Low-Dosage Ozonation in Gaseous VOCs Biolter Promotes Community Diversity and Robustness

Background Ozonation of biolter is known for alleviating clogging and pressure drop issues while maintaining removal performances in bioltration systems treating gaseous VOCs (Volatile Organic Compounds). The implications of ozone on the biolter microbiome, in terms of biodiversity, community structure, metabolic abilities and dominant taxa correlated with performance remain largely unknown. Methods This study investigated two biolter treating high concentration toluene operating in parallel, with one acting as control and the other exposed to low-dosage (200 mg/m 3 ) ozonation. Microbial community diversity, metabolic rates of different carbon sources, functional predictions and microbial co-occurrence networks of both communities were examined. in microbiota


Introduction
Bio ltration is a technology widely applied in abatement of VOCs emissions, known for its low cost (Jorio exposure, concluding an increase in metabolism rate of numerous carbon sources of lower biodegradability, such as γ-hydroxybutyric acid, d-galactonic acid γ -lactone, d-mannitol, d-cellobiose and γ -methyl-d-glucoside etc. (Saingam et al., 2016). Traditionally O 3 is regarded as a strong oxidant that purges microorganisms and lowering overall activity. However, the opposite response was found in lowdosage O 3 exposure. For example, low concentration O 3 (e.g. 120 mg m − 3) was found to improve metabolic activity (Wang et al., 2009). It was shown that despite the decrease in biomass, microbial activity for metabolism of multiple carbon sources increased in the bio lter, implying an inherent change in the microbial level. It is well known that the microbiome of a bio lter is crucial for effective pollutant abatement (Ralebitso-Senior et al., 2012), yet the exact microbiome and functional changes that occur allowing increased microbial activity while maintaining system performance with decrease in biomass remained intriguing and unknown. In this study, 16srRNA sequences of the v4 region in a controlled bio lter and ozonated bio lter operated in parallel was sequenced for investigation of microbial changes leading to the adaptation and performance changes of the microbiome. Using statistical analysis techniques, we attempt to explicate the relevance of dominant taxa treating toluene after addition of O 3 , using latest databases we predict changes in functional and phenotypic characteristics under ozonation, and quantifying relations of microbiome change with removal performances.

Experimental setup
Two lab-scale bio lters named BF1 and BF2, constructed as acrylic cylinder with 12 cm inner diameter and 25 cm height. Each bio lter was packed with porous perlite (0.54 void fraction) to form a 1.6 L lter bed 15.0 cm in height. An air compressor (Hailea ACO-318, Fuzhou, China) was used to feed air into the system. Toluene gas inlet was produced with fresh air passing through a bottle containing liquid toluene. The gas ow rate was controlled by a ow meter (Zenxing LZD-4WB, Xianghu, China). A stainless-steel reactor equipped with a UV lamp (Cnlight ZW23D15W-Z436, Shenyang, China) was used to generate gaseous ozone for BF1. For each bio lter, the packing media was initially mixed with 1.0 L activated sludge collected from a municipal wastewater treatment plant (Xiaojiahe WWTP, Beijing, China). Nutrient solution containing NaNO 3 (20 g/L), Na 2 HPO(1.6 g/L) and KH 2 PO 4 (1.04 g/L) was sprayed directly on lter beds of the two bio lters for su cient humidity and nutrients. The leachate was discharged every day. The two bio lters were operated in parallel for 160 days in total, both operated in identical conditions without ozone for the rst 44 days, BF1 was fed with 200 mg/m 3 gaseous ozone after day 45.

