Environ Sci Pollut Res DOI 10.1007/s11356-015-4385-y
RESEARCH ARTICLE
Effect of multiwalled carbon nanotubes on UASB microbial consortium Tushar Yadav 1 & Alka A. Mungray 1 & Arvind K. Mungray 1
Received: 18 December 2014 / Accepted: 16 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The continuous rise in production and applications of carbon nanotubes (CNTs) has grown a concern about their fate and toxicity in the environment. After use, these nanomaterials pass through sewage and accumulate in wastewater treatment plants. Since, such plants rely on biological degradation of wastes; their activity may decrease due to the presence of CNTs. This study investigated the effect of multiwalled carbon nanotubes (MWCNTs) on upflow anaerobic sludge blanket (UASB) microbial activity. The toxic effect on microbial viability, extracellular polymeric substances (EPS), volatile fatty acids (VFA), and biogas generation was determined. The reduction in a colony-forming unit (CFU) was 29 and 58 % in 1 and 100 mg/L test samples, respectively, as compared to control. The volatile fatty acids and biogas production was also found reduced. The scanning electron microscopy (SEM) and fluorescent microscopy images confirmed that the MWCNT mediated microbial cell damage. This damage caused the increase in EPS carbohydrate, protein, and DNA concentration. Fourier transform infrared (FTIR) spectroscopy results supported the alterations in sludge EPS due to MWCNT. Our observations offer a new insight to understand the nanotoxic effect of MWCNTs on UASB microflora in a complex environment system.
Responsible editor: Robert Duran * Arvind K. Mungray [email protected] 1
Chemical Engineering Department, Sardar Vallabhbhai National Institute of Technology, Ichchhanath, Surat 395007, Gujarat, India
Keywords Nanotoxicity . Multiwalled carbon nanotubes . Extracellular polymeric substances (EPS) . Volatile fatty acids (VFA) . UASB sludge
Introduction Carbon nanotubes (CNTs) are the novel materials with rising commercial interest. The exceptional mechanical, electrical, and physicochemical characteristic features make them one of the extensively used nanomaterials (Kang et al. 2008a). Currently, they have been utilized in several sectors that include energy, construction, biomedical engineering, agriculture, wastewater treatment, etc. (Jia et al. 2005; Sobolev and Gutierrez 2005; De Ibarra et al. 2006; Joseph and Morrison 2006; Lee et al. 2014; Mamba et al. 2014). The increased production and use of CNTs have caused a growing concern about its fate and toxicity in the environment (Lam et al. 2006). The worldwide production of CNT is increasing by approximately 67.1 % that may attain up to 9300 t in 2015 (IRP 2011). In the absence of any well-defined regulation for production, use, and dumping of CNT-containing products, a considerable amount of CNTs will be entering into the environment, creating a potential hazard to human and environmental health (Petersen et al. 2011; Pereira et al. 2014). Presently, some studies are available on the environmental impact of manufactured CNTs. Researchers had worked with bioaccumulation, cytotoxicity, and ecotoxicity of CNTs (Kang et al. 2009; Luongo and Zhang 2010; Chung et al. 2011; Musee et al. 2011; Li et al. 2015), although there is still plenty of information required about the fate, transport, toxicity, and impact of CNTs in the complex environment matrices. The wastewater treatment plants (WWTPs) are designed to treat municipal sewage and industrial wastewater, in which the indigenous microbial community plays a central role in the
Environ Sci Pollut Res
treatment process. The release of CNTs from CNT-based commodities and their accumulation in wastewater treatment plant has been reported (Brar et al. 2010). Few studies modeled the concentration of CNTs in wastewater sludge and effluents, suggesting their concentration in Bμg/L^ (Mueller and Nowack 2008; Gottschalk et al. 2009, 2013); however, their load will increase in the future. In such conditions, their retention in the sludge flocs may decline the working efficiency of wastewater treatment plant (Luongo and Zhang 2010; Musee et al. 2011). Moreover, the various organic and inorganic components of sludge may alter the fate and toxicological effects of CNTs. The sludge component can bind CNTs and cause their stabilization in aqueous media (Hyung et al. 2007) or may assist in other reactions such as reduction/oxidation (Lowry et al. 2012). On the other hand, CNTs may also interact with the noxious waste present in sewage sludge, becoming their carrier and potentially augment the toxic effect (Gupta and Saleh 2013). Some of the recent work reported the effects of CNTs on activated sludge under aerobic conditions. Luongo and Zhang (2010) studied the role of extracellular polymeric substances (EPS) in protecting the microorganisms from MWCNT toxicity and effect on respiration inhibition. They found respiration inhibition to be dose-dependent and were more in sheared mixed liquor compared to the unsheared mixed liquor. Hai et al. (2014) reported the decrease in nutrient removal efficiency, decrease in several enzyme activities and microbial diversity in response to long-term exposure of MWCNTs in activated sludge. In one more work, the antimicrobial activities of MWCNTs functionalized with ethanolamine (EA) groups were investigated against different bacterial species and found toxic (Zardini et al. 2014). Anaerobic digestion, especially upflow anaerobic sludge blanket (UASB) reactors are preferred compare to an activated sludge process nowadays due to many advantages like less area requirement, less sludge generation, broadly applied for high strength wastewater and its biogas generation efficiency (Latif et al. 2011; Abbasi and Abbasi 2012). The microbial communities mediating anaerobic digestion are sensitive to various organic and inorganic toxicants, such as heavy metals, surfactants, and nanoparticles (Chen et al. 2008; Mu et al. 2012). Mu et al. (2012) performed a study to check the sensitivity of anaerobic granular sludge (AGS) toward ZnO NPs. They found that above 100 mg/g of total suspended solids, ZnO NP significantly affects the general physiological activity, EPS content, and methane production of AGS. The toxicity reported was mainly due to the release of Zn+ from ZnO NP. Till date, the effects of CNTs are carried out mostly in an aerobic environment and on isolated species, but still information toward UASB sludge is scarce. There is a shift of
