.96) 3 BTF—Inlet 3 51 653 702 (622–781) 6.39 (6.36–6.42) 4 BTF—Outlet 4 51 754 915 (828–1002) 6.58 (6.55–6.60) 1 Operational taxonomic units observed using the normalized sequences (29,125). 2 The 95% conﬁdence intervals of respective estimations are shown in parenthesis. 3 Sample taken at 20 cm bed height from the gas inlet to the reactor. 4 Sample taken at 105 cm bed height from the gas inlet to the reactor.

At the phylum level, three of the five classes of Proteobacteria concentrate the biggest part of the described catabolic pathways to degrade aromatic compounds. In our study, most of the sequences identified belong to the Proteobacteria phylum, with a relative abundance ranging from 57.0% to 60.1% in every sample. Previous studies on BTFs degrading styrene also found that Proteobacteria was the predominant phylum [16–18]. The relative abundance of the dominant families (with a relative abundance >1% at least in one sample) is presented in Figure6. As it can be seen, there was a shift in the predominant ones from the inoculum (sample 0) to the four samples of day 51 due to the adaptation of the microbial population to styrene as a unique exogenous carbon source. Among the top 22 classified families, those predominant in the inoculum (sample 0) were identified as Rhodocyclaceae, Saprospiraceae and Comamonadaceae, with a relative abundance of 13.0%, 11.3% and 9.8% of total sequences, respectively. Furthermore, the dominant families observed on day 51 (samples 1–4) were dependent on the reactor, since the water trickling of each reactor homogenized the bacterial population through its bed packing. In the TPPB-BTF (samples 1–2), Comamonadaceae, Pseudomonadaceae, Weeksellaceae, and Nocardiaceae were detected in high relative abundances (average values: 23.9%, 10.1%, 8.5%, and 6.5% of total sequences, respectively). In the BTF (samples 3–4), Comamonadaceae, Xanthomonadaceae, Sphingobacteriaceae and Nocardiaceae made up the dominant members (average values: 20.4%, 17.6%, 10.4% and 6.7% of total Sustainability 2020, 12, x FOR PEER REVIEW 10 of 14 sequences, respectively).

Figure 5. Principal coordinates analysis (PCoA) of weighted Unifrac distances for the series Figureconﬁguration 5. Principal TPPB-BTF coordinates+ BTF. analysis Inoculum (PCoA) (sample of 0),weighted TPPB-BTF Unifrac (samples distances 1 and 2), for and the BTF series configuration(samples 3 TPPB-BTF and 4). + BTF. Inoculum (sample 0), TPPB-BTF (samples 1 and 2), and BTF (samples 3 and 4).

At the phylum level, three of the five classes of Proteobacteria concentrate the biggest part of the described catabolic pathways to degrade aromatic compounds. In our study, most of the sequences identified belong to the Proteobacteria phylum, with a relative abundance ranging from 57.0% to 60.1% in every sample. Previous studies on BTFs degrading styrene also found that Proteobacteria was the predominant phylum [16–18]. The relative abundance of the dominant families (with a relative abundance >1% at least in one sample) is presented in Figure 6. As it can be seen, there was a shift in the predominant ones from the inoculum (sample 0) to the four samples of day 51 due to the adaptation of the microbial population to styrene as a unique exogenous carbon source. Among the top 22 classified families, those predominant in the inoculum (sample 0) were identified as Rhodocyclaceae, Saprospiraceae and Comamonadaceae, with a relative abundance of 13.0%, 11.3% and 9.8% of total sequences, respectively. Furthermore, the dominant families observed on day 51 (samples 1–4) were dependent on the reactor, since the water trickling of each reactor homogenized the bacterial population through its bed packing. In the TPPB-BTF (samples 1–2), Comamonadaceae, Pseudomonadaceae, Weeksellaceae, and Nocardiaceae were detected in high relative abundances (average values: 23.9%, 10.1%, 8.5%, and 6.5% of total sequences, respectively). In the BTF (samples 3–4), Comamonadaceae, Xanthomonadaceae, Sphingobacteriaceae and Nocardiaceae made up the dominant members (average values: 20.4%, 17.6%, 10.4% and 6.7% of total sequences, respectively). Sustainability 2020, 12, 6740x FOR PEER REVIEW 1111 of of 14

