Water Research 87 (2015) 127e136

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Effects of octahedral molecular sieve on treatment performance, microbial , and microbial community in expanded granular sludge bed reactor

** Fei Pan a, , Aihua Xu a, Dongsheng Xia a, Yang Yu a, Guo Chen b, Melissa Meyer c, * Dongye Zhao d, Ching-Hua Huang c, Qihang Wu e, Jie Fu c, a School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China b Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA c School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA d Environmental Engineering Program, Department of Civil Engineering, Auburn University, Auburn, AL 36849, USA e Collaborative Innovation Center of Water Quality Safety and Protection in Pearl River Delta, Guangzhou University, Guangzhou 510006, China article info abstract

Article history: This study evaluated the effects of synthesized octahedral molecular sieve (OMS-2) nanoparticles on the Received 18 April 2015 anaerobic microbial community in a model digester, expanded granular sludge bed (EGSB) reactor. The Received in revised form addition of OMS-2 (0.025 g/L) in the EGSB reactors resulted in an enhanced operational performance, i.e., 7 September 2015 COD removal and biogas production increased by 4% and 11% respectively, and effluent volatile fatty acid Accepted 11 September 2015 (VFA) decreased by 11% relative to the control group. The Biolog EcoPlate™ test was employed to Available online 14 September 2015 investigate microbial metabolism in the EGSB reactors. Results showed that OMS-2 not only increased the microbial metabolic level but also significantly changed the community level physiological profiling Keywords: Manganese of the microorganisms. The Illumina MiSeq high-throughput sequencing of 16S rRNA gene indicated Nanoparticles OMS-2 enhanced the microbial diversity and altered the community structure. The largest bacterial Lactic acid bacteria genus Lactococcus, a lactic acid bacterium, reduced from 29.3% to 20.4% by abundance in the presence of High-throughput sequencing 0.25 g/L OMS-2, which may be conducive to decreasing the VFA production and increasing the microbial Anaerobic digestion diversity. OMS-2 also increased the quantities of acetogenic bacteria and Archaea, and promoted the Microbial metabolism acetogenesis and methanogenesis. The X-ray photoelectron spectroscopy illustrated that Mn(IV)/Mn(III) with high redox potential in OMS-2 were reduced to Mn(II) in the EGSB reactors; this in turn affected the microbial community. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2015), catalytic combust exhausts (Wang et al., 2015), and remediate contaminated soil (Xie et al., 2014). However, with the Nanoparticles (NPs) have been increasingly applied in industries increasing uses of nanotechnology, more and more man-made NPs such as manufacture, medical devices and diagnostics, construc- are released into our atmosphere, soil, or water environments tion, electronics, and water/wastewater treatment (Meyer et al., during their life-time. Consequently, this has caused a heated 2009; Reinhart et al., 2010; Yang et al., 2013). Their excellent debate on the environmental and health risks of engineered NPs in physiochemical properties can be attributed to their high surface- the environment (Hristozov and Malsch, 2009). to-volume ratio. Among these NPs, metallic/metal oxide NPs have NPs can reach the wastewater treatment plants (WWTPs) been some of the most important materials. In environmental ap- through discharges from industrial plants and from various con- plications, these NPs have been employed to treat wastewater (Luo sumer products. In typical secondary biological wastewater treat- ment processes, most of the NPs are associated with the biomass. For example, a mass balance for metallic silver NPs (Ag-NPs) in a * Corresponding author. Daniel Lab 304, School of Civil and Environmental En- WWTP indicated that more than 95% of the entering Ag-NPs were gineering, 200 Bobby Dodd Way NW, Atlanta, GA 30332, USA. sequestered into the wastewater biomass and removed along with ** Corresponding author. 1 Fangzhi Rd, Wuhan 430073, China. biomass sedimentation from the effluent stream (Shafer et al., E-mail addresses: [email protected] (F. Pan), [email protected] (J. Fu). http://dx.doi.org/10.1016/j.watres.2015.09.022 0043-1354/© 2015 Elsevier Ltd. All rights reserved. 128 F. Pan et al. / Water Research 87 (2015) 127e136

