Journal of Environmental Science and Health, Part A (2015) 50, 913–921 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2015.1030279
Effects of titanium dioxide and zinc oxide nanoparticles on methane production from anaerobic co-digestion of primary and excess sludge XIONG ZHENG, LIJUAN WU, YINGUANG CHEN, YINGLONG SU, RUI WAN, KUN LIU and HAINING HUANG State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, Shanghai, China
Anaerobic co-digestion of primary and excess sludge is regarded as an efficient way to reuse sludge organic matter to produce methane. In this study, short-term and long-term exposure experiments were conducted to investigate the possible effects of titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) on methane production from anaerobic co-digestion of primary and excess sludge. The data showed that TiO2 NPs had no measurable impact on methane production, even at a high concentration (150 mg/g total suspended solids (TSS)). However, short-term (8 days) exposure to 30 or 150 mg/g-TSS of ZnO NPs significantly decreased methane production. More importantly, these negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time to 105 days. Further studies indicated that the presence of ZnO NPs substantially decreased the abundance of methanogenic archaea, which reduced methane production. Meanwhile, the activities of some key enzymes involved in methane production, such as protease, acetate kinase, and coenzyme F420, were remarkably inhibited by the presence of ZnO NPs, which was also an important reason for the decreased methane production. These results provide a better understanding of the potential risks of TiO2 and ZnO NPs to methane production from anaerobic sludge co-digestion. Keywords: Engineered nanoparticles, enzyme activity, inhibitory effect, methanogenic archaea, primary and excess sludge.
Introduction With the rapid development of nanotechnology, engineered nanomaterials have been widely used in various applications because of their unique physicochemical properties.[1–3] For example, titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) are increasingly used in many products, such as sunscreens, cosmetics, and bottle coatings.[4] The extensive production and application of these nanomaterials inevitably result in their release into the environment, which raises concerns about their potential impacts on human health and ecosystems.[5] Although the current environmental concentrations of nanomaterials are still low,[6] their release into the environment could continue to increase owing to their large-scale
Address correspondence to Yinguang Chen, State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China; E-mail: yg2chen@yahoo. com Received November 7, 2014.
production. It is well-known that wastewater treatment plants (WWTPs) play important roles in pollutant removal and water purification throughout the world. However, recent studies found that WWTPs are becoming substantial reservoirs of large numbers of engineered nanomaterials, such as TiO2 and ZnO NPs.[7] Therefore, it is necessary to consider the potential risks of these nanomaterials to WWTPs. The activated sludge process is the most widely used method for treating wastewater, especially municipal wastewater, in WWTPs. However, this process generates large amounts of primary and excess sludge, which should be treated to avoid contaminating the environment.[8] In recent years, sludge disposal using landfills or incineration has faced increasing public opposition because of stringent environmental control regulations and the scarcity of land. Anaerobic digestion is regarded as a good alternative, as it treats sludge as a valuable resource and can convert sludge organics into methane.[9] Usually, primary sludge has a high carbohydrates concentration, while excess sludge has a high protein content. A previous study reported that anaerobic co-digestion of primary and excess sludge could achieve a better carbon to nitrogen ratio, thus improving the treatment of sludge
914 organics by anaerobic microorganisms.[10] However, it is still unclear whether the presence of nanomaterials can affect the anaerobic sludge co-digestion process. Previous studies have found that the adsorption of activated sludge is one of the most important mechanisms for pollutant removal.[11] Hence, the majority of nanomaterials in influents were observed to be adsorbed on the sludge surface,[12,13] which resulted in the accumulation of much higher concentrations of nanomaterials in biosolids.[14,15] Additionally, the presence of ZnO NPs significantly affected sludge microorganisms, thereby substantially inhibiting wastewater nitrogen and phosphorus removal.[16] Although TiO2 NPs had no negative influences on wastewater treatment after short-term exposure, prolonged exposure to this nanomaterial greatly suppressed biological nitrogen removal from wastewater.[17] Nevertheless, to date, the short-term and long-term effects of TiO2 and ZnO NPs on methane production from anaerobic sludge co-digestion are still unknown. The aim of this study was to evaluate the short-term and long-term effects of TiO2 and ZnO NPs on methane production from the anaerobic co-digestion of primary and excess sludge. First, a scanning electron microscopy (SEM) analysis and the lactate dehydrogenase (LDH) release assay were used to determine whether sludge surface integrity was affected by the presence of these NPs. Then, both short-term and long-term exposure experiments were conducted to investigate the potential impacts of TiO2 and ZnO NPs on methane production from anaerobic sludge co-digestion at the concentrations of 6, 30, and 150 mg g¡1-total suspended solids (TSS). Because methane production is relevant to sludge solubilization, hydrolysis, acidification, and methanation, the effects of these NPs on the abundances of key microorganisms (mainly bacteria and archaea) were studied. Finally, the activities of key enzymes involved in methane production were measured to explore the possible mechanisms for the NPsinduced effects on anaerobic sludge co-digestion.
Materials and methods Characteristics of primary and excess sludge Primary and excess sludge were obtained from the primary and secondary sedimentation tanks, respectively, of a traditional WWTP in Shanghai, China. The sludge was concentrated via settling at 4 C for 24 h before conducting the experiments. The main characteristics of both primary and excess sludge after settlement are shown in Table 1. Primary and excess sludge were mixed at a 1:1 ratio (volatile suspended solids; VSS) to conduct the anaerobic sludge co-digestion.
Engineered nanoparticles and their stock suspensions In this study, commercial TiO2 (anatase, purity > 99.7%) and ZnO (wurtzite, purity > 99.5%) NPs were
Zheng et al. purchased from Sigma-Aldrich (St. Louis, MO, USA). X-ray diffraction analysis was conducted using a D/ Max-RB diffractometer (Rigaku, Tokyo, Japan) equipped with a rotating anode and a Cu Ka radiation source, as shown in Figure A1 (Appendix). By measuring nitrogen adsorption at 77 K using a Tristar 3000 analyzer (Micromeritics, Norcross, GA, USA), the specific surface areas of the TiO2 and ZnO NPs were determined to be 102 § 6 and 35 § 4 m2/g, respectively. Before the experiment, stock suspensions (2000 mg L¡1) were prepared by adding 2000 mg of NPs to 1 L of deionized water (pH 7.0), followed by 1 h of ultrasonication (25 C, 250 W) to disperse the NPs.[18] The primary sizes of the TiO2 and ZnO NPs in the stock suspensions were in the ranges of 150–170 and 120–140 nm, respectively, as determined by dynamic light scattering using an Autosizer 4700 (Malvern Instruments, Malvern, UK).