Figure 6. Heatmap distribution of the most abundant families (relative abundance > 1% at least in Figureone sample) 6. Heatmap of biofilm distribution samples of fromthe most the inoculumabundant families (sample (relative 0) and fromabundance the reactors > 1% at in least the in series one sample)configuration of biofilm TPPB-BTF samples+ BTF from (samples the inoculum 1–4). (sample 0) and from the reactors in the series configuration TPPB-BTF + BTF (samples 1–4). Biofilm samples from both reactors had two predominant families in common, suggesting that styreneBiofilm was thesamples critical from factor, both despite reactors its had concentration, two predominant to shape families the bacterial in common, population, suggesting which that was styrenegreater was in the the TPPB-BTF critical factor, (samples despite 1–2) its concentr than ination, the BTF to shape (samples the bacterial 3–4). The population, highest percentagewhich was greaterof sequences in the thatTPPB-BTF were classified (samples to 1–2) known than families in the BTF across (samples all bioreactor 3–4). The samples highest belonged percentage to the of sequencesComamonadaceae that ,were with relativeclassified percentages to known greater families than across 20% ofall thebioreactor total sequences. samples Indeed, belonged its relativeto the Comamonadaceaeabundance was, morewith relative than doubled percentages from greater the inoculum than 20% (9.8%). of the total In this sequences. regard, Indeed, genes for its relative styrene abundancemonooxygenase, was more which than encodes doubled the from initial the enzyme inocul inum the (9.8%). metabolic In this pathway regard, forgenes degradation for styrene of monooxygenase,styrene, have been which identified encodes in somethe initial species enzyme of the Comamonadaceaein the metabolicfamily. pathway Thus, for theydegradation have been of styrene,reported have as one been with identified the highest in metabolic some species potential of the to degradeComamonadaceae aromatic family. compounds Thus, [19 they,20]. have The otherbeen reportedcommon as dominant one with family the highest detected metabolic in both potent reactorsial to at degrade very similar aromatic percentages compounds in [19,20]. all samples The other(6.6% common1.3%) wasdominantNocardiaceae family. detected Interestingly, in both this reactors family at was very not similar detected percentages as an abundant in all samples family ± (6.6%in the ± inoculum. 1.3%) was SomeNocardiaceae species. Interestingly, belonging to Nocardiaceaethis family wasfamily not havedetected been as described an abundant as capable family in to themineralize inoculum. styrene Some as speciesNocardia belongingspp. and toRhodococcus Nocardiaceaespp. family [16,21 have–23]. been On the described other side, as thecapable styrene to mineralizeconcentration styrene rather as than Nocardia the presence spp. and or absenceRhodococcus of silicone spp. [16,21–23]. oil seems to On play the a other role in side, the proliferation the styrene concentrationof the other dominant rather than families, the suchpresence as Pseudomonadaceae or absence of ,siliconeSphingobacteriaceae oil seems toand playXanthomonadaceae a role in the. proliferationThe Pseudomonas of thegenus other has do beenminant frequently families, studied such inas thePseudomonadaceae aerobic degradation, Sphingobacteriaceae of styrene [24 –and28]. XanthomonadaceaeThis genus belongs. The to thePseudomonasPseudomonadaceae genus hasfamily been frequently and it was studied the second in the in aerobic relative degradation abundance of in styrenethe TPPB-BTF [24–28]. reactor. This genus Furthermore, belongs to it the is remarkable Pseudomonadaceae that there family was and a positive it was tendencythe second between in relative the abundancerelative abundance in the TPPB-BTF of the Pseudomonadaceae reactor. Furthermore,and the it is styrene remarkable concentration. that there was The a relative positive abundance tendency betweenof the Pseudomonadaceae the relative abundancefamily decreasedof the Pseudomonadaceae as styrene concentration and the styrene decreased concentration. through The the relative system abundanceTPPB-BTF +of BTFthe Pseudomonadaceae (11.9%, 8.2%, 3.5% family and decreased 1.7% for samplesas styrene 1, concentration 2, 3, and 4 respectively). decreased through It could the systemsuggest TPPB-BTF that species + BTF from (11.9%, this family 8.2%, are 3.5% favored and 1.7% by high for styrenesamples concentrations 1, 2, 3, and 4 respectively). versus other familiesIt could suggestsuch as Sphingobacteriaceaethat species from thisand familyXanthomonadaceae are favored by. These high styrene two families concentrations show an oppositeversus other tendency families to suchPseudomonadaceae, as Sphingobacteriaceaemore abundant and Xanthomonadaceae in the second reactor. These (BTF)two families than in show the first anreactor opposite (TPPB-BTF) tendency to of Pseudomonadaceae,the series configuration. more abundant Therefore, in their the second abundances reactor were (BTF enhanced) than in the at low first styrene reactor concentrations.(TPPB-BTF) of theRecently, series microorganismsconfiguration. Therefore, belonging their to the abundancSphingobacteriaceaees were enhancedfamily at have low been styrene associated concentrations. with the Recently,biodegradation microorganisms of expanded belonging polystyrene to the [29 Sphingobacteriaceae]. Arnold et al. [30 family] identified have several been associated styrene degraders with the biodegradationbelonging to families of expandedTsukamurellaceae polystyrene, Sphingomonadaceae [29]. Arnold et al., Xanthomonadaceae [30] identified severaland Pseudomonadaceae styrene degradersin belonging to families Tsukamurellaceae, Sphingomonadaceae, Xanthomonadaceae and Pseudomonadaceae in biofilms from a peat biofilter and Portune et al. [16,31] also observed some styrene degraders such Sustainability 2020, 12, 6740 12 of 14 biofilms from a peat biofilter and Portune et al. [16,31] also observed some styrene degraders such as Dokdonella inmobilis and Brevundimonas from families Xanthomonadaceae and Caulobacteraceae in biofilms from BTF. In addition, Liao et al. [18] showed bacterial genera Achromobacter belonging to Alcaligenaceae family as potential degraders of styrene, and Zhong et al. [32] reported the families Pseudomonadaceae, Caulobacteraceae and Sphingomonadaceae as dominant bacterial families in styrene degrading biofilters.