1998). Metallic/metal oxide NPs can affect the microorganisms and microbial ecosystems in WWTPs due to their various nano-toxic effects. The antimicrobial mechanisms of metallic/metal oxide NPs include cell membrane disruption (Lee et al., 2008), release of metal ions (Li et al., 2008), and generation of oxidative stress (e.g., reactive species, ROS) (Xia et al., 2008). Typically, the aerobic biological treatments (e.g., activated sludge and membrane reactor) are the dominant processes in WWTPs. The impacts of metallic/metal oxide NPs on aerobic pro- cesses have been well studied, and the most investigated NPs are Ag-NPs (Choi et al., 2008), nano-ZnO (Zheng et al., 2011), nano-TiO2 (Xia et al., 2006), and nano-zero valent iron (NZVI) (Lee et al., 2008). Due to the growing need for sustainable energy, anaerobic treat- ment has been rapidly developed and applied in past 20 years, of which anaerobic granular sludge (AGS) reactors have been widely used (Rajeshwari et al., 2000). Upflow anaerobic sludge blanket (UASB) and expanded granular sludge bed (EGSB) reactors are the most popular AGS reactors and employed to treat different types of organic wastewater, such as food, beverage, and pulp and paper industry wastewater (van Lier, 2008). Recent research found that NZVI can increase the rate of anaerobic granulation and organic removal in a UASB reactor treating coking wastewater (Liu et al., 2011). Mu and coworkers indicated a high dose of nano-ZnO (>100 mg/g-TSS) decreased the productions of extracellular poly- Fig. 1. Schematic diagram of the expanded granular sludge bed (EGSB) reactor device. 1 feed tank, 2 peristaltic pump, 3 reactor, 4 circulating pump, 5 water bath tank, 6 re- meric substances (EPS) and methane in the UASB reactor (Mu et al., circulating pump, 7 sampling ports, 8 water-sealed bottle, and 9 wet gas flow meter. 2012). However, the impacts of diverse NPs on anaerobic digestion are still largely unknown and the effects of metallic/metal oxide NPs on the anaerobic microbial metabolism and communities shows the schematic diagram of an EGSB reactor. The reactor was remain unexplored. made up of a plexiglass column with an internal diameter of The octahedral molecular sieve (OMS-2) is a form of manganese 70 mm, an overall height of 850 mm, and an effective volume of dioxide having a tunnel size of 0.46 nm 0.46 nm (Ding et al., 3.6 L. The exterior of the reaction zone was coated with a water 2005). OMS-2 NPs exhibit unique features like mixed-valence of jacket and the top of the reactor was equipped with a settling manganese (Mn) and acidic sites, and have extensive applications chamber and a three-phase separator. The EGSB reactor was (Luo et al., 2015; Suib, 2008). However, the potential health risks operated under the mesophilic conditions (approximately 35 C) and environmental impacts of OMS-2 are unknown. The dissimi- and its was maintained by circulating the warm water latory Mn reduction is known to promote the growth of several through the reactor jacket. The influent wastewater entered the microorganisms in anaerobic environment (Nealson and Saffarini, reactor from the bottom via a peristaltic pump. A part of the 1994). The redox circle between different Mn species in OMS-2 effluent returned to the bottom of the reactor by a re-circulating may provide some benefits for the anaerobic microbial community. pump and the other part was discharged through the overflow. A This study aimed to test the effect of synthetic OMS-2 NPs on the liquid upflow velocity of 3.0 m/h was maintained. The produced gas operational performance of the EGSB reactor by evaluating the was separated by the three-phase separator and determined by a chemical oxygen demand (COD) removal, biogas production, and wet gas flow meter. the effluent volatile fatty acid (VFA) . We employed the Biolog EcoPlate™ test (Garland and Mills, 1991) to distinguish 2.3. Inoculated sludge and synthetic wastewater spatial and temporal changes in microbial metabolism. The high- throughput microbial community analysis was further carried out The inoculated sludge, taken from the anaerobic digester of the on the Illumina MiSeq platform. The information will aid in our sewage treatment plant in Wuhan Textile University (Wuhan, understanding of the NPs' effects and the action mechanisms on China), had a grain size of 0.6e2.0 mm, volatile suspended solids anaerobic microorganisms and the digestion process. (VSS) of 7.4 g/L, and suspended solids (SS) of 11.75 g/L. The influent organic wastewater was synthesized using the following stock so- 2. Materials and methods lution: sucrose 90 g/L, NH4HCO3 14.1 g/L, KH2PO4 0.97 g/L, K2HPO4$3H2O 1.62 g/L, and NaHCO3 33.3 g/L. The chemical oxygen 2.1. Preparation of OMS-2 demand (COD) of the stock wastewater was approximately 100 g/L.