Short-term and long-term exposure experiments A previous study reported that the titanium and zinc contents of biosolids in WWTPs (84 in total) ranged from 0.02 to 7.02 and 0.22 to 8.55 mg/g-TSS, respectively.[19] Hence, in this study, 6 mg/g-TSS was chosen as the environmentally relevant concentration of TiO2 or ZnO NPs. Because the release of these NPs into the environment would be expected to increase during large-scale production, the potential risks of higher NP concentrations (30 and 150 mg/g-TSS) were also considered. First, batch experiments (18 d) were conducted to determine the short-term effects of TiO2 and ZnO NPs on anaerobic sludge codigestion. Briefly, 0, 6, 30, or 150 mL of NPs stock suspensions (2000 mg L¡1) were fed into several serum bottles, each containing 140 mL of mixed sludge (approximately 15 g L¡1 of TSS). Then, deionized water was added to each bottle to adjust the final volume to 300 mL, resulting in final NP concentrations of 0, 6, 30, or 150 mg g¡1-TSS. A bottle to which TiO2 or ZnO NPs were not added was used as the control. All bottles were flushed with nitrogen gas for 5 min to remove oxygen, capped with rubber stoppers, sealed, and placed in an air-bath shaker (150 rpm) at 35 § 1 C. To assess the long-term effects of TiO2 and ZnO NPs on anaerobic sludge co-digestion, serum bottles containing 0, 6, 30, or 150 mg/g-TSS of TiO2 or ZnO NPs were flushed with nitrogen gas, capped with rubber stoppers, and shaken in an air-bath shaker (150 rpm, 35 § 1 C) for 105 d. Every day, 15 mL of fermentation mixture was manually withdrawn from each bottle, and the same amount of raw mixed sludge and NPs were supplemented, which resulted in a sludge retention time of 20 d. During the exposure time, methane production was measured every 3 d to evaluate the possible effects of these NPs on anaerobic sludge co-digestion.
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TiO2 and ZnO nanoparticle influence on methane production Table 1. Characteristics of the primary and excess sludge used in this study. Parameter pH Total suspended solids (TSS) (mg/L) Volatile suspended solids (VSS) (mg/L) Total chemical oxygen demand (TCOD) (mg/L) Soluble chemical oxygen demand (SCOD) (mg/L) Total carbohydrate (mg COD/L) Total protein (mg COD/L)
Scanning electron microscopy The sludge surface morphology in the absence and presence of TiO2 and ZnO NPs was observed by SEM. Briefly, 50 mL of fermentation mixture was withdrawn from each bottle at the end of the exposure experiments (18 d for short-term exposure and 105 d for long-term exposure, respectively). These mixtures were centrifuged at 4000 rpm for 5 min, and washed three times each with 0.9% NaCl and 0.1 M phosphate buffer (pH 7.4). The centrifuged pellets were fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde at 4 C for 4 h. Then, the pellets were washed three times with 0.1 M phosphate buffer, dehydrated in increasing ethanol concentrations (50%, 70%, 90%, and 100%, 15 min per step), and dried in air. Finally, the samples were observed via a scanning electron microscope with an energy-dispersive X-ray elemental analysis fitted with an Oxford Inca 300 EDS system (FEI, Eindhoven, Netherlands). Surface structure integrity of sludge microorganisms The surface integrity of sludge microorganisms was analyzed by LDH release assays. A cytotoxicity detection kit (Roche Applied Science, Penzberg, Germany) was used to measure the released LDH according to the manufacturer’s instructions. At the end of the exposure experiments, the mixture was centrifuged at 12,000 rpm for 5 min, and then the supernatant was seeded into the wells of a 96-well plate, followed by the addition of 50 mL of substrate mixture (Roche Applied Science). After incubation at room temperature for 30 min in the dark, 50 mL of stop solution (Roche Applied Science) was added to each well. The absorbance of each sample was recorded at 490 nm using a microplate reader (BioTek, Winooski, VT, USA). Fluorescence in situ hybridization The abundances of bacteria and methanogenic archaea were determined using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. The oligonucleotide probes used in this study were Cy3labelled EUB338 (50 -GCTGCCTCCCGTAGGAGT-30 )
Primary sludge
Excess sludge
6.8 § 0.2 14752 § 864 9836 § 261 13960 § 810 96 § 8 6593 § 350 3952 § 268
6.9 § 0.2 15681 § 942 11354 § 295 14730 § 835 45 § 6 1648 § 125 9825 § 460
for bacteria,[20] and FITC-labeled ARC915 (50 GTGCTCCCCCGCCAATTCCT-30 ) for archaea.[21] The procedure of the FISH analysis was as follows. Briefly, a sludge sample obtained from each serum bottle was fixed in a freshly prepared 4% paraformaldehyde solution for 4 h at 4 C. Then, 10 mL of the fixed sample was mounted on a glass slide using a micropipette. After dehydration with increasing ethanol concentrations (50, 80, and 95%, 5 min per step), the slide was pre-hybridized in 2 £ SSC buffer for 30 min at 37 C. After removal of the prehybridization solution, 20 mL of hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl (pH 7.2), 0.01% SDS) containing 5 ng mL¡1 of each labeled probe was applied to the sample. Hybridizations were performed in a hybridization incubator (ThermoBrite, USA) at 46 C for 2.5 h. The hybridizations were stringently adjusted by adding formamide to the hybridization buffer (5% for EUB338 and 35% for ARC915). A washing step was performed at 48 C for 30 min with washing buffer containing the same components as in the hybridization buffer. Then, the sections hybridized with the probes were observed via a confocal laser scanning microscope, and 10 random fields were analyzed to determine the average abundances of bacteria and methanogenic archaea in the sludge samples. Analytical methods To calculate methane production, the total gas volume during anaerobic sludge co-digestion was measured by releasing the pressure in the serum bottles using a syringe (100 mL) to allow equilibration with the room pressure. The gas component was determined via a gas chromatograph (6890N, Agilent, Santa Clara, CA, USA) equipped with a thermal conductivity detector using nitrogen as a carrier gas. The cumulative methane gas volume was calculated by the following equation: VH;i D VH;i ¡ 1 C .CH;i £ VG;i ¡ CH;i ¡ 1 £ VG;i ¡ 1 /
(1)
where VH, i and VH, i-1 are the cumulative methane gas volumes in the current (i) and previous (i-1) time intervals, respectively, VG, i is the total biogas volume (including the total volume of headspace in the reactor and the syringe)
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Fig. 1. SEM images of the sludge samples in the absence (A and D) and presence of TiO2 (B and E) and ZnO NPs (C and F). The sludge was sampled after the short-term (A–C) and long-term (D–F) exposure, respectively.
at the current time, VG, i-1 is the total volume of biogas after the gas component analysis at the previous time, and CH, i and CH, i-1 are the fractions of methane gas in the total gas at the current and previous time intervals, respectively. Finally, methane production was recorded as the cumulative methane gas volume per gram of VSS (mL/g VSS), and relative methane production was calculated by dividing the methane production during exposure to NPs by that in the control. The procedures for measuring the activities of key enzymes involved in methane production from anaerobic sludge codigestion were as follows. Briefly, protease activity was determined by mixing 3 mL of fermentation mixture with 1 mL of 0.5% azocasein, followed by incubation at 37 C for 90 min. Then, the reaction was terminated by adding 2 mL of 10% trichloroacetic acid. The mixture was centrifuged at
4,000 rpm for 30 min, and the absorbance of 2 mL of the supernatant in 2 mL of 2 M NaOH was measured at 440 nm. Amylase activity was determined at 60 C by mixing 0.25 mL of the fermentation mixture with 0.25 mL of 0.2% soluble starch dissolved in 0.1 M phosphate buffer (pH 7.0). The amount of residual starch was determined after 10 min using a starch-iodine assay. To measure the enzyme activity, 25 mL of the fermentation mixture was obtained, washed, and resuspended in 10 mL of 100 mM sodium phosphate buffer (pH 7.4). The mixture was sonicated at 20 kHz at 4 C for 10 min, and the crude extract was obtained by centrifugation at 10,000 rpm at 4 C for 15 min. Acetate kinase activity was analyzed using the method of Allen et al.,[22] with potassium acetate as the substrate, and coenzyme F420 was assayed by a previously described spectrophotometric method.[23]
Fig. 2. Methane production from anaerobic sludge co-digestion in the absence (the control) and presence of TiO2 and ZnO NPs after the short-term (A) and long-term (B) exposure. Asterisks indicate statistical differences (P < 0.05) from the control.
