Journal of Hazardous Materials 268 (2014) 14–22

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

Dichloromethane removal and microbial variations in a combination of UV pretreatment and biotrickling filtration Yu Jianming a , Liu Wei b , Cheng Zhuowei b,∗ , Jiang Yifeng b , Cai Wenji b , Chen Jianmeng b,∗∗ a b

Collaborative Innovation Center of Green Pharmaceutical Engineering, Zhejiang University of Technology, Hangzhou, China College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• DCM removal performance of the • • • •

combined UV–BTF was much better than the single BTF. Different sections in BTF contributed differently to DCM removal and mineralization. Ozone remaining from UV controlled the secretion of EPS and thus maintained normally. Pyrosequencing analysis showed a drastic change of bacterial community in biofilters. The microbial diversity was higher and biomass distributed evenly in combined BTF.

a r t i c l e

i n f o

Article history: Received 17 July 2013 Received in revised form 15 December 2013 Accepted 19 December 2013 Available online 7 January 2014 Keywords: UV photodegradation Biotrickling filtration Target distribution Extracellular polymeric substances Pyrosequencing Microbial community

a b s t r a c t Biofiltration of hydrophobic and/or recalcitrant volatile organic compounds in industry is currently limited. A laboratory-scale system integrating ultraviolet (UV) photodegradation and a biotrickling filter (BTF) was developed to treat dichloromethane (DCM), and this was compared to BTF alone. A combined UV–BTF approach permitted faster biofilm formation and greater removal than BTF. DCM distribution and its photodegradation intermediates revealed that the lower filter of the UV–BTF contributed more to CO2 production; the upper filter assisted more with DCM removal. The UV–BTF kept secretion of extracellular polymeric substances at a normal level with an evenly distributed biomass. Pyrosequencing analysis showed that the dominant population in the combined biofilter was more diverse than that in BTF alone. Our data provide a foundation for understanding the effect of UV pretreatment on BTF performance and the microbial community. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +86 571 88320881; fax: +86 571 88320882. ∗∗ Corresponding author. Tel.: +86 571 88320226; fax: +86 571 88320882. E-mail addresses: [email protected] (C. Zhuowei), [email protected], [email protected] (C. Jianmeng). 0304-3894/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.12.068

Dichloromethane (DCM) is widely used in industrial operations and as an intermediate/reagent in product synthesis and as a solvent for diverse chemicals [1]. Because of improper handling and disposal practices/poor volatile organic compound (VOC) purification techniques, DCM has frequently been released into the atmosphere. Its toxicity, persistence, and accumulation in the food chain could pose harm for humans and ecosystems. DCM is a priority

Y. Jianming et al. / Journal of Hazardous Materials 268 (2014) 14–22

controlled pollutant according to the US Environmental Protection Agency; thus, better treatment approaches are needed [2]. In the past, industrial VOCs have been treated by physicochemical methods: absorption, condensation, and incineration. Biofiltration is replacing these methods because it costs effective, uses less energy, and offers no secondary pollution [3]. Biofilters can treat gaseous waste streams, especially those containing low concentrations of VOCs at high gas flow rates. Recent studies suggest several factors affect the extent to which biological purification can occur in a biofilter. These are physical and chemical properties, the degree of biodegradability and loading pollutant, as well as temperature and relative humidity [4–7]. Although the efficient removal of hydrophilic and biodegradable VOCs by biofilters has been reported extensively, few studies describe the purification of hydrophobic and recalcitrant VOCs. Technical problems such as large volume biofilters, longer acclimation times, and unstable operations have made this technology problematic for treatment of hydrophobic and recalcitrant VOCs. Recently, urbanization coupled with stringent environmental legislation suggests a need for novel VOC treatments, especially for hydrophobic and recalcitrant VOCs. A recent advance is the combination of single technologies that overcome limitations of individual approaches and remove more VOCs [8]. Ultraviolet (UV) oxidation (including photolytic oxidation and photocatalytic oxidation) and biopurification – including biofilter (BF) and biotrickling filter (BTF) – techniques have been integrated to treat hydrophobic and recalcitrant VOCs. UV oxidation directly converts hydrophobic and recalcitrant VOCs to water-soluble and biodegradable intermediates, potentially offering complete biofilter removal [9,10]. Combining a photocatalytic reactor and a biofilter, Hinojosa-Reyes et al. [11] removed up to 36% more ethylbenzene than with a biofilter alone. Cheng et al. [12] found the elimination capacity (the removal amount of target per volume of media per hour) of biotrickling filtration for ␣-pinene increased by more than 50 g m−3 h−1 when combined with a photolytic reactor (vacuum UV system). Thus, combination systems efficiently removed hydrophobic and recalcitrant VOCs. However, these studies focused on comparing removal characteristics of single and coupled systems, or on longterm operation of a coupled system. The variation in the microbial community and the distribution of target VOCs or their intermediates in the biofilters have not been investigated. Detailed studies focusing on the integration of combined systems are required to better understand their mechanism of action to develop industrial applications for treating hydrophobic and recalcitrant VOCs. Here, we present data from a laboratory-scale combined UV–BTF system that treated treat gaseous DCM. We compared these data with single BTF treatment. To evaluate the performance of both techniques, their comparative removal efficiency, elimination capacity, and mineralization effect were measured over 90 days. The relationship between the distribution of photodegradation intermediates and their removal was analyzed. To understand the effect of UV pretreatment on biofilm formation, changes of extracellular polymeric substances (EPS) and microbial structure were investigated by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). A 454 high-throughput pyrosequencing method that could achieve more sequences than PCR-DGGE was also used to reveal the taxonomic diversity within complex microbial communities. Our work provides insights for the development of UV–BTFs for treating hydrophobic and recalcitrant VOCs industrially.