OMS-2 was synthesized by a reflux method, which has been 2.4. Operation scheme reported elsewhere (Luo et al., 2015). The details in synthesis and characterization of OMS-2 are described in Text S1 and Fig. S1 of Before experiments, the stock wastewater was diluted to the Supporting information (SI). desired concentration of COD and appropriate amounts of trace elements were added, including the following chemicals (all in mg/ 2.2. Experimental setup L): H3BO3 100, CuSO4$5H2O 58, ZnSO4$7H2O 100, NiSO4$6H2O112, MgSO4$7H2O 5000, FeSO4$7H2O 750, yeast 3000, MnSO4$H2O 188, Four groups of EGSB reactors (G1eG4, each group had two NaMo7O24$2H2O 50, CaCl2$2H2O 7500, and FeSO4$7H2O 50. In the duplicate reactors) were employed, of which G1 was the control initial operation stage, reactor pH was adjusted to 7.0e8.0 using group without the addition of OMS-2 NPs and G2eG4 were the NaHCO3. During the operation of the reactors, COD of both influents treatment groups with the addition of variant dose of OMS-2. Fig. 1 and effluents were monitored following the standard method. The F. Pan et al. / Water Research 87 (2015) 127e136 129 volatile fatty acid (VFA) of effluents was analyzed using acid-base abundance statistics, cluster analysis, and principal coordinates titration method. analysis (PCoA) were carried out at the genus level based on the The reactors were operated in 3 stages, i.e., startup (0e61 d), taxonomic and abundance information of OTUs. The detailed in- organic loading increase (62e113 d), and steady operation formation on DNA extraction, PCR amplification, DNA library con- (114e189 d). In the startup operation, 4 groups of EGSB reactors struction and sequencing, and sequences data preparation and were initially (0 d) operated under the COD loading rate of analysis are shown in Text S3 of SI. 2500e3000 g/m3$d, which was then gradually increased to 4200 g/ m3$d. This stage was for the sludge acclimation and the granular 2.7. Statistical analysis sludge emergence. On Day 62, OMS-2 NPs of 0.025, 0.25, and 2.50 g/ L were added into G2eG4 reactors, respectively, however G1, the Bicluster analysis was conducted with the pheatmap package in control group, was not spiked with OMS-2. During the organic R software. Unifrac PCoA was conducted using the Qiime software. loading increase stage, the COD loading rate was slowly increased Alpha diversity analysis was conducted using the Mothur software. to the maximum value of 7500 g/m3$d, which revealed an optimum Principal component analysis (PCA) was conducted with SPSS 19.0 COD loading rate of 4500 g/m3$d. Therefore, the steady operation for Windows (IBM Corporation, Armonk, NY, USA). Statistically was carried out under this COD loading rate. During the steady significant differences between two groups were determined using stage, the granular sludge of >1.4 mm and >2 mm accounted for the metastats order in Mothur. For multiple comparisons, analysis 85.0e87.2%, and 70.6e73.8%, respectively (Fig. S2, SI). The VSS of of variance (ANOVA) followed by Dunnett's test and least signifi- the sludge were 26.47, 27.12, 27.03 and 26.63 g/L, respectively, in cant difference (LSD) multiple comparison were performed using the G1eG4 reactors. SPSS. A value of P < 0.05 was considered significant. Normality and On Day 182, the mixed liquors samples were collected from the homogeneity of the data were checked using the ShapiroeWilk and 4 groups of EGSB reactors and then subjected to morphology Levene tests, respectively, before ANOVA. characterization by an Ultra plus field emission scanning electron microscope (SEM) (Carl Zeiss, Oberkochen, Germany), X-ray 3. Results and discussion photoelectron spectroscopy of OMS-2 by an ESCALab220i-XL elec- tron spectrometer (Thermo VG Scientific, East Grinstead, West 3.1. Operation performance of EGSB reactors Sussex, England), the community level physiological profiling (CLPP) of the microorganisms with the Biolog EcoPlate™ (Biolog The COD removal efficiency, effluent VFA, biogas production Inc., CA, USA), and microbial genera identification by Illumina rate, and COD balances of EGSB reactors in the steady operation MiSeq