TiO2 and ZnO nanoparticle influence on methane production
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Fig. 3. Dissolution of ZnO NPs (A) and the released zinc ions-induced effects on methane production from anaerobic sludge co-digestion (B). Asterisks indicate statistical differences (P < 0.05) from the control.
Carbohydrate measurement was conducted using the phenol-sulfuric method with glucose as the standard.[24] The amount of soluble protein was determined by the Lowry-Folin method using bovine serum albumen as a standard,[25] and total sludge protein was estimated from the corresponding TKN concentration by subtracting the inorganic nitrogen concentration, dividing the difference by 0.16, and then multiplying the result by 1.5. Titanium and zinc concentrations were analyzed by inductively coupled plasma–optical emission spectrometry (Optima 2100 DV, PerkinElmer, Waltham, MA, USA). The total chemical oxygen demand (COD), soluble COD, TSS, and VSS were measured using standard methods.[26] The COD conversion factors for protein and carbohydrate are 1.5 g COD g¡1 protein and 1.06 g COD g¡1 carbohydrate.
Statistical analysis All tests were performed in triplicate, and the results were expressed as means § standard deviation. An analysis of variance was used to test the significance of the results, and P < 0.05 was considered to be statistically significant.
Results and discussion Short-term and long-term effects of nanoparticles on sludge surface structure in anaerobic co-digestion reactors A previous study reported that some nanomaterials might be adsorbed onto bacterial surfaces and then transported across cell membranes, leading to abnormal cell function or death.[5] Therefore, to assess the possible effects of TiO2 and ZnO NPs on anaerobic sludge co-digestion, a SEM analysis was first used to determine their influences on sludge surface morphology after short-term and long-term exposure. Figure 1 shows that the sludge surface in the absence of NPs (the control) was smooth (Fig. 1A and D). In contrast, when TiO2 (Fig. 1B and E) and ZnO NPs (Fig. 1C and F) were present in the sludge co-digestion reactors, large numbers of NPs were aggregated and adsorbed onto the sludge surface. However, the sludge surface morphology was not obviously changed after prolonged exposure to these NPs. To confirm this conclusion, LDH release (a marker of cell membrane damage) assays were conducted to determine the sludge surface integrity. The results indicated that there were no significant differences between the sludge samples from these reactors (p >
Fig. 4. Relative abundances of bacteria (A) and archaea (B) in the absence and presence of TiO2 and ZnO NPs after the long-term exposure. Asterisks indicate statistical differences (P < 0.05) from the control.
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Fig. 5. Schematic pathway for methane production from anaerobic sludge digestion and key enzymes involved in this biological process.
0.05), suggesting that the presence of TiO2 and ZnO NPs had no measurable impact on sludge surface structure during short-term and long-term exposure. In the literature, TiO2 NPs were reported to cause consequential disruptions of important biogeochemical processes of Anabaena variabilis,[27] and the presence of ZnO NPs showed ecotoxicological effects toward different soil organisms.[3] However, other studies observed that TiO2 NPs did not have any negative effects on the viability and morphology of keratinocytes after shortterm exposure.[28] Similarly, it was reported that 500 mg/L of TiO2 NPs did not change the viability and growth of Cupriavidus metallidurans and Bacillus subtilis.[29] Clearly, NP-induced effects might be different for different organisms or environments. Large numbers of studies have confirmed that engineered nanomaterials might cause oxidative damage to the cell membranes of microorganisms[30] or human cells.[31] Nevertheless, reactive oxygen species production is difficult in the absence of oxygen, such as under the anaerobic conditions in this study. Meanwhile, sludge usually contains large amounts of extracellular polymeric substances, and these substances can bind microorganisms together and protect them from adverse environments,[17] which might be one reason for the lack of effects of TiO2 and ZnO NPs on sludge surface integrity. Short-term and long-term effects of nanoparticles on methane production in anaerobic co-digestion reactors Methane production can be achieved by anaerobic codigestion of primary and excess sludge. To determine the
Zheng et al. potential effects of TiO2 and ZnO NPs on sludge co-digestion, short-term and long-term exposure experiments were conducted. Figure 2 shows methane production in the absence (the control) or presence of 6, 30, or 150 mg g¡1TSS of TiO2 and ZnO NPs after short-term and long-term exposure. Compared with the control, the presence of TiO2 NPs had no adverse effects on methane production after either short-term or long-term exposure (P > 0.05) (Figs. 2A and B). However, short-term exposure to 30 or 150 mg/g-TSS of ZnO NPs significantly inhibited methane production by 21.5% and 76.4%, respectively, (P < 0.05). Additionally, long-term exposure to the same concentrations of ZnO NPs caused similar inhibitions of methane production (19.8% and 73.9%, respectively). These results indicated that sludge microorganisms did not adapt to the presence of ZnO NPs during the prolonged exposure time, which resulted in the unchanged inhibitory effects on anaerobic sludge co-digestion. A previous study reported that engineered nanomaterials can exhibit different physicochemical properties and toxicological behaviors than bulk materials under anaerobic conditions.[32] In particular, the dissolution of NPs can release metal ions, and some metal ions, such as copper and zinc ions, might inhibit biological degradation processes. However, the release of titanium ions from TiO2 NPs is difficult under neutral conditions.[15] Thus, no titanium ions were found in wastewater treatment facilities or anaerobic fermentation reactors,[17] which was consistent with the lack of measurable effects of TiO2 NPs on anaerobic sludge co-digestion. In contrast, some studies showed that the toxicity of ZnO NPs might be due to the release of zinc ions,[33–36] and these ions could have inhibitory effects on sludge hydrolysis and the methanation of waste activated sludge.[37] Figure 3 shows that the concentrations of released zinc ions were 3.5, 9.8, or 16.2 mg L¡1 in the presence of 6, 30, or 150 mg g¡1-TSS, respectively, of ZnO NPs, and 9.8 and 16.2 mg L¡1 of zinc ions inhibited methane production from anaerobic sludge co-digestion. These results confirmed that the toxic zinc ions coming from ZnO NPs were responsible for the negative effects of ZnO NPs on anaerobic sludge co-digestion.
Fig. 6. Relative activities of the key enzymes involved in methane production in the presence of ZnO NPs after the short-term (A) and long-term (B) exposure. The presence of TiO2 had no effects on the activities of these key enzymes.