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A custom-made spiral UV reactor composed of quartz glass with an effective volume of 2.25 L had low-pressure mercury vapor lamps (36 W, Electric Light Sources Research Institute, Beijing, China) installed in its center. These lamps emitted at a wavelength of 184.9 nm (0.206 mW cm−2 from 1 m) and produced ozone via photolysis of oxygen. The BTF was made of Plexiglas, and had an internal diameter of 12 cm and height of 70 cm. Ether-based polyurethane foam (PU-foam) with a mean size, stacking density and porosity of 14–18 mm, 155 kg m−3 and 90.8%, respectively, was used as a packing material and the operating volume of the BTF was 6.8 L. Ports sealed with rubber septa were placed at equal intervals along the BTF for sampling the gas and packing material. To generate a DCM-contaminated air stream, compressed air was split into two portions, and the major portion was humidified to the desired relative humidity. The minor air stream was bubbled through liquid DCM (purity 97%, J&K Chemical Company, China) in a sparger to generate the contaminated air stream. Different ratios of these streams, which were controlled by mass flow meters, determined the inlet concentration of DCM and the relative humidity of the mixed air stream. The inoculation for biofilm formation was comprised of acclimated active sludge from a wastewater treatment plant. This was acclimated to DCM for nearly 1 month and a suspension of the prepared DCM degrader (Pandoraea pnomenusa LX-1 isolated by our group) had a volume ratio of 5:1. Packing materials were immersed in the inoculation for 2 days to allow biomass attachment. Packing materials were returned to the biofilter, and the experiment as initiated. The combined UV–BTF and single BTF operated for more than 4 months under the same conditions (Table S1). To maintain adequate nutrients and moisture within the filter bed, a circulating mineral medium was added (constant flow rate = 160 mL min−1 ). Every 2 days, 500 mL of the circulating liquid was replaced with fresh mineral medium (average liquid residence time of 5 days). Experiments were carried out by varying the flow rates of streams to obtain different initial concentrations and the effective contact time in the reactor.

2.2. Extraction and chemical analysis of bound EPS Bound EPS was heat extracted with method similar to that of Comtem et al. [13]. Packing material samples (1 g each) from each biofilter were immersed in sterile saline (0.85%, 10 mL) and vigorously mixed for 10 min with a shaker. Each sample was centrifuged at 3000 rpm for 10 min to remove the supernatant. The residue was immersed in phosphate buffer (pH = 7.4) at 80 ◦ C for 1 h. The heated liquid was cooled to room temperature and centrifuged at 15,000 rpm for 20 min. The obtained supernatant was filtered through a 0.45-␮m membrane and used as the bound EPS fraction for chemical analyses. The total quantity of extracted bound EPS was measured as total organic carbon (TOC) using a TOC analyzer (Shimadzu, Kyoto, Japan). Polysaccharides and proteins in bound EPS were measured using a phenol–sulfuric acid method and a Folin–phenol method [14], respectively. EPS were extracted and measured in triplicate for each sample.