high-throughput sequencing. stage are summarized in Table 1. The average COD removal in G1eG4 reactors was 89.63 ± 1.69%, 93.28 ± 1.87%, 92.47 ± 1.31% and 2.5. Microbial CLPP determination 91.54 ± 1.16%, respectively; the average effluent VFA was 5.04 ± 0.63, 4.49 ± 0.41, 4.50 ± 0.36 and 4.70 ± 0.42 mmol/L; and Biolog EcoPlate™ technique uses carbon source substrates the biogas production rate was 2.20 ± 0.37, 2.44 ± 0.23, 2.43 ± 0.33 distributed in a 96-well plate to evaluate the microbial physiolog- and 2.30 ± 0.40 L/L$d; the calculated COD recovery was 95.6%, ical metabolic characteristics. Each Biolog EcoPlate contains three 101.3%, 101.8% and 97.8%, respectively. These data indicate the EGSB sets of 31 different carbon sources (including 2 amines/amides, 6 reactors were in good condition for digesting the wastewater. amino acids, 7 carbohydrates, 9 carboxylic acids, 3 miscellaneous, The addition of OMS-2 enhanced the COD removal efficiency as and 4 polymers) (Table S1, SI). The procedure for Biolog EcoPlate™ well as the biogas production rate, with the greatest enhancement assay is described in Text S2 of SI. occurring at the lowest dose (0.025 g/L). Furthermore, the addition of OMS-2 lowered the VFA contents in the effluent. The accumu- 2.6. Illumina high-throughput sequencing lation of the VFA produced by acidogenic bacteria is known to negatively influence the treatment efficiency of EGSB digestion by The mixed liquors samples were subjected to DNA extraction impacting methanogenesis (Zhang et al., 2008). using E.Z.N.A. Soil DNA kits (Omega Bio-Tek, Norcross, GA, USA). The effects of metallic/metal oxide NPs on anaerobic reactors are The V4 region of 16S rRNA genes was amplified using the following diverse. Researchers found nano-CuO and nano-ZnO can inhibit primers: Forward 50-AYTGGGYDTAAAGNG-30 and Reverse 50- biogas and methane production during anaerobic digestion of TACNVGGGTATCTAATCC-3'. The extracted DNA fragments and cattle manure (Luna-delRisco et al., 2011). Ag-NPs were found to adapters with tags were incubated and connected by the ligase, and have negligible impact on anaerobic digestion and methanogenic then enriched by PCR to amplify the DNA library. The DNA library assemblages because of little to no silver ion release (Yang et al., (10 nM) was progressively and quantitatively diluted to 4e5pM 2012). On the contrary, under illumination nano-TiO2 can and subjected to the DNA sequencing on an Illumina MiSeq ma- enhance gas production in the fermentation of waste chine with 2 250 bp reads. activated sludge (WAS), which was attributed to the ability of nano- The obtained sequence fragments were assembled using the TiO2 to facilitate the removal of organic matter, promote the growth Flash software (http://www.genomics.jhu.edu/software/FLASH/ of photosynthetic bacteria, and inhibit the activity of hydrogen- index.shtml). The valid sequences were extracted according to the uptake enzymes (Zhao and Chen, 2011). index information. The Qiime (Caporaso et al., 2010) (version 1.7.0, http://qiime.org/) and Mothur (Schloss et al., 2009) (version 1.31.2, 3.2. Microbial metabolism http://www.mothur.org/) were used to obtain high-quality se- quences for subsequent analysis. The high-quality sequences were The average well color development (AWCD) of the Biolog classified to operational taxonomic units (OTUs) using Qiime soft- EcoPlate was used to represent the average microbial metabolic ware. The taxonomic information of each OTU was obtained by activity (Choi and Dobbs, 1999). Fig. 2a shows the AWCD changes comparing the sequences database following the blast method in over time for the 4 groups of EGSB reactors. The AWCD curves show Qiime (Altschul et al., 1990). The rarefaction curve was constructed a similar pattern for the 4 cases. The microbial activity in the initial by random sampling of the sequences, and the rank abundance 24 h was very low, but increased quickly thereafter. After 120 h, the curve was drawn to reflect the species distribution pattern. Species microbial activity reached a relatively stable phase. The microbial 130 F. Pan et al. / Water Research 87 (2015) 127e136