technology from aerobic to anaerobic because of the energy concern in terms of methane. Any work in this direction would be very helpful to understand the response of UASB microflora toward MWCNTs in complex environmental matrix. One study reports the positive effect of single-walled CNT in anaerobic granular sludge (Li et al. 2015). However, no information was still available for the response of UASB flocs to MWCNT exposure. Various wastewater treatment studies support the difference in properties of granular sludge and floccular sludge (Pekin et al. 2010). In a well-established UASB reactor, synthesized granules naturally show resistant to various chemical and physical stresses, and protect inhabitant microflora. On the other hand, floccular sludge remains more susceptible due to the loose mass of EPS that can be penetrated easily by stress factors such as MWCNTs. Floccular sludge is common during initial start-up and in commercial full-scale UASB-based WWTP; therefore, our work presents much more realistic scenario regarding the problem. In this study, we investigated the effect of MWCNTs on UASB microbial flocs. Study was focused to explore microbial cell damage, the biochemical changes occurring in EPS, volatile fatty acid (VFA) production, and biogas generation when exposed to MWCNT. The parameters were selected on the basis of their utility in routine sludge analysis. Scanning electron microscopy (SEM) and fluorescent microscopy were used to observe the morphological variations and physical damage to microbial cells, respectively. Fourier transform infrared (FTIR) spectroscopy was used to confirm the biochemical changes occurred in EPS due to the MWCNT exposure.
Materials and methods Characterization of MWCNTs Commercially available MWCNT was purchased from Reinste Nano Ventures Pvt. Ltd., India (purity 95 %, number of walls 3–15, length 1–10 μm, outer diameter 5–20 nm and inner diameter 2–6 nm, as mentioned by the supplier). MWCNTs were characterized before use as mentioned in previous literature (Jaisi et al. 2008). The metal impurities were examined by using SEM with energy-dispersive X-ray spectroscopy (SEM-EDS). The field emission gun SEM (FEGSEM) was used for morphology analysis. Preparation of MWCNT stock suspension For stock suspension, the MWCNT (1.0 g/L) was ultrasonicated (sweep-mode, 80 % power, 45 kHz, 25 °C) in deionized (DI) water for 60 min. The dispersed MWCNTs were stable in suspension for several days. This suspension
Environ Sci Pollut Res
was used for subsequent toxicity experiments after 10-min sonication just prior to experiments. Preparation of test samples Preparation of sludge The anaerobic sludge (floccular, COD=55,506 mg/L, VSS= 22.95 g/L, pH≈8) was collected from a lab-scale UASB reactor and analyzed. The test samples exposed to two different concentrations of MWCNTs (1 and 100 mg/L), and one taken as control (0 mg/L). The concentration of MWCNT was selected on the basis of previous studies as well as the futuristic high emission scenario. A designated quantity of MWCNTs was added to every flask except the control flasks that was added with only DI water. Subsequently, nitrogen was purged into all flasks in order to create anaerobic condition. Flasks were made airtight and placed in a shaker incubator maintained at 37 °C, 150 rpm. Aliquots of sludge were collected at different time intervals from every flask for analysis. Preparation of FEG-SEM samples The FEG-SEM, model JSM-7600 F with resolution 1.0 nm (15 kV), 1.5 nm (1 kV), and magnification×25 to 1,000,000, was used to image the morphology of MWCNTs and to study the test samples. FEG-SEM imaging was done to detect the morphology of MWCNT, its status in sludge, and its interaction with microbial flocs. Sample preparation was done according to Kumar et al. (2011) and analyzed. Microbial inhibition test For total culturable microflora from the exposed sludge, triplicates of 10 mL of sludge samples were taken. These samples were exposed to 1- and 100-mg/L MWCNT concentrations, incubated overnight in a shaker incubator at 37 °C, 150 rpm in anaerobic condition. One milliliter from each aliquot diluted in 9 mL of 1× PBS (pH≈8). The suspension was vortexed for 5 min and serially diluted. The spread plate method was used for plating. Agar medium (1 g glucose, 0.5 g yeast extract, 0.5 g K2HPO4, 15 g agar) supplemented with sludge extract was prepared to imitate natural nutrient conditions for the microbial consortia (Rodrigues et al. 2013). All the plates were incubated in an anaerobic chamber for 48 h at 37 °C, and observations were made. A fluorescent test was used to confirm the cell damage after 24-h incubation (37 °C, 150 rpm, anaerobic) with MWCNT. The triplicates of control and test sludge were suspended in phosphate buffer, washed twice using the centrifuge (6000 rpm, 15 min) and suitably diluted. The fluorescent dye propidium iodide (10 μg/mL) added to the samples and incubated for 15 min in a closed chamber. After incubation,