2.3. Bacterial community study 2. Materials and methods 2.1. Experimental set-up and operation The integrated system consisting of a UV reactor and a BTF used in this study (Fig. S1) has been described previously [12].

2.3.1. DNA extraction and PCR To analyze biofilter bacterial communities, biofilm DNA (0.3 g) sampled at the indicated times was extracted using a Fast DNA® SPIN Kit for soil (QBIOgene, Carlsbad, CA) following the manufacturer’s instructions.

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2.3.2. PCR-DGGE analysis Partial sequences of the 16S rRNA gene including the V3 region (corresponding to regions in Escherichia coli) were amplified from the obtained DNA using the primer set 341f (5 -CG-clampCCTACGGGAGGCAGCAG-3 ) and 534r (5 -ATTACCGCGGCTGCTGG3 ). PCR was performed in a PTC-200 DNA Engine Thermal Cycle (BioRad, Hercules, CA) using a previously published specific cycling method [15]. PCR products were confirmed with electrophoresis on 1.5% agarose gels stained with Goldview II. DGGE was performed with the Bio-Rad DcodeTM system (BioRad). Gels were stained with GelRed before visualizing/analyzing on a Gel Doc XR system (BioRad). Selected bands were excised, eluted in sterile buffer overnight and then directly re-amplified with the above primer sets. Finally, PCR products were sequenced by TaKaRa Biotechnology (Dalian, China).

2.3.3. Pyrosequencing and data analysis Amplicon pyrosequencing was performed by Majorbio Pharmaceutical Technology Co., Ltd. (Shanghai, China) using a 454/Roche GS-FLX Titanium instrument (Roche, Nutley, NJ). The pretreated sequences obtained from pyrosequencing and the V1–V3 region of the clone library were aligned using NAST according to the criterion of 75% identity over 50% of the sequence length. Sequences more than 97% similar were classified into one operational taxonomic unit (OTU) based on the distance matrix using MOTHUR software (http://www.mothur.org./wiki/Main page). Rarefaction curves, a Shannon diversity index, a Chao1 species richness estimator and a principal component analysis (PCA) were generated in MOTHUR for each sample. Taxonomy classification was performed using BLASTN software with an e-score cutoff of 0.01 against version 10.18 of the RDP database and the Greengenes web service (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi). After phylogenetic allocation of the sequences at the phylum, class and genus level, the relative abundance of a given phylogenetic group was set as the number of sequences affiliated with that group divided by the total number of sequences per sample. Venn diagrams with shared and unique OTUs were used to depict the similarities among the microbial communities.

2.4. Analytical methods DCM was measured with a gas chromatograph (Agilent 6890) equipped with an electron capture detector. Gas samples were collected using a six-way valve with a gas sampling loop and then transferred into a HP-5 capillary column (30 m × 0.32 mm × 0.25 ␮m, J&W Scientific). The operating conditions of the GC were as follows: injector: 250 ◦ C, oven: 70 ◦ C for 3.5 min and detector: 300 ◦ C. Analysis of CO2 concentration was conducted on a GC (Agilent 6890) equipped with a thermal conductivity detector and a silica HP-Plot-Q capillary column (30 m × 0.32 mm × 20 ␮m, J&W Scientific). The operating conditions were as follows: injector: 90 ◦ C, oven: 40 ◦ C for 4 min and detector: 100 ◦ C. DCM and photodegradation intermediate distribution in the biofilters were measured as follows: packing materials (2 g) from different sections of the filter beds were immersed in deionized water and vigorously mixed for 1 h by a shaker. Each mixture was centrifuged at 15,000 rpm for 20 min and the obtained supernatant was filtered through a 0.22-␮m membrane. Cl ions and water-soluble organic carbon values were measured with an ion chromatograph (IC, Dionex model ICS 2000) and a TOC analyzer (TOC-VCPH, Shimadzu, Kyoto, Japan), respectively.

Fig. 1. Overall performance of the single BTF (a) and the combined BTF (b) during the start up and stable operational periods.