Table 1 COD removal efficiency, effluent VFA, biogas production rate and COD balances in steady operation stage.

G1 G2 G3 G4

** ** ** COD removal efficiency (%) 89.63 ± 1.69 93.28 ± 1.87 92.47 ± 1.31 91.54 ± 1.16 ** ** VFA (mmol/L) 5.04 ± 0.63 4.49 ± 0.41 4.50 ± 0.36 4.70 ± 0.42 * Biogas production rate (L/L$d) 2.20 ± 0.37 2.44 ± 0.23 2.43 ± 0.33 2.30 ± 0.40 COD balance Influent (A) (g/L$d) 4.5 4.5 4.5 4.5 Effluent (B) (g/L$d) 0.47 0.30 0.34 0.38 Biogas (Ca) (g/L$d) 3.84 4.26 4.25 4.02 B þ C (g/L$d) 4.31 4.56 4.58 4.40 Recovery (B þ C)/A (%) 95.6 101.3 101.8 97.8

* ** P < 0.05, P < 0.01, significant differences were compared with G1 (n ¼ 14). a Calculated by taking into account 70% methane content (V/V) in biogas. metabolic activity of G2eG4 was notably higher than that of G1, metabolic level with G3, G2 was not subjected to sequencing). indicating OMS-2 played a favorable role in promoting microbial Good's coverage is an estimator, which estimates the probability metabolism. This accounts for the enhanced COD removal by the that the next read will belong to a found OTU. The high Good's addition of OMS-2 in the EGSB reactors (Table 1). The microbial coverage for three samples indicated the good sequencing depth metabolic levels in G2 and G3 were similar, while that in G4 was (Table 2). The microbial phylotype richness levels were reflected lowered, suggesting the high dose of OMS-2 may inhibit the mi- by using the Chao1/Ace estimator, and the Simpson/Shannon crobial metabolism. diversity index (Table 2), which revealed that the G3 sample had There is no significant difference in VSS among the EGSB re- the highest microbial diversity of the three samples. The distri- actors, suggesting the microbial biomass in G1eG4 was compara- bution of sequences and OTUs, rarefaction curves, and rank ble. Thus, the enhanced microbial metabolism in G2eG4 may be abundance curves also illustrated a much higher microbial di- attributed to alteration of microbial community. In order to study versity in the G3 sample (Text S4, Figs. S3, S4, SI). PCoA indicated the impact of OMS-2 on the microbial CLPP, the optical density (OD) that the microbial communities in the three samples differed data of 31 carbon sources obtained from Biolog EcoPlate assay significantly, and that different of OMS-2 had during 168 h of incubation (n ¼ 7) were normalized by the AWCD of different impacts on the microbial community in EGSB reactors each microplate to remove inoculum density effects and subjected (Text S4, Fig. S5, SI). to PCA (Garland, 1997). Two principal components (PC1 and PC2) The identified microbes consisted mainly of 2 domains, i.e., were extracted, and Fig. 2bee shows carbon sources loading to PC1 Bacteria and Archaea. The Archaea play a unique role in methano- and PC2, which reflects the metabolic characteristics of the mi- genesis by converting hydrogen gas, carbon dioxide, and acetic acid crobial communities. The assembled carbon sources imply a similar to methane in an anaerobic environment (Pimentel et al., 2012). consumption pattern by the microorganisms. The distributions of The addition of OMS-2 significantly elevated the Archaea abun- the carbon sources are evidently different between G1 and G2eG4. dance (Fig. 3a), which corresponded to the biogas production rate In G1, the carbon sources are mainly in the upper left and lower (Table 1), i.e., OMS-2 promoted the production of biogas (mainly right areas, while in G2eG4, the carbon sources are mostly located methane). in the upper right and lower left regions. Evidently, the addition of Besides the Archaea, the microbial community was dominated OMS-2 greatly changed the microbial communities. by Bacteria (abundance >99.5%) in the EGSB reactors, which were Generally, a highly-utilized substrate has a higher positive mainly assigned to 19 phyla (abundance 0.1%) (Fig. 3b). OMS-2 loading on PC1, while those of lower utilization show a lower suppressed most of the phyla in a dose-dependent manner, positive or negative loading on PC1 (Fig. 2bee, Tables S2 & S3, SI). including Acidobacteria, Actinobacteria, BRC1, Chloroflexi, Firmicutes, The microbes in G1 preferred to utilize amino acids (A4, B4, D4 and Fusobacteria, Gemmatimonadetes, OD1, Planctomycetes, Spirochaetes, F4), carbohydrates (C2, D2, E2 and G1), and D-malic acid (H3), while Tenericutes, WPS-2 and WS3. However, OMS-2 promoted the rich- showed a relatively weaker utilization of amines/amides (G4 and ness of Bacteroidetes, Nitrospirae, and Verrucomicrobia in a dose- H4), polymers (E1 and F1), carboxylic acids (C3, E3, F3 and G3), dependent manner. For Cyanobacteria and Proteobacteria, a low some carbohydrates (A2, B2 and H1), and D,L-a-glycerol-phosphate dose of OMS-2 increased the richness while a high dose of OMS-2 (H2) (Fig. 2b, Table S3, SI). The addition of proper amounts of OMS-2 reduced the richness. (0.025 g/L and 0.25 g/L) noticeably increased the microbial utili- Firmicutes represent the most abundant phylum, accounting for zation capacity on amines/amides (G4 and H4), carboxylic acids over 35% of the species richness. Of the Firmicutes, Lactobacillales (B3, C3, D3, F2, F3, and H3), Tween 40 (C1), pyruvic acid methyl and Clostridiales dominated the assigned orders. OMS-2 distinctly (B1), D-xylose (B2), and L-phenylalanine (C4) (Fig. 2c, d, reduced the abundance of Lactobacillales, yet promoted Clostridiales Table S3, SI). In other words, OMS-2 promoted the types of mi- (Fig. 3c). The Lactobacillales or lactic acid bacteria (LAB) are a clade crobes that metabolize the listed substances. However, increasing of bacteria that are commonly found in decomposing plants and the OMS-2 dose to 2.5 g/L decreased the microbial utilization ca- milk products, and produce lactic acid as the major metabolic end pacity on amines/amides (G4 and H4), amino acids (B4, C4 and F4), product of carbohydrate fermentation (Salvetti et al., 2013). The carbohydrates (B2, E2 and G1), carboxylic acids (C3 and H3), and accumulation of lactic acid inhibits the growth of other microor- pyruvic acid methyl ester (B1) compared to that in G1 and G2 ganisms. The inhibition on the LAB by OMS-2 may pose positive (Fig. 2e, Table S3, SI). Thus, higher doses of OMS-2 might cause an effects on the enhancement of microbial diversity (Table 2) and the inhibitory effect on the growth of the corresponding microbes. alleviation of VFA accumulation (Table 1) in the EGSB reactors. Many species in the order Clostridiales, such as Phascolarctobacte- 3.3. Microbial community analysis rium spp., Christensenellaceae fam. spp., and Clostridium spp., have relatively strong metabolic activity for carbohydrates, amino acids Samples collected from G1, G3 and G4 were subjected to and carboxylic acids (Chou et al., 2011; Morotomi et al., 2012; Illumina MiSeq high-throughput sequencing (Due to the similar Watanabe et al., 2012). The elevation of these bacteria by OMS-2 F. Pan et al. / Water Research 87 (2015) 127e136 131