samples were vortexed slightly to disperse the microbial cells. The cells for imaging were taken from dispersed suspension in order to improve the clarity of images obtained. Thereafter, aliquots were taken on a clean slide and observed under the fluorescent microscope. The red fluorescent spots were counted for nonviable bacterial cells in control as well as in test slides. Analysis of EPS Test samples (with MWCNTs) and control (sludge with no MWCNTs) in triplicates were incubated for 15 days at 37± 1 °C, 150 rpm, on shaker under anaerobic condition. Samples from day 15 were extracted according to Yang and Li (2009) and analyzed. The overall EPS extracted was examined for total carbohydrate, protein, and deoxyribonucleic acid (DNA). Total carbohydrate content quantified by the phenolsulfuric acid method (Dubois et al. 1956), and glucose was used as a standard. Total protein was measured according to the method of Lowry (Lowry et al. 1951) using bovine serum albumin as standard. For the quantification of DNA, absorbance (A) at 260 nm (Chen and Cooper 2002) was observed with the help of UV–vis spectrophotometer (DR 6000, Hach, USA). The following equation was used to quantify Unknown DNA concentration ðμg=mLÞ ¼ 50 μg=mL measured A260 where concentration of pure double-stranded DNA with an A260 of 1.0=50 μg/mL. The extracted EPS was also analyzed using a FTIR spectrophotometer (Shimadzu, 8400S with DRS) for alterations produced due to the presence of MWCNTs. Analysis of VFA and biogas production The triplicates of sludge samples were prepared with concentration 100 and 1 mg/L of MWCNT and control. Samples were purged with nitrogen gas to create anaerobic conditions and kept airtight at 37±1 °C for 15 days on shaker (150 rpm). VFA was measured by the modified Montgomery method (Montgomery et al. 1962; Siedlecka et al. 2008). The UV– vis spectrophotometer (DR 6000, Hach) was used to take absorbance at 495 nm. Acetic acid (1200 mg/L CH3COOH) was used as a standard. For the effect of MWCNTs on biogas production, 250-mL airtight flasks were used. Four setups were prepared in triplicates. Test set contained 100 mL anaerobic sludge and 100 mL of dextrose (0.4 g) solution containing MWCN∁Ts (1 and 100 mg/L), to get a final volume of 200 mL. A set of blank and control was also kept for comparison. The blank set (100 mL sludge and DI water to final volume 200 mL) was
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placed to facilitate biogas production from any biodegradable organic matter already present in the sludge. The control set (100 mL sludge and 100 mL of DI water containing 0.4 g dextrose) was used to compare biogas production with test sample (Garcia et al. 2012). These setups were maintained for 25 days at 37±1 °C on an orbital shaker (REMI, India) at 150 rpm. Biogas was collected in gas collection bags and weighed carefully.
Results and discussion Characterization of MWCNTs The purity of MWCNT is imperative for any eco-toxicological analyses. Several assumptions have been published to elucidate the possible causes of CNT toxicity, and one is the occurrence of the residual catalyst used during the CNT fabrication processes (Kang et al. 2008a). Figure 1a shows the analytical details of MWCNTs on SEM-EDS. The major peak obtained of carbon that represents CNTs. Analysis indicated a metal content of 0.52 % (by weight), with magnesium and aluminum as the major metals, oxygen 1.20 %. The MWCNT sample was approximately 98.28 % pure. Morphological analysis of MWCNTs was carried out in FEG-SEM. Figure 1b represents the cluster of MWCNTs. The measured average diameter of MWCNTs was 60 nm. Figure 2 designates the status of MWCNTs in UASB sludge. It is a well-known fact that the high surface area to volume ratio of NPs causes their aggregation in aqueous media. In addition to aggregation, several other alterations in the open environment modify physiochemical properties, reactivity, fate, transport, and biological interactions of NPs (Zhang 2014). As expected, the nanotubes seem to form aggregates in sludge assisted by thick, viscous organic matrix, even though the pointed ends can be seen clearly emerging out from
the aggregates. Such structures may entangle the microbial flocs around and damage the cells present in the close vicinity. Microbial inhibition analysis In the present study, the decrease in CFU count (Table 1, 29 % in 1 mg/L and 58 % in 100 mg/L test samples) was observed among test samples with respect to control that represents the inhibition of growth. The factors responsible for CNT-induced toxicity may involve (i) oxidative stress, (ii) residual catalyst, and (iii) direct membrane penetration (Kang et al. 2007, 2008a, b; Pasquini et al. 2013). Under aerobic conditions, reactive oxygen species (ROS) may generate by nanoparticle activity. Here, due to anaerobic conditions, the toxicity due to oxidative stress might not be significant. Moreover, the MWCNT used here was more than 98 % pure that reduces the chances of residual catalyst-based toxicity. Our results suggest the third possibility, i.e., the direct physical contact between the bacterial cells and MWCNTs that caused membrane damage. The images obtained in SEM analysis supported this fact. Figure 3a, b represents the SEM images of test (100 mg/L MWCNT) samples. Test samples were showing cells with a rough surface and damaged by the presence of MWCNTs. Earlier, Young et al. (2012) presented the concept of the cell fracture/breakage in an isolated culture of E. coli caused by the MWCNT stress while shaking and incubation. Here, as well as in Fig. 3a, the cell damage was evident. In Fig. 3b, a bacterial cell can be seen covered by MWCNTs, which could be the initial stage of cell damage/breakage. The phenomenon of cell damage was also supported by the fluorescent test results as shown in Fig. 4a, b. Table 1 summarizes the average number of fluorescent spots counted that corresponds to the number of nonviable/damaged cells in control and test (100 mg/L MWCNT) samples. Propidium iodide is a fluorescent dye that exclusively stains cells with a damaged membrane. The test samples expressed a higher number of damaged cells as compared to control. The larger diameter
Fig. 1 a SEM-EDS analysis of MWCNTs. b Morphology of MWCNTs observed in FEG-SEM image
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Fig. 2 Status of MWCNTs in UASB sludge. The pointed ends of MWCNTs are visible within clusters
due to multiple graphene layers makes MWCNTs highly stiff and rigid. Therefore, during incubation, the cell membrane damage occurs caused by MWCNT piercing leading to cell death or reduced viability.