3. Results and discussion 3.1. Performance of BTF and UV–BTF The removal efficiencies (REs) of DCM by the single (BTF) and combined (UV–BTF) systems with the same inlet concentrations over 4 months are presented in Fig. 1. With single BTF, DCM RE reached 54.57% with an inlet concentration of 420 mg m−3 on the 16th day, suggesting the formation of a biofilm. However, with increased inlet concentration, RE decreased to 36.33% on the 19th day, indicating that biofilm formation was incomplete or biofilm microorganisms did not adapt to the sudden increase of the inlet concentration. Over time, the biofilm adapted to variations of inlet concentrations, and RE increased rapidly with increased inlet concentrations. For example, RE increased to 51.54% from 40.21% with an inlet concentration of 750 mg m−3 in 3 days. Biofilm formation was more rapid in the combined UV–BTF than in the single BTF. After BTF incubation, REs increased to 81.78% on the 10th day with the same DCM concentration. Because UV photodegradation reduced the DCM inlet load to the biofilter, the biofilm quickly acclimated to inlet concentration variations and overall RE improved. When DCM increased from 425 to 650 mg m−3 and from 650 to 800 mg m−3 , REs reached 85.35% and 82.57%, respectively, in just 3 days. Some intermediates such as carbonyl compounds and volatile fatty acids, which were detected in our previous study [16], were directly introduced into the subsequent BTF with the remaining DCM. These components not only accelerated the development of the stable microbial consortium but also improved the mass transfer of the parent compound. These data suggest that UV treatment

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of DCM may positively affect microorganism growth in the subsequent BTF, substantially shortening time to biofilm formation [17,18]. Steady-state experiments at different total empty bed residence times (EBRTs), i.e., 22, 34 and 46 s, were performed by gradually increasing DCM. At an EBRT of 46 s, for DCM below 410 mg m−3 , more than 46% of DCM was removed and a maximum elimination capacity of 46.51 g m−3 h was reached in the single BTF. During BTF operation at EBRTs of 22 and 34 s, inlet loads varied from 43.31 to 98.52 g m−3 h−1 . With low DCM concentrations, RE approached 44%. And high DCM concentrations (∼600 mg m−3 ) yielded Res of ∼23%. This decreased removal capacity could be attributed to insufficient contact time between the gas pollutant and the biofilm [19]. Substrate toxicity and high inlet concentration may also explain the poor performance at high DCM concentrations [20,21]. Because various conditions destabilize or lower REs for DCM using the BTF alone as discussed above, DCM removal in the combined system was investigated with the same EBRTs. As shown in Fig. 1, maximum elimination capacities of 60.9, 66.5 and 68.5 g m−3 h−1 were obtained at EBRTs of 22, 34 and 46 s, respectively, under which DCM RE exceeded 66%. The combined technique was better than single BTF, which has issues with hydrophobicity and higher target load, decreasing performance [9,10]. UV photodegradation directly converted DCM to watersoluble or easily biodegradable intermediates (aldehyde, formic acid, acetic acid, for example) by combining molecular ozone and hydroxyl radical under UV light (184.9 nm) [16]. This altered waste gas and reduced substrate toxicity due to higher pollutant loads. Therefore, UV photodegradation improved subsequent BTF. Ideal treatment reduces pollutants to H2 O and CO2 with no secondary pollution. Therefore, we measured CO2 in inlet and outlet gaseous streams (Fig. 1). More (1.5–2 times) CO2 was produced more by the combined system. DCM removal and CO2 production for UV–BTF and BTF were correlated and fitted line slopes were ∼0.251 and 0.422, respectively. Theoretically, 1 mg of DCM generated 0.518 mg of CO2 via chemical oxidation. Therefore, combined UV–BTF and single BTF mineralized 81.46% and 48.45%, respectively, incoming DCM vapor under test conditions. Thus, UV pretreatment allowed greater conversion of target compounds than BTF alone and subsequent biopurification accounted for more than 50% of CO2 production. 3.2. Target distribution in combined UV–BTF and single BTF Biofilter target distribution is important to understanding removal capacity and microbial structures. Thus, the mass distributions of various intermediates during DCM removal were measured for the combined and single systems. Because instantaneous quantification of intermediates in the gaseous stream is not possible, accumulation was measured over 2 h in a liquid film that was captured between pieces of packing material. DCM, CO2 , Cl ions and TOC were measured to estimate mass distributions in each system. Table 1 shows these data obtained for BTF and UV–BTF at various EBRTs and inlet concentrations. Compared with single BTF, the DCM load entering the biofilter in the combined system was lower; microorganism toxicity at higher pollutant loads was mild. Moreover, UV pretreatment directly converted DCM to water-soluble and biodegradable intermediates. Variations in pollutant concentration and type improved BTF after UV treatment of DCM. Based on data in Table 1, the contribution for each system unit to DCM removal and mineralization was calculated. For single BTF, ∼58.4% of DCM was removed by the lower section, and 61.5% of CO2 was generated by the upper section. Thus, both sections had similar roles in DCM removal and mineralization. In contrast, each section’s contribution was different during the removal process in the combined system. The