Microbial sampling
Microbial samples were taken from both bio lters at day 66, 80, 94 and 160, packing media were taken from depths of 1 cm, 7 cm, 15 cm of the lter bed, bio lms were detached and suspended in phosphate buffer saline (PBS) by sonication at 425 W, 21-25 kHz for 10 min (Ningbo Science Biotechnology SCIENTZ-IID, Ningbo, China). The sonicated suspension was centrifuged at 10000 × g for 1 min and resuspended in 5 ml PBS. To exclude dead cells within the community, a uorescent dye (propidium monoazide, PMA) was used to treat the microbial suspension by inactivating DNA of cells of damaged cell membrane as well as exposed DNA (Guo and Zhang, 2014). PMA (Biotum, PMA™ dye, USA) stock was prepared by dissolving 1 mg PMA in 100 µL of 20% dimethyl sulfoxide (DMSO) and stored at − 20 °C; 2.5 µL 20 mmol/L PMA solution was added into 500 µL microbial suspension. The mixture was incubated at room temperature for 5 min and occasionally mixed. The tubes were placed horizontally on ice and exposed to a 650 W halogen light, at 20 cm distance, for 4 min. Then, DNA from PMA-treated aliquot are isolated with the FastDNA® SPIN Kit for Soil (MP Biomedicals, USA) following the manufacturer's instruction.
Each sample from a particular bio lter at a particular time were divided into 3 identical samples after PMA treatment and before DNA extraction, therefore each sample was represented as triplets to counter systematic biases from DNA isolation and sequencing procedures.

Metabolic activity analysis
Suspension from soni cation containing detached microbiome from the packing media as described in part 2.2 is diluted in PBS to obtain optical density at 600 nm wavelength (O.D. 600 ) at 0.05. Then, the ECO plate (Biolog, Inc, USA), with 31 various sources of carbon substrates mixed with the tetrazolium dye, was prepared for the determination. 150 µL of the microbial dilutions was inoculated to each well of the ECO plate and incubated at 30 °C. The plate was observed for the absorbance at 600 nm regularly during 3 days period by the microplate reader (Molecular devices, Spectramax M5, USA). Absorbance over time from wells of containing carbon source of a same group (e.g. amino acid) is averaged and deducted with the absorbance from the control well to avoid systematic error, and obtaining average metabolic rates of different groups of carbon sources.

Sequencing results
10,000-25,000 sequences of 250 bp were obtained, samples with under 13,000 sequences were discarded and others are rare ed at 13,000 sequences, resulting in 24 samples. 3296 OTUs were identi ed after denoising.

Biodiversity
Biodiversity index comparing the two bio lters are shown in Fig. 1. Intra-group diversity spanned over 400 in both groups of observed diversity, but median and average diversity of ozone bio lter is consistently higher than those of the control samples, indicating an increase in phylogenetic diversity range and average evenness.

Community differences and propensity of variables
Clear separation of microbiome composition is seen between the two bio lters, whereas intra-group differences also uctuated considerably with time, relative abundance of major phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes and TM7 were added as constraining variable in Fig. 2b to show overall compositional difference of OTUs level and abundance differences of major phyla. In addition to prediction of microbiome functions, microbiome phenotypic metabolizing activity from Biolog ECO plates were adopted as constraining variable to show inclination of communities for different carbon sources in Fig. 2b. Proteobacteria remained dominant in relative abundance, ozone stimulated its presence despite it consisting mostly of gram-negative species, along with Firmicutes, consisting mostly of gram-positive species. Actinobacteria is another known gram-positive bacteria seen in bio lters, decreased with ozonation. Metabolization ability of groups of amino acids and carboxylic acids increased in microbiome under ozonation, each containing a consortium of numerous compounds from the groups, while metabolization rates for complex carbohydrates decreased, metabolization data of individual compounds are provided in supplementary material 1.