4. Conclusions The operation of two biotrickling filters connected in series, one of them working as a TPPB, has been proven to be robust for the efficient treatment of styrene emissions under typical industrial conditions. The series configuration TPPB-BTF + BTF has been demonstrated as the best alternative 3 1 since nearly complete removal of 30 g m− h− both in stationary and oscillating emissions was obtained. In addition, this approach allowed for reducing the silicone oil quantity to a minimum single dose of 3 1 0.83 m per kg h− of styrene emitted. However, the effect of silicone oil on the process performance could only be corroborated during the first weeks, and it was mainly associated with a faster start-up and biofilm development of the TPPB-BTF when compared to the BTF. In fact, the conventional BTF achieved slightly better performance than the TPPB-BTF after two months of operation. Therefore, from an industrial point of view, the use of silicone oil should be carefully evaluated due its high cost. Microbial community analysis showed enrichment in identified styrene degraders belonging to Comamonadaceae, Nocardiaceae and Pseudomonadaceae families with their relative abundances through the system modulated by the styrene concentration.

Author Contributions: Conceptualization and Methodology, P.S.-V., J.Á.-H., P.F., J.M.P.-R., P.M. and C.G.; Investigation, P.F., J.Á.-H., P.M., J.M.P.-R. and C.G.; Resources, P.M.; Writing—Original Draft Preparation, P.S.-V. and P.F.; Writing—Review & Editing, J.Á.-H. and C.G.; Visualization, P.M. and C.G.; Project Administration, C.G.; Funding Acquisition, C.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the FEDER/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación/Project CTM2017-88042-R and by the Ministerio de Economía y Competitividad/Project CTM2014-54517-R with FEDER funds. P. Ferrero had an FPI contract from the Ministerio de Economía y Competitividad, Spain. Acknowledgments: We would like to thank the Unidad de Genómica del Servei Central de Suport a la Investigació Experimental at the Universitat de València for performing the high-throughput sequencing. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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