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upper section contributed more to DCM removal (∼68.2%) and the lower one accounted for 80% of the entire CO2 produced by the biofilter. Because photodegradation intermediates, such as aldehydes, ketones, and volatile fatty acids, were biodegraded and mineralized more easily than others [22,23], microorganisms used these to generate more CO2 in the lower section of the biofilter in preference to DCM. Also, photodegradation intermediates were hydrophilic and easily absorbed by biofilter’s liquid film. Studies suggest that the presence of such hydrophilic compounds can enhance removal of hydrophobic and recalcitrant compounds by biofiltration [24,25]. Addition of organic compounds (aldehydes and ketones) to water can increase absorption of some hydrophobic VOCs [26]. Because DCM is a typical hydrophobic compound, the presence of photodegradation intermediates in the liquid film may promote mass transfer between gas and liquid phases, allowing subsequent biodegradation. Thus, it is found UV pretreatment changed the function of each biofilter section, unevenly distributing compounds within it. 3.3. Characteristics of bound EPS Bound EPS are composed of a variety of organic compounds. The differences of the total EPS and the ratio of extracellular protein (PN) to extracellular polysaccharide (PS), denoted PN/PS, between the single and combined biofilters are shown in Fig. 2. The combined UV–BTF had less bound EPS than the single BTF. Single filter EPS increased to 94.2 mg TOC/g VSS at 115 days, about twice that obtained at 35 days. However, EPS of the combined UV–BTF was almost constant during the experiment and was ∼1.1 times greater than the initial value after 115 days. Because the PS of both biofilters narrowly fluctuated (14–16 mg TOC/g VSS for the single BTF and 12–13.5 mg TOC/g VSS for the combined UV–BTF), proteins for each were different according to PN/PS (Fig. 2b). In the final operation stage, PN/PS for the lower section of the single BTF exceeded 0.6, indicating excessive secretion of PN. PN has been reported to be a chief component of EPS and is critical to biofilm formation [27]. More PN would increase microorganism bonding, increasing the adherence of the aging biofilm to the packing material. Overgrown biofilm accumulated along the filter, decreasing performance [28]. Ozone remaining after UV photodegradation was introduced into the biofilter where it was toxic to EPS secretion. Ozone is a strong oxidant, capable of destroying aging biomass structures, causing more biofilm renewing. Therefore, the biomass in the biofilter was maintained at a normal level (average values for the UV–BTF and single BTF were 10 mg VSS g−1 media and 14 mg VSS g−1 media, respectively), regulating its activity. Wang et al. [29] and Cheng’s group [12] reported that ozone (added or produced by UV photodegradation) had a similar role during VOC biofiltration. The biofilm was thickest along the filter bed due to substrate and metabolic product concentrations [30]. Generally, a thicker biofilm appeared near the biofilter inlet, and the biomass is unevenly distributed [31]. Here, we report that the remaining ozone helped distribute the biomass. At the 75th day, the biofilm thickness in the lower and upper sections of the combined UV–BTF was 2.10 and 2.02 mm, respectively, and in the single filter were 2.35 and 1.84 nm, respectively. Aside from creating ozone, photodegradation intermediates positively affected microorganisms. Various compounds allowed diverse microorganisms to inhabit the biofilter, producing unique EPS components. Studies suggest that one type of EPS might promote/inhibit another EPS secreted by a different microorganism [32]. Thus, the total EPS would be normally maintained without secretion. However, for the single BTF, the stream contained only one component (DCM) and the microorganisms were simple. No regulatory mechanism of EPS secretion during biofilm development was noted. Previous studies indicate that bioreactor

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Table 1 The distributions of various components along the filter bed. EBRT (s)