Fig. 2. Biology EcoPlate assay results: (a) Average well color development (AWCD), and (b)e(e) principal component analysis (PCA) on microbial metabolism. The details on the substrate designation are given in Table S1 (SI).

was favorable for the improvement of the organics removal of the Proteobacteria is complex and should be discussed at the genus EGSB reactors (Table 1). level (see below). The Proteobacteria are a large group of bacteria and the main The Bacteroidetes are well known degraders of polymeric orders include Caulobacterales, Rhodospirillales, Burkholderiales, organic matter (Thomas et al., 2011). Exposure to OMS-2 greatly Desulfovibrionales, Enterobacteriales and Pseudomonadales (Fig. 3c). increased the richness of Bacteroidetes, which is corresponded to Due to the wide variety of metabolism, the effect of OMS-2 on the above microbial CLPP, i.e., the addition of OMS-2 enhanced the 132 F. Pan et al. / Water Research 87 (2015) 127e136

Table 2 can inhibit the growth of other microorganisms by secreting Alpha richness and diversity estimators of the bacteria phylotype in G1, G3 and G4 various antibiotics (Ventura et al., 2007). Thus the inhibition of samples. Actinobacteria may be beneficial for the growth of other bacteria. In Group Chao1a Acea Simpsonb Shannonb Coveragec addition, some of the Acidobacteria are acidophilic, and their G1 (Control) 2737 2837 0.052 4.62 0.987 abundance is associated with the acidic environment (Naether G3 (OMS-2, 0.25 g/L) 3327 3684 0.076 4.77 0.985 et al., 2012). The declining trend of Acidobacteria abundance indi- G4 (OMS-2, 2.5 g/L) 2996 2808 0.047 4.41 0.987 cated that the groups exposed to OMS-2 were less acidic, which a Chao1/Ace richness estimator: the total number of OTUs estimated by infinite agrees with the lowered effluent VFA content and LAB abundance sampling. A higher number indicates higher richness. (Table 1, Fig. 3c). b Simpson/Shannon diversity index: an index to characterize species diversity. A The microbial community was further investigated at the genus higher value represents more diversity. c Good's coverage: estimated probability that the next read will belong to an OTU level, where a heat-map was employed to describe the microbial that has already been found. compositions in G1, G3 and G4 samples. As shown in Fig. 3d, 73 bacterial genera were identified at an abundance level of 0.2%. Based on the biclustering, the distribution of bacterial genera was microbial ability to metabolize polymers (Fig. 2). The major genus relatively similar in G3 and G4, but different from the control group in phylum Verrucomicrobia is Akkermansia, which also showed good (G1). The classified genera can be divided into two branches based degrading ability on macromolecular substances, such as glyco- on the species richness: B1eB45 (Low abundance, LA) and conjugates (Derrien et al., 2004). The increase in Verrucomicrobia B46eB73 (High abundance, HA). In the LA branch, the abundance of abundance by OMS-2 favored the overall microbial metabolism of genera B3eB15 increased with OMS-2 dose, while B19eB45 organic substrates. exhibited the opposite trend. For B1, B2, B16eB18, a low dose of The bacterial phyla which were inhibited by OMS-2, including OMS-2 (0.25 g/L) elevated the bacterial abundance; however, a high Acidobacteria, Actinobacteria, Chloroflexi, Fusobacteria, Gemmati- dose of OMS-2 (2.5 g/L) inhibited an increase in species richness. In monadetes, Spirochaetes, and Tenericutes, showed relatively low the HA branch, B46eB49 were the most abundant species, followed fermenting capacity for organic substrates. Thus, the decrease in by B69eB73. OMS-2 apparently inhibited B50eB54, but promoted their abundance might pose a positive effect on the fermentation of B63eB68. The abundances of B55eB62 kept relatively stable with organics for the whole microbial community. The Actinobacteria the addition of OMS-2.