TiO2 and ZnO nanoparticle influence on methane production Changes in microbial community structures involved in anaerobic co-digestion reactors Previous studies observed that TiO2 and ZnO NPs altered the bacterial composition of soil and reduced microbial populations after 60 d of exposure.[4] It is well-known that large numbers of microorganisms are involved in anaerobic sludge co-digestion to produce methane. For example, bacteria are mainly responsible for the decomposition of carbohydrates, proteins, and other organic substances that lead to the production of volatile fatty acids.[38] Subsequently, methanogenic archaea can use these acidification products (such as acetate) or hydrogen/carbon dioxide to produce methane.[9] Clearly, methane production is closely related to the structure of the bacteria and methanogenic archaea communities. Therefore, the possible effects of TiO2 and ZnO NPs on the abundances of bacteria and methanogenic archaea involved in sludge co-digestion reactors need to be investigated. In this study, FISH was conducted to quantitatively determine the abundances of bacteria and methanogenic archaea following long-term exposure to 6, 30, or 150 mg g¡1-TSS of TiO2 or ZnO NPs. Figure 4 indicates that in the absence of NPs, methanogenic archaea and bacteria accounted for 42.5% and 50.3%, respectively, of the total number of microorganisms, suggesting that the ratio of archaea to bacteria in the control was 0.84:1. After longterm exposure to TiO2 NPs, the abundances of archaea and bacteria were similar to those of the control, indicating that 6, 30, or 150 mg/g-TSS of TiO2 NPs had no significant effect on the ratio of archaea to bacteria. However, the abundances of archaea and bacteria were 28.4% and 66.1%, respectively, after long-term exposure to 30 mg/g-TSS of ZnO NPs, resulting in a lower (0.43:1) ratio of archaea to bacteria. Furthermore, when the concentration of ZnO NPs increased to 150 mg/g-TSS, the abundance of archaea was only 6.8%, and the ratio of archaea to bacteria significantly decreased to 0.08:1 (P < 0.05). Therefore, these data indicated that the presence of ZnO NPs inhibited the growth of methanogenic archaea, and thus significantly decreased the ratio of archaea to bacteria, which might be responsible for the lower methane production.
Short-term and long-term effects of nanoparticles on the activities of key enzymes related to methane production from anaerobic sludge co-digestion Primary and excess sludge contain significant amounts of organic matter that can be used by anaerobic microorganisms to produce valuable products.[10] However, this anaerobic co-digestion process depends upon a series of efficient biochemical reactions catalyzed by some important enzymes (Fig. 5). For example, proteases can hydrolyze proteins into peptides, and amylase is able to hydrolyze carbohydrates into glucose and maltose during
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sludge hydrolysis. Acetate kinase (AK) is responsible for the transformations of monosaccharides and amino acids into acetate, whereas coenzyme F420 plays an important role in the conversion of acetate to methane.[37] Clearly, methane production may be affected by these key enzymes. Figure 6 shows the relative protease, amylase, AK, and coenzyme F420 activities after short-term and long-term exposure to 6, 30, or 150 mg g¡1-TSS of TiO2 or ZnO NPs. The data indicated that both short-term and longterm exposure to TiO2 NPs had no adverse effects on the protease, amylase, AK, and coenzyme F420 activities, which is consistent with the lack of measurable impacts of TiO2 NPs on methane production from anaerobic sludge co-digestion. However, compared with the control, the relative protease activity was significantly inhibited after short-term exposure to 150 mg g¡1-TSS of ZnO NPs, which might be an important reason for the inhibition of sludge hydrolysis by ZnO NPs. Meanwhile, the activities of AK and coenzyme F420 were also substantially suppressed, which is adverse to acidification and methanation processes. Moreover, after long-term exposure, protease, AK, and coenzyme F420 activities were still suppressed by 150 mg g¡1-TSS of ZnO NPs. These results suggested that prolonged exposure did not enable the enzyme activities of the sludge microorganisms to recover, which might explain why the negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time. Previous studies also found that the dissolution of ZnO NPs was able to release some zinc ions, which were responsible for the toxicity of ZnO NPs toward anaerobic fermenting microorganisms in waste activated sludge.[37] A previous study showed that methanogenic archaea were highly sensitive to metal ions, such as zinc ions,[39] which might be one reason for the inhibition of the anaerobic co-digestion of primary and excess sludge by ZnO NPs.
Conclusion Our data showed that short-term and long-term exposure to 6, 30, or 150 mg g¡1-TSS of TiO2 NPs did not affect sludge hydrolysis, acidification, and methane production during anaerobic sludge co-digestion. However, 30 or 150 mg g¡1-TSS of ZnO NPs significantly inhibited methane production after short-term exposure. Further experiments indicated that the presence of ZnO NPs decreased the abundance of methanogenic archaea, and thus lowered the ratio of archaea to bacteria, which was an important reason for the inhibition of methane production. Meanwhile, both short-term and long-term exposure to ZnO NPs substantially suppressed the relative activities of key enzymes related to methane production (proteases, AK, and coenzyme F420), which reduced methane production.
920 These results also confirmed that the negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time.
Zheng et al.
[15]
Funding [16]
This work was financially supported by the National HiTech Research and Development Program of China (863 Program) (2011AA060903), the National Natural Science Foundation of China (51425802, 41301558, and 51278354), the Program of Shanghai Subject Chief Scientist (15XD1503400), and the Fundamental Research Funds for the Central Universities (2014KJ006).
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treatment plant: Influence of agglomeration and surfactants on clearing efficiency. Environ. Sci. Technol. 2008, 42, 5828–5833. Kiser, M. A.; Westerhoff, P.; Benn, T.; Wang, Y.; Perez-Rivera, J.; Hristovski, K. Titanium nanomaterial removal and release from wastewater treatment plants. Environ. Sci. Technol. 2009, 43, 6757–6763. Zheng, X.; Wu, R.; Chen, Y. Effects of ZnO nanoparticles on wastewater biological nitrogen and phosphorus removal. Environ. Sci. Technol. 2011, 45, 2826–2832. Zheng, X.; Chen, Y.; Wu, R. Long-term effects of titanium dioxide nanoparticles on nitrogen and phosphorus removal from wastewater and bacterial community shift in activated sludge. Environ. Sci. Technol. 2011, 45, 7284–7290. Mu, H.; Zheng, X.; Chen, Y.; Chen, H.; Liu, K. Response of anaerobic granular sludge to a shock load of zinc oxide nanoparticles during biological wastewater treatment. Environ. Sci. Technol. 2012, 46, 5997–6003. USEPA. Targeted National Sewage Sludge Survey Sampling and Analysis Technical Report. http://water.epa.gov/scitech/ wastetech/biosolids/upload/2009_01_15_biosolids_tnsss-tech.pdf (accessed Jan 2011). Mann, A.J.; Hahnke, R.L.; Huang, S.X.; Werner, J.; Xing, P.; Barbeyron, T.; Huettel, B.; Stuber, K.; Reinhardt, R.; Harder, J.; Glockner, F.O.; Amann, R. I.; Teeling, H. The genome of the alga-associated marine flavobacterium Formosa agariphila KMM 3901T reveals a broad potential for degradation of algal polysaccharides. Appl. Environ. Microbiol. 2013, 79, 6813–6822. Stahl, D.A.; Flesher, B.; Mansfield, H.R.; Montgomery, L. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 1988, 54, 1079–1084. Allen, S.H.; Kellermeyer, R.W.; Stjernholm, R.L.; Wood, H.G. Purification and properties of enzymes involved in the propionic acid fermentation. J. Bacteriol. 1964, 87, 171–187. Delafontaine, M.J.; Naveau, H.P.; Nyns, E.J. Fluorimetric monitoring of methanogenesis in anaerobic digesters. Biotechnol. Lett. 1979, 1, 71–73. Herbert, D.; Philipps, P.J.; Strange, R.E. Carbohydrate analysis. Methods Enzymol. 1971, 5B, 265–277. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. APHA. Standard Methods for the Examination of Water and Wastewater, 20th Ed.; American Public Health Association: Washington, DC, 1998. Cherchi, C.; Gu, A.Z. Impact of titanium dioxide nanomaterials on nitrogen fixation rate and intracellular nitrogen storage in Anabaena variabilis. Environ. Sci. Technol. 2010, 44, 8302–8307. Kocbek, P.; Teskac, K.; Kreft, M.E.; Kristl, J. Toxicological aspects of long-term treatment of Keratinocytes with ZnO and TiO2 nanoparticles. Small 2010, 6, 1908–1917. Simon-Deckers, A.; Loo, S.; Mayne-L’Hermite, M.; Herlin-Boime, N.; Menguy, N.; Reynaud, C.; Gouget, B.; Carriere, M. Size-, composition- and shape-dependent toxicological impact of metal oxide nanoparticles and carbon nanotubes toward bacteria. Environ. Sci. Technol. 2009, 43, 8423–8429. Adams, L.K.; Lyon, D.Y.; Alvarez, P.J.J. Comparative eco-toxicity of nanoscale TiO2, SiO2, and ZnO water suspensions. Water Res. 2006, 40, 3527–3532. Limbach, L.K.; Wick, P.; Manser, P.; Grass, R.N.; Bruinink, A.; Stark, W.J. Exposure of engineered nanoparticles to human lung epithelial cells: Influence of chemical composition and catalytic activity on oxidative stress. Environ. Sci. Technol. 2007, 41, 4158– 4163. Yang, Y.; Zhang, C.Q.; Hu, Z.Q. Impact of metallic and metal oxide nanoparticles on wastewater treatment and anaerobic digestion. Environ. Sci. Process Impacts 2013, 15, 39–48.