34

46

22

34

46

a b c

Inlet

BTF down inlet

C-DCMa (mg)

C-DCM (mg)

Cl− (mg)

TOC (mg)

BTF up inlet C-DCM (mg)

C–CO2 b (mg)

Cl− (mg)

TOC (mg)

C-DCM (mg)

C–CO2 (mg)

TOC (mg)

0.0547c 0.0688 0.0826 0.0551 0.0687 0.0818 0.0555 0.0667 0.0822 Single system Inlet C-DCM (mg) 0.0559 0.0681 0.0816 0.0559 0.0673 0.0831 0.0548 0.0675 0.0821

0.0416 0.0511 0.0672 0.0423 0.0523 0.0669 0.0434 0.0519 0.0670

0.0775 0.1021 0.0914 0.0742 0.0962 0.0876 0.0710 0.0855 0.0875

0.0109 0.0116 0.0108 0.0114 0.0147 0.0142 0.0118 0.0144 0.0149

0.0298 0.0387 0.0548 0.0273 0.0369 0.0523 0.0243 0.0334 0.0474

0.0150 0.0147 0.0168 0.0197 0.0200 0.0205 0.0272 0.0292 0.0287

0.0685 0.0728 0.0740 0.0875 0.0904 0.0849 0.1022 0.1094 0.1148

0.0208 0.0269 0.0248 0.0189 0.0254 0.0245 0.0207 0.0221 0.0254

0.0214 0.0332 0.0476 0.0185 0.0314 0.0429 0.0174 0.0284 0.0403

0.0034 0.0026 0.0025 0.0069 0.0057 0.0051 0.0101 0.0097 0.0087

0.0176 0.0197 0.0168 0.0158 0.0184 0.0176 0.0098 0.0087 0.0124

BTF up inlet C-DCM (mg) 0.0418 0.0539 0.0699 0.0409 0.0524 0.0705 0.0405 0.0518 0.0671

C–CO2 (mg) 0.0054 0.0061 0.0067 0.0087 0.0076 0.0069 0.0101 0.0094 0.0087

Cl− (mg) 0.0829 0.0828 0.0678 0.0842 0.0864 0.0724 0.0829 0.0910 0.0864

TOC (mg) 0.0082 0.0072 0.0043 0.0059 0.0065 0.0047 0.0038 0.0054 0.0052

BTF up outlet C-DCM (mg) 0.0311 0.0439 0.0608 0.0308 0.0432 0.0591 0.0299 0.0434 0.0561

C–CO2 (mg) 0.0090 0.0084 0.0095 0.0120 0.0113 0.0112 0.0180 0.0184 0.0156

TOC (mg) 0.0142 0.0141 0.0104 0.0117 0.0121 0.0123 0.0064 0.0052 0.0098

C-DCM refers to the amount of carbon containing by DCM. C-CO2 refer to the amount of carbon contained by CO2 . The value is the average value of three measurements and the standard deviations values are omitted.

BTF up outlet

Y. Jianming et al. / Journal of Hazardous Materials 268 (2014) 14–22

22

Combined system

Y. Jianming et al. / Journal of Hazardous Materials 268 (2014) 14–22

112

1200

Down section of biofilter in single BTF

(a)

Up section of biofilter in single BTF Down section of biofilter in combined UV-BTF Up section of biofilter in combined UV-BTF

900

80

Number of OTUs

EPS content (mg TOC/gVSS)

96

19

64 48

3% C-BTF 3% INO 3% S-BTF

600

300

32 16

0

0

0

35

75

95

115

Down section of biofilter in single BTF

(b)

m(PN)/m(PS)

0.6

0.3

35

75

95

6000

8000

10000

Fig. 3. Rarefaction curves base on pyrosequencing of INO, S-BTF and C-BTF bacterial communities.