Fig. 3. Microbial community analysis in G1, G3 and G4 EGSB reactors. (a) Distribution of domains Bacteria and Archaea. (b) Distribution of bacterial phyla (abundance 0.1%). (c) Distribution of bacterial orders (abundance 1%). (d) Heat-map of the classified genera (abundance 0.2%). (e) Distribution of bacterial trophic types. (f) Distribution of acetogenic bacteria. (g) Most sensitive/resistant bacterial genera in G3 and G4 samples. The counted bacteria genera for (e)e(g) were B1eB73 shown in (d). F. Pan et al. / Water Research 87 (2015) 127e136 133

With respect to taxonomy, these bacteria (B1eB73) came from function of Akkermansia spp. population in the biofilm. Our results 12 phyla with most belonging to Firmicutes (23 genera) and Pro- showed the increase of Akkermansia spp. population may enhance teobacteria (21 genera) (Fig. S6, SI). Based on the metabolic types of the microbial metabolism of sucrose in EGSB reactors. The genus typical species in anaerobic and dark environment, these bacteria from the family S24-7 in the phylum Bacteroidetes also showed an can be divided into chemolithotrophs and chemoorganotrophs increase in abundance. It is well known that the members of (Fig. 3e). At a low dose of OMS-2, chemolithotrophic bacteria phylum Bacteroidetes are degraders of polymeric organic matter. largely increased, while at a high dose of OMS-2 these bacteria Therefore, the increase of S24-7 gen. spp. is beneficial for the showed a declining trend. For chemoorganotrophic bacteria, the enhancement of microbial metabolism of organic substrates. The effect of OMS-2 was the opposite, i.e., a low dose of OMS-2 other genera that showed an increasing trend in abundance come decreased the bacterial abundance while a high dose increased from either Firmicutes or Proteobacteria (Fig. 3g). the abundance. The increase in chemolithotrophic bacteria has The sensitive genera were all from either the phyla Firmicutes or many benefits for anaerobic digestion and enhances the efficiency Proteobacteria, with the most sensitive genera being Lactococcus and stability of EGSB reactors. These bacteria can utilize various and Comamonas (Fig. 3g). Lactococcus spp. are typical LAB that fermentation products as reducing equivalents (e.g. ammonia and produce lactic acid as the major or only product of glucose sulfur compounds) or as carbon sources to fix carbon (e.g. carbon fermentation (Oliveira et al., 2005), and their inhibition allowed for dioxide and small organic acids) (Fernandez et al., 2008; Mekjinda the growth of other microorganisms and increase of the microbial and Ritchie, 2015). This can explain the Biolog EcoPlate™ results diversity. The Comamonas can break down a wide variety of organic which portray that a low dose of OMS-2 (0.025 g/L and 0.25 g/L) acids, but not metabolize carbohydrates (Willems and De Vos, noticeably increased the microbial utilization capacity of small 2006). Thus, the reduction of Comamonas did not impact the mi- molecular substrates, such as carboxylic acids and pyruvic acid crobial metabolic ability on carbohydrates in EGSB reactors. methyl ester (Fig. 2). In particular, the acetogens (acetogenic bac- teria) increased with the addition of OMS-2 (Fig. 3f), which pro- 3.4. Mechanism discussion moted acetogenesis. The chemoorganotrophic bacteria are the most abundant and Based on the above results, we can see that the addition of OMS- play a critical role in hydrolysis and acidogenesis. Fig. 3g shows the 2 NPs can affect EGSB reactors, resulting in an enhanced treatment ten genera from G3 and G4 that displayed either the largest in- performance (i.e. higher COD removal and biogas production, and crease or decrease in abundance. Of the resistant genera, Akker- lower VFA production, Table 1), an enhanced microbial metabolism mansia had the largest abundance increase of 6.6% and 14.7% in G3 (Fig. 2), and a higher microbial diversity (Table 2). These improve- and G4 samples, respectively. Akkermansia is in the phylum Ver- ments in EGSB reactors may be caused by the alteration of micro- rucomicrobia and was first proposed in 2004 with the type species bial community via the modulation of OMS-2 (Fig. 3): (1) The Akkermansia muciniphila, which showed a strong mucin-degrading competing bacteria (e.g., Lactobacillales and Actinobacteria)were ability (Derrien et al., 2004). So far, no study has reported the inhibited, contributing to the reduction of VFA production and