TiO2 and ZnO nanoparticle influence on methane production [33] Liu, G.Q.; Wang, D.M.; Wang, J.M.; Mendoza, C. Effect of ZnO particles on activated sludge: Role of particle dissolution. Sci. Total Environ. 2011, 409, 2852–2857. [34] Franklin, N.M.; Rogers, N.J.; Apte, S.C.; Batley, G.E.; Gadd, G. E.; Casey, P.S. Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): The importance of particle solubility. Environ. Sci. Technol. 2007, 41, 8484–8490. [35] Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H. B.; Yeh, J.I.; Zink, J.I.; Nel, A.E. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2008, 2, 2121–2134.
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[36] Wong, S.W.Y.; Leung, P.T.Y.; Djurisic, A.B.; Leung, K.M.Y. Toxicities of nano zinc oxide to five marine organisms: Influences of aggregate size and ion solubility. Anal. Bioanal. Chem. 2010, 396, 609–618. [37] Mu, H.; Chen, Y. Long-term effect of ZnO nanoparticles on waste activated sludge anaerobic digestion. Water Res. 2011, 45, 5612–5620. [38] Yuan, H.; Chen, Y.; Zhang, H.; Jiang, S.; Zhou, Q.; Gu, G. Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline conditions. Environ. Sci. Technol. 2006, 40, 2025–2029. [39] Zayed, G.; Winter, J. Inhibition of methane production from whey by heavy metals - Protective effect of sulfide. Appl. Microbiol. Biotechnol. 2000, 53, 726–731.
Appendix
Figure A1. X-ray diffraction (XRD) patterns of TiO2 (A) and ZnO NPs (B) used in this study.
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Effects of titanium dioxide and zinc oxide nanoparticles on methane production from anaerobic co-digestion of primary and excess sludge XIONG ZHENG, LIJUAN WU, YINGUANG CHEN, YINGLONG SU, RUI WAN, KUN LIU and HAINING HUANG State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, Shanghai, China
Anaerobic co-digestion of primary and excess sludge is regarded as an efficient way to reuse sludge organic matter to produce methane. In this study, short-term and long-term exposure experiments were conducted to investigate the possible effects of titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) on methane production from anaerobic co-digestion of primary and excess sludge. The data showed that TiO2 NPs had no measurable impact on methane production, even at a high concentration (150 mg/g total suspended solids (TSS)). However, short-term (8 days) exposure to 30 or 150 mg/g-TSS of ZnO NPs significantly decreased methane production. More importantly, these negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time to 105 days. Further studies indicated that the presence of ZnO NPs substantially decreased the abundance of methanogenic archaea, which reduced methane production. Meanwhile, the activities of some key enzymes involved in methane production, such as protease, acetate kinase, and coenzyme F420, were remarkably inhibited by the presence of ZnO NPs, which was also an important reason for the decreased methane production. These results provide a better understanding of the potential risks of TiO2 and ZnO NPs to methane production from anaerobic sludge co-digestion. Keywords: Engineered nanoparticles, enzyme activity, inhibitory effect, methanogenic archaea, primary and excess sludge.
Introduction With the rapid development of nanotechnology, engineered nanomaterials have been widely used in various applications because of their unique physicochemical properties.[1–3] For example, titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) are increasingly used in many products, such as sunscreens, cosmetics, and bottle coatings.[4] The extensive production and application of these nanomaterials inevitably result in their release into the environment, which raises concerns about their potential impacts on human health and ecosystems.[5] Although the current environmental concentrations of nanomaterials are still low,[6] their release into the environment could continue to increase owing to their large-scale
Address correspondence to Yinguang Chen, State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China; E-mail: yg2chen@yahoo. com Received November 7, 2014.
production. It is well-known that wastewater treatment plants (WWTPs) play important roles in pollutant removal and water purification throughout the world. However, recent studies found that WWTPs are becoming substantial reservoirs of large numbers of engineered nanomaterials, such as TiO2 and ZnO NPs.[7] Therefore, it is necessary to consider the potential risks of these nanomaterials to WWTPs. The activated sludge process is the most widely used method for treating wastewater, especially municipal wastewater, in WWTPs. However, this process generates large amounts of primary and excess sludge, which should be treated to avoid contaminating the environment.[8] In recent years, sludge disposal using landfills or incineration has faced increasing public opposition because of stringent environmental control regulations and the scarcity of land. Anaerobic digestion is regarded as a good alternative, as it treats sludge as a valuable resource and can convert sludge organics into methane.[9] Usually, primary sludge has a high carbohydrates concentration, while excess sludge has a high protein content. A previous study reported that anaerobic co-digestion of primary and excess sludge could achieve a better carbon to nitrogen ratio, thus improving the treatment of sludge
914 organics by anaerobic microorganisms.[10] However, it is still unclear whether the presence of nanomaterials can affect the anaerobic sludge co-digestion process. Previous studies have found that the adsorption of activated sludge is one of the most important mechanisms for pollutant removal.[11] Hence, the majority of nanomaterials in influents were observed to be adsorbed on the sludge surface,[12,13] which resulted in the accumulation of much higher concentrations of nanomaterials in biosolids.[14,15] Additionally, the presence of ZnO NPs significantly affected sludge microorganisms, thereby substantially inhibiting wastewater nitrogen and phosphorus removal.[16] Although TiO2 NPs had no negative influences on wastewater treatment after short-term exposure, prolonged exposure to this nanomaterial greatly suppressed biological nitrogen removal from wastewater.[17] Nevertheless, to date, the short-term and long-term effects of TiO2 and ZnO NPs on methane production from anaerobic sludge co-digestion are still unknown. The aim of this study was to evaluate the short-term and long-term effects of TiO2 and ZnO NPs on methane production from the anaerobic co-digestion of primary and excess sludge. First, a scanning electron microscopy (SEM) analysis and the lactate dehydrogenase (LDH) release assay were used to determine whether sludge surface integrity was affected by the presence of these NPs. Then, both short-term and long-term exposure experiments were conducted to investigate the potential impacts of TiO2 and ZnO NPs on methane production from anaerobic sludge co-digestion at the concentrations of 6, 30, and 150 mg g¡1-total suspended solids (TSS). Because methane production is relevant to sludge solubilization, hydrolysis, acidification, and methanation, the effects of these NPs on the abundances of key microorganisms (mainly bacteria and archaea) were studied. Finally, the activities of key enzymes involved in methane production were measured to explore the possible mechanisms for the NPsinduced effects on anaerobic sludge co-digestion.