Effect of MWCNTs on EPS EPS biochemical test The UASB biomass is a complex dynamic structure of bacterial flocs surrounded by a matrix of EPS. Major components of EPS are humic-like substances, proteins, and polysaccharides secreted by microbial cells. EPS form a protective layer around microbial cells. They protect cells from the unfavorable external environment such as turbulence, dehydration, antibiotics, and biocides (Wingender et al. 1999). They serve as carbon and energy reserves during starvation, promote cell aggregation, as well as vital for the microstructure of methanogenic granular sludge (Liu and Fang 2002). In anaerobic sludge, EPS are responsible for producing microbial flocs and keeping colonies, cells, and other particulate materials together, making the sludge flocculent in appearance. Even though the components and quantity of EPS generated by Table 1 Average number of CFUs/mL obtained in microbial inhibition analysis and average number of spot count observed in fluorescent assay after incubation with MWCNT
the bacterium is a genetically determined feature, media components and growth environment affects them greatly. Figure 5 represents the average concentration of carbohydrate, protein, and DNA obtained from control and test EPS samples. The concentration of EPS carbohydrates was found to increase in test sample numbers 1 and 2 at the end of 15-day incubation as compared to control. The carbohydrates increased to 114.76 and 122.97 mg/L in the test samples 1 and 2, respectively. It suggests that due to the influence of MWCN Ts, EPS from the microbial flocs released into the bulk liquor. The protein concentration also found higher in test sample 1 (24 mg/L) and 2 (34.7 mg/L) than measured for control (22 mg/L). The result satisfies the observations made by Hou et al. (2014) on EPS for CuO NP toxicity in activated sludge flocs. The increase in DNA concentration was also found in test samples. Few earlier works suggests that extracellular DNA may be found in EPS as a result of cellular autolysis in the presence of chemical toxins or it may be produced on purpose in order to assist the biofilm formation (Thomas et al. 2008; Mann et al. 2009; Pammi et al. 2013). However, in our case, physical damage was evident from fluorescent assay and SEM images that helped to conclude that DNA release was majorly due to the cell damage as a result of MWCNT penetration. The increases in DNA concentration observed were 27.01 and 30.61 mg/L for test samples 1 and 2, respectively. The single factor ANOVA (p< 0.05) expressed with mean±standard deviation showed the significant increase in the concentration of EPS carbohydrates, proteins, and DNA in the test samples, due to the presence of MWCWNT. Results confirm the severe damage to cell membrane integrity, especially in the higher concentration of MWCNT.
FTIR analysis of EPS samples The FTIR spectrum was analyzed to verify the changes that may have occurred in EPS biochemical composition due to MWCNT. Figure 6 shows the FTIR spectra of extracted EPS from control and test sludge samples. The samples of the 24th hour (Fig. 6a) and day 15 (Fig. 6b) were investigated. As shown in the figure, the absorption band near 3403–
Sample
Microbial inhibition Average number of CFUs/ ml
Fluorescent assay Average number of spot count
Controla Test 1b Test 2c
800±1.73 566±2.51 333±1.52
28±10.14 – 73.67±10.01
a
Control=no MWCNT added
b
Test 1=1 mg/L MWCNT
c
Test 2=100 mg/L MWCNT
Environ Sci Pollut Res Fig. 3 FEG-SEM images of test samples (100 mg/L MWCNTs): a damaged bacterial cells; b a single bacterial cell entangled in MWCNT aggregate
3446 cm−1 represents the stretching vibration of both hydroxyl (of carbohydrates) and amino groups (of proteins). The peak around 2347 cm−1 attributed to groups like –NH2, −NH, =NH–. The absorption band near 1604 and 1404 cm−1 are related to the stretching vibration of primary amides and RCOO– groups respectively in the presence of proteins. The band near 1119–1120 cm−1 correlated with the C–O stretching vibrations from the carbohydrates and aromatics. The peaks around 669 cm−1 reflect the presence of unsaturated bonds in the sample (Guibaud et al. 2005). Among control, there were small peaks present in the 24th hour as well as the 15th day sample. Though on comparison, the test samples showed enlarged peaks since the 24th hour that was found further increased on the 15th day EPS. The peak at 1404 cm−1 suggests the presence of plentiful lipid components in EPS that was increased due to the effect of MWCNTs in the test samples. The elevated peaks near 1655–1604 and 3400 cm−1 in the test samples were indicative of an increased level of proteins in EPS. The range from 1054 to 1125 cm−1 represents carbohydrates in EPS, which were found with higher absorption intensity in both the test samples. The peaks 1120 and 1055 cm−1 from the test samples lie in the region 1250–1000 cm−1, suggestive of vibrations along the sugar-phosphate string, provide marker bands responsive to nucleic acid backbone conformation (Banyay et al. 2003). These peaks were increased among the test samples as compared to the control that may be due to the more nucleic acid in the exposed samples, a resultant of cell damage. Fig. 4 Fluorescent microscopy images (×40) of damaged cells: a control sample with no MWCNT; b sample treated with 100 mg/L MWCNT
The overall results obtained in FTIR spectra of the 24th hour and day 15 showed considerable dominant peaks among the test samples as compared to control suggestive of increased EPS components in the presence of MWCNTs. This result was found consistent with the outcome of biochemical analysis of EPS protein, carbohydrate, and DNA described elsewhere in this text. Effect of MWCNTs on VFAs and biogas production During steady operating conditions, the acidogenic and acetogenic bacteria produce hydrogen and acetic acid, and methanogens instantly convert it to methane. On the contrary, during overload conditions or in the presence of toxins or inhibitory substances, the microbial activity reduces. In the present study, decreasing pattern of VFA was observed in all the samples (Fig. 7a). The results were analyzed using oneway repeated measure ANOVA with Origin 2015 statistical software. There is a significant effect (p
RESEARCH ARTICLE
Effect of multiwalled carbon nanotubes on UASB microbial consortium Tushar Yadav 1 & Alka A. Mungray 1 & Arvind K. Mungray 1
Received: 18 December 2014 / Accepted: 16 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The continuous rise in production and applications of carbon nanotubes (CNTs) has grown a concern about their fate and toxicity in the environment. After use, these nanomaterials pass through sewage and accumulate in wastewater treatment plants. Since, such plants rely on biological degradation of wastes; their activity may decrease due to the presence of CNTs. This study investigated the effect of multiwalled carbon nanotubes (MWCNTs) on upflow anaerobic sludge blanket (UASB) microbial activity. The toxic effect on microbial viability, extracellular polymeric substances (EPS), volatile fatty acids (VFA), and biogas generation was determined. The reduction in a colony-forming unit (CFU) was 29 and 58 % in 1 and 100 mg/L test samples, respectively, as compared to control. The volatile fatty acids and biogas production was also found reduced. The scanning electron microscopy (SEM) and fluorescent microscopy images confirmed that the MWCNT mediated microbial cell damage. This damage caused the increase in EPS carbohydrate, protein, and DNA concentration. Fourier transform infrared (FTIR) spectroscopy results supported the alterations in sludge EPS due to MWCNT. Our observations offer a new insight to understand the nanotoxic effect of MWCNTs on UASB microflora in a complex environment system.