Functional characteristics and traits caused by ozonation
Phenotypic traits of microbiome from both bio lters are shown in Fig. 3. In contrast of the greater intra-group differences seen in biodiversity and OTU level community differences, results of functional traits are relatively clustered, with expected increases in microbes capable of mobility, bio lm formation and stress tolerance with induction of ozonation, yet median proportion of gram-positive strains dropped from 42-27% after ozonation, in contrast with common understandings.
Metacyc pathways predicted by PICRUSt2 that changed substantially with an ALDEx effect size of over 2.5 is shown in Fig. 4. Among 15 pathways highly speci c to ozonation, mycolate biosynthesis, taxadiene biosynthesis (engineered), superpathway of heme biosynthesis from glycine, (5Z)-dodec-5-enoate biosynthesis are crucial pathways for assembling of proteins for cell membrane synthesis, although gram-negative bacteria increased with ozonation, repair and synthesis of cell membrane to mitigate oxidative distruption of ozone is seen in microbiome, along with production of anti-oxidants such as heme production and UDP-glucose-derived O-antigen biosynthesis. Of all enriched pathways after ozonation, phynylacetate degradation I (aerobic) pathway is the most signi cant with effect size of over Decrease in TCA cycle participation is seen in ozonation lter, and pathways for degradation of other metabolites in VOCs degrading pathways such as 3-phenylpropanoate and inositol degradation is seen, indicating a motley ununiform changes in pathways after ozonation, no decisive changes affecting overall functional ability are seen between groups, but a mixture of degradation inclination changes favoring different parts of the VOCs degradation pathways. Both correlation of OTU and Genus level are chosen to investigate possible intra-genus differentiation and radicality of differences. 9 out of 20 genera are monotonously correlated with removal e ciency in both ozone and control bio lter, whereas only 3 out of 20 OTUs are monotonously correlated with removal e ciency, indicating a high speci city and different reaction of OTUs under same genus towards ozonation. genus Rhodococcus was reported in numerous studies to be dominant in degrdation of xenobiotics in bio lter systems (Portune, Perez et al. 2015, Allievi, Silveira et al. 2018), all four OTUs from the genus Rhodococcus present in this system are strongly proportional with removal e ciency in control bio lter but all negatively correlates in ozonated bio lter, suggesting a shift of degrading contribution by Rhodococcus and more towards OTUs of genera Devosia, Aquamicrobium and Rhizobiales with high positive correlation with performance, similar trends of these three genus are seen in combined genus level.

Correlation of system performance and dominant taxa
3.7 Topological analysis and co-occurrence network construction of microbiome 91 and 107 nodes, 146 and 256 links, 5.1 and 3.6 average path distance were found for control and ozone bio lters respectively, with the increase of average degree from 3.2 to 4.7 under identical speci cations for network construction. A more connected and even network is seen in ozone microbiome, smaller path distance coupled with smaller centralization of betweenness is commonly seen as higher stability as major hubs are more diverged and less nodes are likely to be affected under shocks.
Major hubs in control bio lter such as OTU68 and OTU309 are substantially irrelevant in ozone bioi lter, indicating a radical change in microbial network and hub distribution, yet OTU26 corresponding to the genus Pandoreae remain highly relevant in betweenness and degree count in both system, genus Pandoreae was reported to be highly enriched in species capable of effective degradation of xenobiotics