Materials and methods Characteristics of primary and excess sludge Primary and excess sludge were obtained from the primary and secondary sedimentation tanks, respectively, of a traditional WWTP in Shanghai, China. The sludge was concentrated via settling at 4 C for 24 h before conducting the experiments. The main characteristics of both primary and excess sludge after settlement are shown in Table 1. Primary and excess sludge were mixed at a 1:1 ratio (volatile suspended solids; VSS) to conduct the anaerobic sludge co-digestion.
Engineered nanoparticles and their stock suspensions In this study, commercial TiO2 (anatase, purity > 99.7%) and ZnO (wurtzite, purity > 99.5%) NPs were
Zheng et al. purchased from Sigma-Aldrich (St. Louis, MO, USA). X-ray diffraction analysis was conducted using a D/ Max-RB diffractometer (Rigaku, Tokyo, Japan) equipped with a rotating anode and a Cu Ka radiation source, as shown in Figure A1 (Appendix). By measuring nitrogen adsorption at 77 K using a Tristar 3000 analyzer (Micromeritics, Norcross, GA, USA), the specific surface areas of the TiO2 and ZnO NPs were determined to be 102 § 6 and 35 § 4 m2/g, respectively. Before the experiment, stock suspensions (2000 mg L¡1) were prepared by adding 2000 mg of NPs to 1 L of deionized water (pH 7.0), followed by 1 h of ultrasonication (25 C, 250 W) to disperse the NPs.[18] The primary sizes of the TiO2 and ZnO NPs in the stock suspensions were in the ranges of 150–170 and 120–140 nm, respectively, as determined by dynamic light scattering using an Autosizer 4700 (Malvern Instruments, Malvern, UK).
Short-term and long-term exposure experiments A previous study reported that the titanium and zinc contents of biosolids in WWTPs (84 in total) ranged from 0.02 to 7.02 and 0.22 to 8.55 mg/g-TSS, respectively.[19] Hence, in this study, 6 mg/g-TSS was chosen as the environmentally relevant concentration of TiO2 or ZnO NPs. Because the release of these NPs into the environment would be expected to increase during large-scale production, the potential risks of higher NP concentrations (30 and 150 mg/g-TSS) were also considered. First, batch experiments (18 d) were conducted to determine the short-term effects of TiO2 and ZnO NPs on anaerobic sludge codigestion. Briefly, 0, 6, 30, or 150 mL of NPs stock suspensions (2000 mg L¡1) were fed into several serum bottles, each containing 140 mL of mixed sludge (approximately 15 g L¡1 of TSS). Then, deionized water was added to each bottle to adjust the final volume to 300 mL, resulting in final NP concentrations of 0, 6, 30, or 150 mg g¡1-TSS. A bottle to which TiO2 or ZnO NPs were not added was used as the control. All bottles were flushed with nitrogen gas for 5 min to remove oxygen, capped with rubber stoppers, sealed, and placed in an air-bath shaker (150 rpm) at 35 § 1 C. To assess the long-term effects of TiO2 and ZnO NPs on anaerobic sludge co-digestion, serum bottles containing 0, 6, 30, or 150 mg/g-TSS of TiO2 or ZnO NPs were flushed with nitrogen gas, capped with rubber stoppers, and shaken in an air-bath shaker (150 rpm, 35 § 1 C) for 105 d. Every day, 15 mL of fermentation mixture was manually withdrawn from each bottle, and the same amount of raw mixed sludge and NPs were supplemented, which resulted in a sludge retention time of 20 d. During the exposure time, methane production was measured every 3 d to evaluate the possible effects of these NPs on anaerobic sludge co-digestion.
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TiO2 and ZnO nanoparticle influence on methane production Table 1. Characteristics of the primary and excess sludge used in this study. Parameter pH Total suspended solids (TSS) (mg/L) Volatile suspended solids (VSS) (mg/L) Total chemical oxygen demand (TCOD) (mg/L) Soluble chemical oxygen demand (SCOD) (mg/L) Total carbohydrate (mg COD/L) Total protein (mg COD/L)
Scanning electron microscopy The sludge surface morphology in the absence and presence of TiO2 and ZnO NPs was observed by SEM. Briefly, 50 mL of fermentation mixture was withdrawn from each bottle at the end of the exposure experiments (18 d for short-term exposure and 105 d for long-term exposure, respectively). These mixtures were centrifuged at 4000 rpm for 5 min, and washed three times each with 0.9% NaCl and 0.1 M phosphate buffer (pH 7.4). The centrifuged pellets were fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde at 4 C for 4 h. Then, the pellets were washed three times with 0.1 M phosphate buffer, dehydrated in increasing ethanol concentrations (50%, 70%, 90%, and 100%, 15 min per step), and dried in air. Finally, the samples were observed via a scanning electron microscope with an energy-dispersive X-ray elemental analysis fitted with an Oxford Inca 300 EDS system (FEI, Eindhoven, Netherlands). Surface structure integrity of sludge microorganisms The surface integrity of sludge microorganisms was analyzed by LDH release assays. A cytotoxicity detection kit (Roche Applied Science, Penzberg, Germany) was used to measure the released LDH according to the manufacturer’s instructions. At the end of the exposure experiments, the mixture was centrifuged at 12,000 rpm for 5 min, and then the supernatant was seeded into the wells of a 96-well plate, followed by the addition of 50 mL of substrate mixture (Roche Applied Science). After incubation at room temperature for 30 min in the dark, 50 mL of stop solution (Roche Applied Science) was added to each well. The absorbance of each sample was recorded at 490 nm using a microplate reader (BioTek, Winooski, VT, USA). Fluorescence in situ hybridization The abundances of bacteria and methanogenic archaea were determined using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. The oligonucleotide probes used in this study were Cy3labelled EUB338 (50 -GCTGCCTCCCGTAGGAGT-30 )
Primary sludge
Excess sludge
6.8 § 0.2 14752 § 864 9836 § 261 13960 § 810 96 § 8 6593 § 350 3952 § 268
6.9 § 0.2 15681 § 942 11354 § 295 14730 § 835 45 § 6 1648 § 125 9825 § 460
for bacteria,[20] and FITC-labeled ARC915 (50 GTGCTCCCCCGCCAATTCCT-30 ) for archaea.[21] The procedure of the FISH analysis was as follows. Briefly, a sludge sample obtained from each serum bottle was fixed in a freshly prepared 4% paraformaldehyde solution for 4 h at 4 C. Then, 10 mL of the fixed sample was mounted on a glass slide using a micropipette. After dehydration with increasing ethanol concentrations (50, 80, and 95%, 5 min per step), the slide was pre-hybridized in 2 £ SSC buffer for 30 min at 37 C. After removal of the prehybridization solution, 20 mL of hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl (pH 7.2), 0.01% SDS) containing 5 ng mL¡1 of each labeled probe was applied to the sample. Hybridizations were performed in a hybridization incubator (ThermoBrite, USA) at 46 C for 2.5 h. The hybridizations were stringently adjusted by adding formamide to the hybridization buffer (5% for EUB338 and 35% for ARC915). A washing step was performed at 48 C for 30 min with washing buffer containing the same components as in the hybridization buffer. Then, the sections hybridized with the probes were observed via a confocal laser scanning microscope, and 10 random fields were analyzed to determine the average abundances of bacteria and methanogenic archaea in the sludge samples. Analytical methods To calculate methane production, the total gas volume during anaerobic sludge co-digestion was measured by releasing the pressure in the serum bottles using a syringe (100 mL) to allow equilibration with the room pressure. The gas component was determined via a gas chromatograph (6890N, Agilent, Santa Clara, CA, USA) equipped with a thermal conductivity detector using nitrogen as a carrier gas. The cumulative methane gas volume was calculated by the following equation: VH;i D VH;i ¡ 1 C .CH;i £ VG;i ¡ CH;i ¡ 1 £ VG;i ¡ 1 /
(1)
where VH, i and VH, i-1 are the cumulative methane gas volumes in the current (i) and previous (i-1) time intervals, respectively, VG, i is the total biogas volume (including the total volume of headspace in the reactor and the syringe)
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Fig. 1. SEM images of the sludge samples in the absence (A and D) and presence of TiO2 (B and E) and ZnO NPs (C and F). The sludge was sampled after the short-term (A–C) and long-term (D–F) exposure, respectively.