Responsible editor: Robert Duran * Arvind K. Mungray [email protected] 1
Chemical Engineering Department, Sardar Vallabhbhai National Institute of Technology, Ichchhanath, Surat 395007, Gujarat, India
Keywords Nanotoxicity . Multiwalled carbon nanotubes . Extracellular polymeric substances (EPS) . Volatile fatty acids (VFA) . UASB sludge
Introduction Carbon nanotubes (CNTs) are the novel materials with rising commercial interest. The exceptional mechanical, electrical, and physicochemical characteristic features make them one of the extensively used nanomaterials (Kang et al. 2008a). Currently, they have been utilized in several sectors that include energy, construction, biomedical engineering, agriculture, wastewater treatment, etc. (Jia et al. 2005; Sobolev and Gutierrez 2005; De Ibarra et al. 2006; Joseph and Morrison 2006; Lee et al. 2014; Mamba et al. 2014). The increased production and use of CNTs have caused a growing concern about its fate and toxicity in the environment (Lam et al. 2006). The worldwide production of CNT is increasing by approximately 67.1 % that may attain up to 9300 t in 2015 (IRP 2011). In the absence of any well-defined regulation for production, use, and dumping of CNT-containing products, a considerable amount of CNTs will be entering into the environment, creating a potential hazard to human and environmental health (Petersen et al. 2011; Pereira et al. 2014). Presently, some studies are available on the environmental impact of manufactured CNTs. Researchers had worked with bioaccumulation, cytotoxicity, and ecotoxicity of CNTs (Kang et al. 2009; Luongo and Zhang 2010; Chung et al. 2011; Musee et al. 2011; Li et al. 2015), although there is still plenty of information required about the fate, transport, toxicity, and impact of CNTs in the complex environment matrices. The wastewater treatment plants (WWTPs) are designed to treat municipal sewage and industrial wastewater, in which the indigenous microbial community plays a central role in the
Environ Sci Pollut Res
treatment process. The release of CNTs from CNT-based commodities and their accumulation in wastewater treatment plant has been reported (Brar et al. 2010). Few studies modeled the concentration of CNTs in wastewater sludge and effluents, suggesting their concentration in Bμg/L^ (Mueller and Nowack 2008; Gottschalk et al. 2009, 2013); however, their load will increase in the future. In such conditions, their retention in the sludge flocs may decline the working efficiency of wastewater treatment plant (Luongo and Zhang 2010; Musee et al. 2011). Moreover, the various organic and inorganic components of sludge may alter the fate and toxicological effects of CNTs. The sludge component can bind CNTs and cause their stabilization in aqueous media (Hyung et al. 2007) or may assist in other reactions such as reduction/oxidation (Lowry et al. 2012). On the other hand, CNTs may also interact with the noxious waste present in sewage sludge, becoming their carrier and potentially augment the toxic effect (Gupta and Saleh 2013). Some of the recent work reported the effects of CNTs on activated sludge under aerobic conditions. Luongo and Zhang (2010) studied the role of extracellular polymeric substances (EPS) in protecting the microorganisms from MWCNT toxicity and effect on respiration inhibition. They found respiration inhibition to be dose-dependent and were more in sheared mixed liquor compared to the unsheared mixed liquor. Hai et al. (2014) reported the decrease in nutrient removal efficiency, decrease in several enzyme activities and microbial diversity in response to long-term exposure of MWCNTs in activated sludge. In one more work, the antimicrobial activities of MWCNTs functionalized with ethanolamine (EA) groups were investigated against different bacterial species and found toxic (Zardini et al. 2014). Anaerobic digestion, especially upflow anaerobic sludge blanket (UASB) reactors are preferred compare to an activated sludge process nowadays due to many advantages like less area requirement, less sludge generation, broadly applied for high strength wastewater and its biogas generation efficiency (Latif et al. 2011; Abbasi and Abbasi 2012). The microbial communities mediating anaerobic digestion are sensitive to various organic and inorganic toxicants, such as heavy metals, surfactants, and nanoparticles (Chen et al. 2008; Mu et al. 2012). Mu et al. (2012) performed a study to check the sensitivity of anaerobic granular sludge (AGS) toward ZnO NPs. They found that above 100 mg/g of total suspended solids, ZnO NP significantly affects the general physiological activity, EPS content, and methane production of AGS. The toxicity reported was mainly due to the release of Zn+ from ZnO NP. Till date, the effects of CNTs are carried out mostly in an aerobic environment and on isolated species, but still information toward UASB sludge is scarce. There is a shift of
technology from aerobic to anaerobic because of the energy concern in terms of methane. Any work in this direction would be very helpful to understand the response of UASB microflora toward MWCNTs in complex environmental matrix. One study reports the positive effect of single-walled CNT in anaerobic granular sludge (Li et al. 2015). However, no information was still available for the response of UASB flocs to MWCNT exposure. Various wastewater treatment studies support the difference in properties of granular sludge and floccular sludge (Pekin et al. 2010). In a well-established UASB reactor, synthesized granules naturally show resistant to various chemical and physical stresses, and protect inhabitant microflora. On the other hand, floccular sludge remains more susceptible due to the loose mass of EPS that can be penetrated easily by stress factors such as MWCNTs. Floccular sludge is common during initial start-up and in commercial full-scale UASB-based WWTP; therefore, our work presents much more realistic scenario regarding the problem. In this study, we investigated the effect of MWCNTs on UASB microbial flocs. Study was focused to explore microbial cell damage, the biochemical changes occurring in EPS, volatile fatty acid (VFA) production, and biogas generation when exposed to MWCNT. The parameters were selected on the basis of their utility in routine sludge analysis. Scanning electron microscopy (SEM) and fluorescent microscopy were used to observe the morphological variations and physical damage to microbial cells, respectively. Fourier transform infrared (FTIR) spectroscopy was used to confirm the biochemical changes occurred in EPS due to the MWCNT exposure.