Discussion
The performance of pollutant removal is the main concern for improvements attempts on alleviating clogging issues, ozonation was reported in numerous studies to improve or maintain VOCs removal performance while signi cantly reducing biomass growth rates (Xi et al., 2015, Maldonado-Diaz and Arriaga, 301 2015; Wang et al., 2009). Results from our study for a period of 160 days is consistent with previous studies that no statically signi cant changes were seen in terms of removal e ciency treating at a relative higher loading rate ( uctuating between 20-70 mg/L/h) compared to usual full-scale applications, showing to be a suitable candidate for full-scale application with the ease of retro tting and exible manipulation as ozone can be simply mixed with the inlet gas ow. Oxidative stress such as ozonation is known to reduce diversity at higher dosage, but our results show the opposite, possibly due to a low-enough dosage to allow subsistence of strains more sensitive to oxidative stress while also stimulating the ones more robust, hence providing a more diverse microbiome. Given the diverse nature of xenobiotics degradation pathways, and the complexity of pollutant composition frequently seen in fullscale application, ozonation provided possibility as a method for steady stimulation of microbiome diversity, which was reported to correlate directly with community stability and resistance to shocks (Hillebrand et al., 2008), which are most commonly seen as spikes in loading rates during periods spanning from hours to months in full-scale operation depending on the particular scenario (Yang et al., 2008). Microbial co-occurrence network shown in Fig. 6 also support this hypothesis with a more decentralized microbiome network after ozone dosage that may be less likely to see changes in community structure as radical as it would be in centralized network of the control bio lter microbiome.
After ozonation, Proteobacteria increaed in relative abundance, which was widely reported to contain dominant degrader of xenobiotics in both bio lter and wastewater treatments (Allievi, Silveira et al. 2018, Van der Heyden, De Mulder et al. 2019), reported for higher presence in treatment of more complex pollutant and higher loading stress, inducing higher functional diversity (Friedrich and Lipski 2010). In contrast, this could be a phenotypic embodiment of larger community diversity seen in sequencing results, the ability to better metabolize carbon sources other than the only provided toluene, while degradation of xenobiotics such as toluene had highly diverse prokaryotic pathways, a more even and less specialized phenotypic functional pro le may be desirable in full-scale applications when composition of pollutants is largely uctuated, though this decrease of complex carbohydrate degradation and increases in other compounds caused by ozonation did not impinge removal e ciency of bio lter.
Increase in stress tolerance of microbiome is highly desirable in bio lters as common applications of bio lters include emission of periodic fermentation, and highly diverse pollutant seen in chemical engineering industries and pharmaceutical industries depending on the production agenda. Such variations in inlet introduce shocks and hinder performances of bio lter by eliminating degrading strains un t for the new environment (Cabrol and Malhautier 2011), with the consistently increased stress tolerant strains after ozonation while maintain performance, such shocks in full-scale situations could be mitigated to an extent and is highly desirable. Increase in the bio lm forming population could be an issue in traditional bio lters with clogging issues mentioned above, but ozonation of bio lters was shown to greatly reduce growth rate of overall biomass and elevating pressure drop, the increase in bio lm formation population can hence be considered uncorrelated for steady operation of bio lters after inoculation phase.
Ozonation of bio lter has proven to alleviate clogging issues while maintaining performance, this study revealed other potential improvements to the microbiome such as higher biodiversity and functional stability, attested by a more connected and robust topological network with less centralized distribution and lower average path, a a higher percentage of microbiome being stress tolerant while phenotypically achieving higher metabolization rate for a variety of carbon sources, all contribute to shaping a more robust and shock-resistant microbiome. Improvements of the microbiome is another positive aspect of ozone application along with ease of retro tting and solving clogging issues in full-scale application.

Conclusion
Shifts in major degrading species corresponding to performance and increase in community biodiversity could explain the consistent performance commonly seen in ozonation of bio lters despite the decrease in biomass. Increased presence of stress tolerant microbes in the microbiome coupled with decentralization of the co-occurrence network suggest that ozonation could not only provide amelioration for clogging issues but also provides a microbiome more robust to loading shocks seen in full-scale bio lters.

Declarations
Availability of data and materials: Sequence data of all samples are available at: GenBank BioProject accession number PRJNA656689. Codes used for statistical analysis and plotting can be accessed at: https://github.com/myhyeung/Low-Dose-Ozonation-in-Gaseous-VOCs-Bio lter-Promotes-Community-Diversity-and-Robustness.      Top 20 taxa in average relative abundance in all samples, and their Pearson correlation with removal e ciency (RE%) and mineralization rate (MR%) in two bio lters. (a) Genus level. (b) OTU level, named for the lowest taxa rank identi ed with con dence over 97%, numbers are appended after names if different OTUs share the same lowest rank name.