at the current time, VG, i-1 is the total volume of biogas after the gas component analysis at the previous time, and CH, i and CH, i-1 are the fractions of methane gas in the total gas at the current and previous time intervals, respectively. Finally, methane production was recorded as the cumulative methane gas volume per gram of VSS (mL/g VSS), and relative methane production was calculated by dividing the methane production during exposure to NPs by that in the control. The procedures for measuring the activities of key enzymes involved in methane production from anaerobic sludge codigestion were as follows. Briefly, protease activity was determined by mixing 3 mL of fermentation mixture with 1 mL of 0.5% azocasein, followed by incubation at 37 C for 90 min. Then, the reaction was terminated by adding 2 mL of 10% trichloroacetic acid. The mixture was centrifuged at
4,000 rpm for 30 min, and the absorbance of 2 mL of the supernatant in 2 mL of 2 M NaOH was measured at 440 nm. Amylase activity was determined at 60 C by mixing 0.25 mL of the fermentation mixture with 0.25 mL of 0.2% soluble starch dissolved in 0.1 M phosphate buffer (pH 7.0). The amount of residual starch was determined after 10 min using a starch-iodine assay. To measure the enzyme activity, 25 mL of the fermentation mixture was obtained, washed, and resuspended in 10 mL of 100 mM sodium phosphate buffer (pH 7.4). The mixture was sonicated at 20 kHz at 4 C for 10 min, and the crude extract was obtained by centrifugation at 10,000 rpm at 4 C for 15 min. Acetate kinase activity was analyzed using the method of Allen et al.,[22] with potassium acetate as the substrate, and coenzyme F420 was assayed by a previously described spectrophotometric method.[23]
Fig. 2. Methane production from anaerobic sludge co-digestion in the absence (the control) and presence of TiO2 and ZnO NPs after the short-term (A) and long-term (B) exposure. Asterisks indicate statistical differences (P < 0.05) from the control.
TiO2 and ZnO nanoparticle influence on methane production
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Fig. 3. Dissolution of ZnO NPs (A) and the released zinc ions-induced effects on methane production from anaerobic sludge co-digestion (B). Asterisks indicate statistical differences (P < 0.05) from the control.
Carbohydrate measurement was conducted using the phenol-sulfuric method with glucose as the standard.[24] The amount of soluble protein was determined by the Lowry-Folin method using bovine serum albumen as a standard,[25] and total sludge protein was estimated from the corresponding TKN concentration by subtracting the inorganic nitrogen concentration, dividing the difference by 0.16, and then multiplying the result by 1.5. Titanium and zinc concentrations were analyzed by inductively coupled plasma–optical emission spectrometry (Optima 2100 DV, PerkinElmer, Waltham, MA, USA). The total chemical oxygen demand (COD), soluble COD, TSS, and VSS were measured using standard methods.[26] The COD conversion factors for protein and carbohydrate are 1.5 g COD g¡1 protein and 1.06 g COD g¡1 carbohydrate.
Statistical analysis All tests were performed in triplicate, and the results were expressed as means § standard deviation. An analysis of variance was used to test the significance of the results, and P < 0.05 was considered to be statistically significant.
Results and discussion Short-term and long-term effects of nanoparticles on sludge surface structure in anaerobic co-digestion reactors A previous study reported that some nanomaterials might be adsorbed onto bacterial surfaces and then transported across cell membranes, leading to abnormal cell function or death.[5] Therefore, to assess the possible effects of TiO2 and ZnO NPs on anaerobic sludge co-digestion, a SEM analysis was first used to determine their influences on sludge surface morphology after short-term and long-term exposure. Figure 1 shows that the sludge surface in the absence of NPs (the control) was smooth (Fig. 1A and D). In contrast, when TiO2 (Fig. 1B and E) and ZnO NPs (Fig. 1C and F) were present in the sludge co-digestion reactors, large numbers of NPs were aggregated and adsorbed onto the sludge surface. However, the sludge surface morphology was not obviously changed after prolonged exposure to these NPs. To confirm this conclusion, LDH release (a marker of cell membrane damage) assays were conducted to determine the sludge surface integrity. The results indicated that there were no significant differences between the sludge samples from these reactors (p >
Fig. 4. Relative abundances of bacteria (A) and archaea (B) in the absence and presence of TiO2 and ZnO NPs after the long-term exposure. Asterisks indicate statistical differences (P < 0.05) from the control.
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Fig. 5. Schematic pathway for methane production from anaerobic sludge digestion and key enzymes involved in this biological process.
0.05), suggesting that the presence of TiO2 and ZnO NPs had no measurable impact on sludge surface structure during short-term and long-term exposure. In the literature, TiO2 NPs were reported to cause consequential disruptions of important biogeochemical processes of Anabaena variabilis,[27] and the presence of ZnO NPs showed ecotoxicological effects toward different soil organisms.[3] However, other studies observed that TiO2 NPs did not have any negative effects on the viability and morphology of keratinocytes after shortterm exposure.[28] Similarly, it was reported that 500 mg/L of TiO2 NPs did not change the viability and growth of Cupriavidus metallidurans and Bacillus subtilis.[29] Clearly, NP-induced effects might be different for different organisms or environments. Large numbers of studies have confirmed that engineered nanomaterials might cause oxidative damage to the cell membranes of microorganisms[30] or human cells.[31] Nevertheless, reactive oxygen species production is difficult in the absence of oxygen, such as under the anaerobic conditions in this study. Meanwhile, sludge usually contains large amounts of extracellular polymeric substances, and these substances can bind microorganisms together and protect them from adverse environments,[17] which might be one reason for the lack of effects of TiO2 and ZnO NPs on sludge surface integrity. Short-term and long-term effects of nanoparticles on methane production in anaerobic co-digestion reactors Methane production can be achieved by anaerobic codigestion of primary and excess sludge. To determine the
Zheng et al. potential effects of TiO2 and ZnO NPs on sludge co-digestion, short-term and long-term exposure experiments were conducted. Figure 2 shows methane production in the absence (the control) or presence of 6, 30, or 150 mg g¡1TSS of TiO2 and ZnO NPs after short-term and long-term exposure. Compared with the control, the presence of TiO2 NPs had no adverse effects on methane production after either short-term or long-term exposure (P > 0.05) (Figs. 2A and B). However, short-term exposure to 30 or 150 mg/g-TSS of ZnO NPs significantly inhibited methane production by 21.5% and 76.4%, respectively, (P < 0.05). Additionally, long-term exposure to the same concentrations of ZnO NPs caused similar inhibitions of methane production (19.8% and 73.9%, respectively). These results indicated that sludge microorganisms did not adapt to the presence of ZnO NPs during the prolonged exposure time, which resulted in the unchanged inhibitory effects on anaerobic sludge co-digestion. A previous study reported that engineered nanomaterials can exhibit different physicochemical properties and toxicological behaviors than bulk materials under anaerobic conditions.[32] In particular, the dissolution of NPs can release metal ions, and some metal ions, such as copper and zinc ions, might inhibit biological degradation processes. However, the release of titanium ions from TiO2 NPs is difficult under neutral conditions.[15] Thus, no titanium ions were found in wastewater treatment facilities or anaerobic fermentation reactors,[17] which was consistent with the lack of measurable effects of TiO2 NPs on anaerobic sludge co-digestion. In contrast, some studies showed that the toxicity of ZnO NPs might be due to the release of zinc ions,[33–36] and these ions could have inhibitory effects on sludge hydrolysis and the methanation of waste activated sludge.[37] Figure 3 shows that the concentrations of released zinc ions were 3.5, 9.8, or 16.2 mg L¡1 in the presence of 6, 30, or 150 mg g¡1-TSS, respectively, of ZnO NPs, and 9.8 and 16.2 mg L¡1 of zinc ions inhibited methane production from anaerobic sludge co-digestion. These results confirmed that the toxic zinc ions coming from ZnO NPs were responsible for the negative effects of ZnO NPs on anaerobic sludge co-digestion.