Materials and methods Characterization of MWCNTs Commercially available MWCNT was purchased from Reinste Nano Ventures Pvt. Ltd., India (purity 95 %, number of walls 3–15, length 1–10 μm, outer diameter 5–20 nm and inner diameter 2–6 nm, as mentioned by the supplier). MWCNTs were characterized before use as mentioned in previous literature (Jaisi et al. 2008). The metal impurities were examined by using SEM with energy-dispersive X-ray spectroscopy (SEM-EDS). The field emission gun SEM (FEGSEM) was used for morphology analysis. Preparation of MWCNT stock suspension For stock suspension, the MWCNT (1.0 g/L) was ultrasonicated (sweep-mode, 80 % power, 45 kHz, 25 °C) in deionized (DI) water for 60 min. The dispersed MWCNTs were stable in suspension for several days. This suspension
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was used for subsequent toxicity experiments after 10-min sonication just prior to experiments. Preparation of test samples Preparation of sludge The anaerobic sludge (floccular, COD=55,506 mg/L, VSS= 22.95 g/L, pH≈8) was collected from a lab-scale UASB reactor and analyzed. The test samples exposed to two different concentrations of MWCNTs (1 and 100 mg/L), and one taken as control (0 mg/L). The concentration of MWCNT was selected on the basis of previous studies as well as the futuristic high emission scenario. A designated quantity of MWCNTs was added to every flask except the control flasks that was added with only DI water. Subsequently, nitrogen was purged into all flasks in order to create anaerobic condition. Flasks were made airtight and placed in a shaker incubator maintained at 37 °C, 150 rpm. Aliquots of sludge were collected at different time intervals from every flask for analysis. Preparation of FEG-SEM samples The FEG-SEM, model JSM-7600 F with resolution 1.0 nm (15 kV), 1.5 nm (1 kV), and magnification×25 to 1,000,000, was used to image the morphology of MWCNTs and to study the test samples. FEG-SEM imaging was done to detect the morphology of MWCNT, its status in sludge, and its interaction with microbial flocs. Sample preparation was done according to Kumar et al. (2011) and analyzed. Microbial inhibition test For total culturable microflora from the exposed sludge, triplicates of 10 mL of sludge samples were taken. These samples were exposed to 1- and 100-mg/L MWCNT concentrations, incubated overnight in a shaker incubator at 37 °C, 150 rpm in anaerobic condition. One milliliter from each aliquot diluted in 9 mL of 1× PBS (pH≈8). The suspension was vortexed for 5 min and serially diluted. The spread plate method was used for plating. Agar medium (1 g glucose, 0.5 g yeast extract, 0.5 g K2HPO4, 15 g agar) supplemented with sludge extract was prepared to imitate natural nutrient conditions for the microbial consortia (Rodrigues et al. 2013). All the plates were incubated in an anaerobic chamber for 48 h at 37 °C, and observations were made. A fluorescent test was used to confirm the cell damage after 24-h incubation (37 °C, 150 rpm, anaerobic) with MWCNT. The triplicates of control and test sludge were suspended in phosphate buffer, washed twice using the centrifuge (6000 rpm, 15 min) and suitably diluted. The fluorescent dye propidium iodide (10 μg/mL) added to the samples and incubated for 15 min in a closed chamber. After incubation,
samples were vortexed slightly to disperse the microbial cells. The cells for imaging were taken from dispersed suspension in order to improve the clarity of images obtained. Thereafter, aliquots were taken on a clean slide and observed under the fluorescent microscope. The red fluorescent spots were counted for nonviable bacterial cells in control as well as in test slides. Analysis of EPS Test samples (with MWCNTs) and control (sludge with no MWCNTs) in triplicates were incubated for 15 days at 37± 1 °C, 150 rpm, on shaker under anaerobic condition. Samples from day 15 were extracted according to Yang and Li (2009) and analyzed. The overall EPS extracted was examined for total carbohydrate, protein, and deoxyribonucleic acid (DNA). Total carbohydrate content quantified by the phenolsulfuric acid method (Dubois et al. 1956), and glucose was used as a standard. Total protein was measured according to the method of Lowry (Lowry et al. 1951) using bovine serum albumin as standard. For the quantification of DNA, absorbance (A) at 260 nm (Chen and Cooper 2002) was observed with the help of UV–vis spectrophotometer (DR 6000, Hach, USA). The following equation was used to quantify Unknown DNA concentration ðμg=mLÞ ¼ 50 μg=mL measured A260 where concentration of pure double-stranded DNA with an A260 of 1.0=50 μg/mL. The extracted EPS was also analyzed using a FTIR spectrophotometer (Shimadzu, 8400S with DRS) for alterations produced due to the presence of MWCNTs. Analysis of VFA and biogas production The triplicates of sludge samples were prepared with concentration 100 and 1 mg/L of MWCNT and control. Samples were purged with nitrogen gas to create anaerobic conditions and kept airtight at 37±1 °C for 15 days on shaker (150 rpm). VFA was measured by the modified Montgomery method (Montgomery et al. 1962; Siedlecka et al. 2008). The UV– vis spectrophotometer (DR 6000, Hach) was used to take absorbance at 495 nm. Acetic acid (1200 mg/L CH3COOH) was used as a standard. For the effect of MWCNTs on biogas production, 250-mL airtight flasks were used. Four setups were prepared in triplicates. Test set contained 100 mL anaerobic sludge and 100 mL of dextrose (0.4 g) solution containing MWCN∁Ts (1 and 100 mg/L), to get a final volume of 200 mL. A set of blank and control was also kept for comparison. The blank set (100 mL sludge and DI water to final volume 200 mL) was