Up section of biofilter in single BTF Down section of biofilter in combined UV-BTF Up section of biofilter in combined UV-BTF

0.0

4000

Number of sequences

Time(days) 0.9

2000

115

Time(days) Fig. 2. EPS contents (a) and PN/PS (b) values of different biofilms from the single BTF and combined BTF at day 75.

highest diversity (Shannon = 5.02) among the three communities. The Shannon index of BTF (4.52) was smaller than 4.89 of INO. Hierarchical cluster analysis was used to identify the differences within the three bacterial community structures (Fig. 4). BTF and UV–BTF belonged to two different clusters, suggesting distinctions of community structure between the combined and single BTFs despite the fact that they shared the same source of microbial consortia at the biofilm formation stage (INO). These data were supported by principal component analysis (PCA, Fig. S2): principal components 1 and 2 were 70.83% and 11.9% of the total community, respectively. Because the only carbon source of the single BTF was DCM, its microbial structure did not change over time, and it contained similar microorganisms to the inoculums for biofilter start-up; that is, BTF and INO fall into one cluster. In contrast, the outlet from the UV reactor contained not only DCM but also photodegradation intermediates, providing various carbon sources for microbial growth and development. Therefore, in the PCA, UV–BTF belonged to a different cluster far away from the other samples.

performance is determined largely by the microbial diversity and community stability, which is closely related to EPS amount and secretion [19,28]. 3.4. Comparison of bacterial communities developed in biofilters We investigated the bacterial communities residing in the two BTFs to understand the effects of UV pretreatment on these communities. The diversity and composition of the bacterial communities were characterized using the 454 pyrosequencing approach with universal bacterial 16S rRNA gene primers. After removing incorrectly identified or poor-quality sequences, a total of 31,639 sequence reads (average length = 454 bp) were obtained from three samples (10,496 reads for the microbial inoculum [INO], 10,073 reads for the biofilm sampled from the single BTF, and 11,070 reads for the biofilm sampled from the combined UV–BTF at 75 days). We obtained 946 (INO), 847 (BTF) and 1002 (UV–BTF) OTUs at a distance of 3%. However, new bacterial phylotypes emerged even after 10,000 reads (Fig. 3). The OTUs estimated by the Chao1 estimator were 1552 (INO), 1197 (BTF) and 1688 (UV–BTF) with infinite sampling, indicating that UV–BTF was richer than INO and BTF. The Shannon diversity index indicates not only species richness (number of species present) but also the species abundance for each community. The UV–BTF system had the

Fig. 4. Hierarchical cluster analysis of INO, S-BTF and C-BTF bacterial communities.

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(a) Bacterial phylum

UV-BTF

BTF

INO

Acidobacteria

Actinobacteria Bacteroidetes Chlorobi Cyanobacteria

Planctomycetes Proteobacteria Others

Unclassified bacteria

(b) Bacterial class

INO

BTF

UV-BTF Acido bacteria Actinobacteria Alphap roteoba cteria Betaproteobacteria Deltap roteobacteria Gammaproteobacteria Chlorob ia Sphingobacteria Flavobacteria Cytop hagia Bacilli Gemmatimonadetes Unclassified class Others

Fig. 5. Taxonomic classification of pyrosequences from bacterial communities of INO, S-BTF and C-BTF at the (a) phylum, and (b) class levels.

PCA indicated that the bacterial communities in the biofilters were largely determined by carbon source, and that addition of some water-soluble and readily biodegradable compounds would affect bacterial development in the treatment of hydrophobic and recalcitrant compounds. To identify the phylogenetic diversity of bacterial communities in the BTFs, we assigned qualified reads to known phyla, classes and genera (Fig. 5 and Table 2). The UV–BTF sample was highly diverse, reflected by the 17 identified bacterial phyla compared with the 13 for INO and 12 for BTF (Fig. 5a). The three largest phyla were Acidobacteria, Bacteroidetes and Proteobacteria. Although their total contents accounted for similar percentages in the samples (INO:

91.06%, BTF: 92.42% and UV–BTF: 90.47%), distribution differences existed of the three largest phyla existed in total community composition. The relative abundance of Proteobacteria was higher in INO (74.41%) and BTF (77.75%), and lower in UV–BTF (63.41%). The sum of Acidobacteria and Bacteroidetes in UV–BTF exceeded those in INO and BTF. These results imply that introduction of various carbon sources formed by UV pretreatment selectively enrich Acidobacteria and Bacteroidetes in biofilms. The class-level identification of the bacterial communities in the samples is illustrated in Fig. 5b. Pyrosequencing detected 40 bacterial classes in all three communities and most of the sequences belonged to 12 classes. Data at the genus level permitted inference

Table 2 Taxonomic classification of pyrosequences from bacterial communities of INO, S-BTF and C-BTF at the genus levels. Phyla