Fig. 6. Relative activities of the key enzymes involved in methane production in the presence of ZnO NPs after the short-term (A) and long-term (B) exposure. The presence of TiO2 had no effects on the activities of these key enzymes.
TiO2 and ZnO nanoparticle influence on methane production Changes in microbial community structures involved in anaerobic co-digestion reactors Previous studies observed that TiO2 and ZnO NPs altered the bacterial composition of soil and reduced microbial populations after 60 d of exposure.[4] It is well-known that large numbers of microorganisms are involved in anaerobic sludge co-digestion to produce methane. For example, bacteria are mainly responsible for the decomposition of carbohydrates, proteins, and other organic substances that lead to the production of volatile fatty acids.[38] Subsequently, methanogenic archaea can use these acidification products (such as acetate) or hydrogen/carbon dioxide to produce methane.[9] Clearly, methane production is closely related to the structure of the bacteria and methanogenic archaea communities. Therefore, the possible effects of TiO2 and ZnO NPs on the abundances of bacteria and methanogenic archaea involved in sludge co-digestion reactors need to be investigated. In this study, FISH was conducted to quantitatively determine the abundances of bacteria and methanogenic archaea following long-term exposure to 6, 30, or 150 mg g¡1-TSS of TiO2 or ZnO NPs. Figure 4 indicates that in the absence of NPs, methanogenic archaea and bacteria accounted for 42.5% and 50.3%, respectively, of the total number of microorganisms, suggesting that the ratio of archaea to bacteria in the control was 0.84:1. After longterm exposure to TiO2 NPs, the abundances of archaea and bacteria were similar to those of the control, indicating that 6, 30, or 150 mg/g-TSS of TiO2 NPs had no significant effect on the ratio of archaea to bacteria. However, the abundances of archaea and bacteria were 28.4% and 66.1%, respectively, after long-term exposure to 30 mg/g-TSS of ZnO NPs, resulting in a lower (0.43:1) ratio of archaea to bacteria. Furthermore, when the concentration of ZnO NPs increased to 150 mg/g-TSS, the abundance of archaea was only 6.8%, and the ratio of archaea to bacteria significantly decreased to 0.08:1 (P < 0.05). Therefore, these data indicated that the presence of ZnO NPs inhibited the growth of methanogenic archaea, and thus significantly decreased the ratio of archaea to bacteria, which might be responsible for the lower methane production.
Short-term and long-term effects of nanoparticles on the activities of key enzymes related to methane production from anaerobic sludge co-digestion Primary and excess sludge contain significant amounts of organic matter that can be used by anaerobic microorganisms to produce valuable products.[10] However, this anaerobic co-digestion process depends upon a series of efficient biochemical reactions catalyzed by some important enzymes (Fig. 5). For example, proteases can hydrolyze proteins into peptides, and amylase is able to hydrolyze carbohydrates into glucose and maltose during
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sludge hydrolysis. Acetate kinase (AK) is responsible for the transformations of monosaccharides and amino acids into acetate, whereas coenzyme F420 plays an important role in the conversion of acetate to methane.[37] Clearly, methane production may be affected by these key enzymes. Figure 6 shows the relative protease, amylase, AK, and coenzyme F420 activities after short-term and long-term exposure to 6, 30, or 150 mg g¡1-TSS of TiO2 or ZnO NPs. The data indicated that both short-term and longterm exposure to TiO2 NPs had no adverse effects on the protease, amylase, AK, and coenzyme F420 activities, which is consistent with the lack of measurable impacts of TiO2 NPs on methane production from anaerobic sludge co-digestion. However, compared with the control, the relative protease activity was significantly inhibited after short-term exposure to 150 mg g¡1-TSS of ZnO NPs, which might be an important reason for the inhibition of sludge hydrolysis by ZnO NPs. Meanwhile, the activities of AK and coenzyme F420 were also substantially suppressed, which is adverse to acidification and methanation processes. Moreover, after long-term exposure, protease, AK, and coenzyme F420 activities were still suppressed by 150 mg g¡1-TSS of ZnO NPs. These results suggested that prolonged exposure did not enable the enzyme activities of the sludge microorganisms to recover, which might explain why the negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time. Previous studies also found that the dissolution of ZnO NPs was able to release some zinc ions, which were responsible for the toxicity of ZnO NPs toward anaerobic fermenting microorganisms in waste activated sludge.[37] A previous study showed that methanogenic archaea were highly sensitive to metal ions, such as zinc ions,[39] which might be one reason for the inhibition of the anaerobic co-digestion of primary and excess sludge by ZnO NPs.
Conclusion Our data showed that short-term and long-term exposure to 6, 30, or 150 mg g¡1-TSS of TiO2 NPs did not affect sludge hydrolysis, acidification, and methane production during anaerobic sludge co-digestion. However, 30 or 150 mg g¡1-TSS of ZnO NPs significantly inhibited methane production after short-term exposure. Further experiments indicated that the presence of ZnO NPs decreased the abundance of methanogenic archaea, and thus lowered the ratio of archaea to bacteria, which was an important reason for the inhibition of methane production. Meanwhile, both short-term and long-term exposure to ZnO NPs substantially suppressed the relative activities of key enzymes related to methane production (proteases, AK, and coenzyme F420), which reduced methane production.
920 These results also confirmed that the negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time.
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[15]
Funding [16]
This work was financially supported by the National HiTech Research and Development Program of China (863 Program) (2011AA060903), the National Natural Science Foundation of China (51425802, 41301558, and 51278354), the Program of Shanghai Subject Chief Scientist (15XD1503400), and the Fundamental Research Funds for the Central Universities (2014KJ006).
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Appendix
Figure A1. X-ray diffraction (XRD) patterns of TiO2 (A) and ZnO NPs (B) used in this study.
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