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placed to facilitate biogas production from any biodegradable organic matter already present in the sludge. The control set (100 mL sludge and 100 mL of DI water containing 0.4 g dextrose) was used to compare biogas production with test sample (Garcia et al. 2012). These setups were maintained for 25 days at 37±1 °C on an orbital shaker (REMI, India) at 150 rpm. Biogas was collected in gas collection bags and weighed carefully.
Results and discussion Characterization of MWCNTs The purity of MWCNT is imperative for any eco-toxicological analyses. Several assumptions have been published to elucidate the possible causes of CNT toxicity, and one is the occurrence of the residual catalyst used during the CNT fabrication processes (Kang et al. 2008a). Figure 1a shows the analytical details of MWCNTs on SEM-EDS. The major peak obtained of carbon that represents CNTs. Analysis indicated a metal content of 0.52 % (by weight), with magnesium and aluminum as the major metals, oxygen 1.20 %. The MWCNT sample was approximately 98.28 % pure. Morphological analysis of MWCNTs was carried out in FEG-SEM. Figure 1b represents the cluster of MWCNTs. The measured average diameter of MWCNTs was 60 nm. Figure 2 designates the status of MWCNTs in UASB sludge. It is a well-known fact that the high surface area to volume ratio of NPs causes their aggregation in aqueous media. In addition to aggregation, several other alterations in the open environment modify physiochemical properties, reactivity, fate, transport, and biological interactions of NPs (Zhang 2014). As expected, the nanotubes seem to form aggregates in sludge assisted by thick, viscous organic matrix, even though the pointed ends can be seen clearly emerging out from
the aggregates. Such structures may entangle the microbial flocs around and damage the cells present in the close vicinity. Microbial inhibition analysis In the present study, the decrease in CFU count (Table 1, 29 % in 1 mg/L and 58 % in 100 mg/L test samples) was observed among test samples with respect to control that represents the inhibition of growth. The factors responsible for CNT-induced toxicity may involve (i) oxidative stress, (ii) residual catalyst, and (iii) direct membrane penetration (Kang et al. 2007, 2008a, b; Pasquini et al. 2013). Under aerobic conditions, reactive oxygen species (ROS) may generate by nanoparticle activity. Here, due to anaerobic conditions, the toxicity due to oxidative stress might not be significant. Moreover, the MWCNT used here was more than 98 % pure that reduces the chances of residual catalyst-based toxicity. Our results suggest the third possibility, i.e., the direct physical contact between the bacterial cells and MWCNTs that caused membrane damage. The images obtained in SEM analysis supported this fact. Figure 3a, b represents the SEM images of test (100 mg/L MWCNT) samples. Test samples were showing cells with a rough surface and damaged by the presence of MWCNTs. Earlier, Young et al. (2012) presented the concept of the cell fracture/breakage in an isolated culture of E. coli caused by the MWCNT stress while shaking and incubation. Here, as well as in Fig. 3a, the cell damage was evident. In Fig. 3b, a bacterial cell can be seen covered by MWCNTs, which could be the initial stage of cell damage/breakage. The phenomenon of cell damage was also supported by the fluorescent test results as shown in Fig. 4a, b. Table 1 summarizes the average number of fluorescent spots counted that corresponds to the number of nonviable/damaged cells in control and test (100 mg/L MWCNT) samples. Propidium iodide is a fluorescent dye that exclusively stains cells with a damaged membrane. The test samples expressed a higher number of damaged cells as compared to control. The larger diameter
Fig. 1 a SEM-EDS analysis of MWCNTs. b Morphology of MWCNTs observed in FEG-SEM image
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Fig. 2 Status of MWCNTs in UASB sludge. The pointed ends of MWCNTs are visible within clusters
due to multiple graphene layers makes MWCNTs highly stiff and rigid. Therefore, during incubation, the cell membrane damage occurs caused by MWCNT piercing leading to cell death or reduced viability.