Class

Genus

Acidobacteria Actinobacteria Proteobacteria

Acidobacteria Actinobacteria ˛-proteobacteria ˇ-proteobacteria

Acidobacterium Mycobacterium Novosphingobium Acidovorax Hydrogenophaga Dokdonella Rhodanobacter Xanthomonas Flavobacterium Flexibacter Gemmatimonas Filimonas Others genera

-proteobacteria

Bacteroidetes Gemmatimonadetes Others Sum

Flavobacteria Sphingobacteria Gemmatimonadetes –

Relative abundance (%)a INO

S-BTF

C-BTF

0.91 0.89 – – – 1.14 – – – – 0.54 – 18.80 100

0.90 0.74 – 0.74 – 1.53 – – – – 0.60 – 24.51 100

2.34 0.58 1.56 5.92 1.55 0.58 0.69 2.27 1.45 3.20 0.85 0.65 10.19 100

Genera

Relative abundance (%) INO

S-BTF

C-BTF

Pandoraea Methylophilus Pseudomonas Sinobacter

46.56 – – –

44.10 0.81 – 0.68

35.07 1.01 2.55 0.82

Steroidobacter Unclassified genera

– 31.16

– 25.33

0.55 28.17

a Relative abundance was defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample. Genera making up less than 0.5% of total composition in all three libraries were classified as “others”.

Y. Jianming et al. / Journal of Hazardous Materials 268 (2014) 14–22

of community functions. The dominant population in INO, BTF and UV–BTF was the inoculated DCM degrader Pandoraea, with a relative abundance of 46.56, 44.10 and 35.07%, respectively. Pandoraea belonged to ˇ-proteobacteria, a class of Proteobacteria, comprising the largest group of the samples. DGGE profiling revealed that Pandoraea was the predominant strain in the biofilters; its band intensity was the brightest (Fig. S3). These results indicate that the inoculator Pandoraea grew well in both BTF and UV–BTF, contributing to removal of DCM. As shown above, the UV–BTF was supplemented with various carbon sources as well as DCM, allowing potential microbial diversity development. Thus, Pandoraea decreased with processing time and was lower than that in INO and BTF. In UV–BTF, more secondary bacteria groups were identified, such as Novosphingobium (1.56%), Acidovorax (5.92%), Xanthomonas (2.27%) and Flexibacter (3.32%). Together they formed a dominant community structure with the photodegradation intermediates as their major carbon sources. Among these secondary genera, some were nonexistent and others were only present in small amounts in either INO or BTF. The relative abundance of Acidovorax, reported to degrade organic acids [33], was higher in UV–BTF than in BTF, which might be related to organic acid formation acids during DCM photodegradation. Aldehydes and ketones in the UV–BTF also promoted the growth of Pseudomonas, most species of which can degrade aldehydes and ketones [34,35]. The higher microbial diversity of UV–BTF improved VOC removal compared to BTF. Greater biodiversity is known to increase ecological stability, and bacterial community evenness helps to maintain the high performance of a bioreactor. 4. Conclusions DCM removal by microbial communities in single BTF and combined UV–BTF systems were investigated. REs and CO2 production of the combined system was greater with same inlet concentration and EBRT than single BTF. Such enhanced removal capability was attributable to microbial growth differences between BTF and UV–BTF. With UV–BTF, ozone remaining from UV photodegradation efficiently controlled biomass. EPS in the UV–BTF system was maintained at a normal level because PN, a component of EPS, was not secreted much in the absence of ozone. The bacterial communities in the systems were analyzed by pyrosequencing technology, revealing that the microbial populations in UV–BTF were more diverse than those in BTF. Photodegradation intermediates in UV–BTF allowed various microorganisms of different phyla to inhabit the biofilter and such microbial diversity contributed to biofilm development and improved DCM removal by UV–BTF. Our results support previous findings that UV pretreatment not only enhances pollutant removal but also positively affected microbial communities. Acknowledgment This study was sponsored by the National Natural Science Foundation of China (21207115), the International S&T Cooperation Program of China (2011DFA92660), and the Ph.D. Programs Foundation of Ministry of Education of China (No. 20093317110003). We thank anonymous reviewers for helpful comments on the manuscript. We also thank LetPub for its linguistic assistance during the preparation of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at.http://dx.doi.org/10.1016/j.jhazmat. 2013.12.068

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