Fig. 4. Changes of OMS-2 before and after EGSB experiments: (a) SEM image of fresh OMS-2, (b) SEM image of used OMS-2, (c) XPS spectra of fresh OMS-2, and (d) XPS spectra of used OMS-2. 134 F. Pan et al. / Water Research 87 (2015) 127e136 increase of microbial diversity; (2) The bacteria with strong of OMS-2 were measured before and after the EGSB experiments. degrading ability (e.g., Akkermansia and Bacteroidetes) were pro- For Mn 2p3/2, two peaks centered at 642.9 and 641.8 eV were ob- moted, and those of relatively lower metabolic ability (e.g. Acid- tained in the fresh OMS-2 NPs with a peak area ratio of 67.5:32.5 obacteria and Actinobacteria) were suppressed which elevated the (Fig. 4c), which were assigned to Mn(IV) and Mn(III) oxidation overall microbial metabolic capacity; (3) The increase of chemo- states, respectively (Luo et al., 2015). After the EGSB experiments, lithotrophic bacteria, especially acetogens, accelerated the con- the two Mn species were still present in the OMS-2 NPs and sumption of fermentation products (e.g., ammonia and VFA) and another peak ascribed to the Mn(II) oxidation state was detected; acetogenesis; and (4) The Archaea abundance was enhanced, which their atomic ratio was 33.3:33.6:33.1 (Fig. 4d). The XPS results promoted the methanogenesis. showed that the Mn(IV)/Mn(III) with high redox potential were The modulation on the microbial community by OMS-2 is reduced to Mn(II) during the digestion process, which provided possibly associated with the function of manganese oxide as the large reducing equivalents to the anaerobic microorganisms. extracellular electron accepter. OMS-2 contains various oxidation Dissimilatory Mn(IV)-reducing microorganisms can be sepa- states of manganese such as Mn(II), Mn(III) and Mn(IV) (Suib, rated into two major groups: those that support growth by 2008). It is well known that a wide phylogenetic diversity of mi- conserving energy from electron transfer to Mn(IV) (known as croorganisms are capable of dissimilatory Mn(IV) reduction, i.e., Mn(IV)-respiring microorganisms, FMR) and those that do not microorganisms transfer electrons to external Mn(IV), reducing it (Non-FMR). FMR are phylogenetically dispersed throughout the to Mn(II) without assimilating the manganese (Lovley, 2006). Bacteria and Archaea, and grow by oxidizing organic compounds, Mn(IV)/Mn(III) oxides are relatively stable and insoluble, while hydrogen or S0 with the reduction of Mn(IV) (Lovley et al., 2004). Mn(II) oxide can be solubilized in acid. An examination of the SEM The addition of OMS-2 into the EGSB reactors can greatly promote images of OMS-2 before and after the experiments revealed that the FMR growth. On the other hand, although Non-FMR do not gain some of the nanofibers became bent, and heads became irregularly energy from Mn(IV) reduction, the terminal electron-accepting (Fig. 4a, b), indicating OMS-2 was partially solubilized. To further process (TEAP) of Mn(IV) reduction can impact the metabolic investigate the changes of Mn species in the OMS-2, the XPS spectra process. For example, many Lactococcus spp. are fermentative

Fig. 5. Schematic plot of action mechanisms of OMS-2 on the microbial community and performance of EGSB reactors. F. Pan et al. / Water Research 87 (2015) 127e136 135

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