Effect of MWCNTs on EPS EPS biochemical test The UASB biomass is a complex dynamic structure of bacterial flocs surrounded by a matrix of EPS. Major components of EPS are humic-like substances, proteins, and polysaccharides secreted by microbial cells. EPS form a protective layer around microbial cells. They protect cells from the unfavorable external environment such as turbulence, dehydration, antibiotics, and biocides (Wingender et al. 1999). They serve as carbon and energy reserves during starvation, promote cell aggregation, as well as vital for the microstructure of methanogenic granular sludge (Liu and Fang 2002). In anaerobic sludge, EPS are responsible for producing microbial flocs and keeping colonies, cells, and other particulate materials together, making the sludge flocculent in appearance. Even though the components and quantity of EPS generated by Table 1 Average number of CFUs/mL obtained in microbial inhibition analysis and average number of spot count observed in fluorescent assay after incubation with MWCNT
the bacterium is a genetically determined feature, media components and growth environment affects them greatly. Figure 5 represents the average concentration of carbohydrate, protein, and DNA obtained from control and test EPS samples. The concentration of EPS carbohydrates was found to increase in test sample numbers 1 and 2 at the end of 15-day incubation as compared to control. The carbohydrates increased to 114.76 and 122.97 mg/L in the test samples 1 and 2, respectively. It suggests that due to the influence of MWCN Ts, EPS from the microbial flocs released into the bulk liquor. The protein concentration also found higher in test sample 1 (24 mg/L) and 2 (34.7 mg/L) than measured for control (22 mg/L). The result satisfies the observations made by Hou et al. (2014) on EPS for CuO NP toxicity in activated sludge flocs. The increase in DNA concentration was also found in test samples. Few earlier works suggests that extracellular DNA may be found in EPS as a result of cellular autolysis in the presence of chemical toxins or it may be produced on purpose in order to assist the biofilm formation (Thomas et al. 2008; Mann et al. 2009; Pammi et al. 2013). However, in our case, physical damage was evident from fluorescent assay and SEM images that helped to conclude that DNA release was majorly due to the cell damage as a result of MWCNT penetration. The increases in DNA concentration observed were 27.01 and 30.61 mg/L for test samples 1 and 2, respectively. The single factor ANOVA (p< 0.05) expressed with mean±standard deviation showed the significant increase in the concentration of EPS carbohydrates, proteins, and DNA in the test samples, due to the presence of MWCWNT. Results confirm the severe damage to cell membrane integrity, especially in the higher concentration of MWCNT.
FTIR analysis of EPS samples The FTIR spectrum was analyzed to verify the changes that may have occurred in EPS biochemical composition due to MWCNT. Figure 6 shows the FTIR spectra of extracted EPS from control and test sludge samples. The samples of the 24th hour (Fig. 6a) and day 15 (Fig. 6b) were investigated. As shown in the figure, the absorption band near 3403–
Sample
Microbial inhibition Average number of CFUs/ ml
Fluorescent assay Average number of spot count
Controla Test 1b Test 2c
800±1.73 566±2.51 333±1.52
28±10.14 – 73.67±10.01
a
Control=no MWCNT added
b
Test 1=1 mg/L MWCNT
c
Test 2=100 mg/L MWCNT
Environ Sci Pollut Res Fig. 3 FEG-SEM images of test samples (100 mg/L MWCNTs): a damaged bacterial cells; b a single bacterial cell entangled in MWCNT aggregate
3446 cm−1 represents the stretching vibration of both hydroxyl (of carbohydrates) and amino groups (of proteins). The peak around 2347 cm−1 attributed to groups like –NH2, −NH, =NH–. The absorption band near 1604 and 1404 cm−1 are related to the stretching vibration of primary amides and RCOO– groups respectively in the presence of proteins. The band near 1119–1120 cm−1 correlated with the C–O stretching vibrations from the carbohydrates and aromatics. The peaks around 669 cm−1 reflect the presence of unsaturated bonds in the sample (Guibaud et al. 2005). Among control, there were small peaks present in the 24th hour as well as the 15th day sample. Though on comparison, the test samples showed enlarged peaks since the 24th hour that was found further increased on the 15th day EPS. The peak at 1404 cm−1 suggests the presence of plentiful lipid components in EPS that was increased due to the effect of MWCNTs in the test samples. The elevated peaks near 1655–1604 and 3400 cm−1 in the test samples were indicative of an increased level of proteins in EPS. The range from 1054 to 1125 cm−1 represents carbohydrates in EPS, which were found with higher absorption intensity in both the test samples. The peaks 1120 and 1055 cm−1 from the test samples lie in the region 1250–1000 cm−1, suggestive of vibrations along the sugar-phosphate string, provide marker bands responsive to nucleic acid backbone conformation (Banyay et al. 2003). These peaks were increased among the test samples as compared to the control that may be due to the more nucleic acid in the exposed samples, a resultant of cell damage. Fig. 4 Fluorescent microscopy images (×40) of damaged cells: a control sample with no MWCNT; b sample treated with 100 mg/L MWCNT
The overall results obtained in FTIR spectra of the 24th hour and day 15 showed considerable dominant peaks among the test samples as compared to control suggestive of increased EPS components in the presence of MWCNTs. This result was found consistent with the outcome of biochemical analysis of EPS protein, carbohydrate, and DNA described elsewhere in this text. Effect of MWCNTs on VFAs and biogas production During steady operating conditions, the acidogenic and acetogenic bacteria produce hydrogen and acetic acid, and methanogens instantly convert it to methane. On the contrary, during overload conditions or in the presence of toxins or inhibitory substances, the microbial activity reduces. In the present study, decreasing pattern of VFA was observed in all the samples (Fig. 7a). The results were analyzed using oneway repeated measure ANOVA with Origin 2015 statistical software. There is a significant effect (p
