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Simultaneous Cr(VI) bio-reduction and methane production by anaerobic granular sludge
Accepted Manuscript Simultaneous Cr(VI) Bio-reduction and Methane Production by Anaerobic Granular sludge Qian Hu, Jiaji Sun, Dezhi Sun, Lan Tian, Yanan Ji, Bin Qiu PII: DOI: Reference:
S0960-8524(18)30573-X https://doi.org/10.1016/j.biortech.2018.04.060 BITE 19840
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 February 2018 12 April 2018 14 April 2018
Please cite this article as: Hu, Q., Sun, J., Sun, D., Tian, L., Ji, Y., Qiu, B., Simultaneous Cr(VI) Bio-reduction and Methane Production by Anaerobic Granular sludge, Bioresource Technology (2018), doi: https://doi.org/10.1016/ j.biortech.2018.04.060
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Simultaneous Cr(VI) Bio-reduction and Methane Production by Anaerobic Granular sludge Qian Hua, Jiaji Sunb, Dezhi Suna, Lan Tiana, Yanan Jia, Bin Qiua* a
Beijing Key Laboratory for Source Control Technology of Water Pollution, College of Environmental
Science and Engineering, Beijing Forestry University, Beijing, 100083 China b
School of Environment, Beijing Normal University, Beijing 100875, China
* Corresponding author. E-mail address: [email protected]
Abstract Wastewater containing toxic hexavalent Cr (Cr(VI)) were treated with well-organized anaerobic granular sludge in this study. Results showed that the anaerobic granular sludge rapidly removed Cr(VI), and 2000 µg∙L−1 Cr(VI) was completely eliminated within 6 min, which was much faster than the reported duration of removal by reported artificial materials. Sucrose added as a carbon source acted as an initial electron donor to reduce Cr(VI) to Cr(III). This process was considered as the main mechanism of Cr(VI) removal. Methane production by anaerobic granular sludge was improved by the addition of Cr(VI) at a concentration lower than 500 µg∙L−1. Anaerobic granular sludge had a well-organized structure, which presented good resistance against toxic Cr(VI). Trichoccus accelerated the degradation of organic substances to generate acetates with a low Cr(VI) concentration, thereby enhancing methane production by acetotrophic methanogens.
Keywords:
Bio-reduction; Cr(VI) removal; anaerobic granular sludge; methane production
1. Introduction Hexavalent Cr (Cr(VI)) has been widely used in different industries (Gu et al., 2013) but has been detected as the main toxic substance in wastewater. Cr(VI) in wastewater has been recognized as a serious environmental problem because of its high toxicity and mobility in aqueous environments (Qiu et al., 2014b; Zhu et al., 2012; Liang et al., 2017). By contrast, Cr(III) is relatively nontoxic and immobile in aqueous solutions. Thus, the reduction of Cr(VI) to Cr(III) has been considered as an effective way to treat Cr(VI) in wastewater. Various materials, including activated carbon and magnetic carbon (Qiu et al., 2014a; Qiu et al., 2015), zero valent iron (Mitra et al., 2011), polymers (Qiu et al., 2014b; Qiu et al., 2014c; Qiu et al., 2014d), and biomass (Lin & Wang, 2012; Wang & Lee, 2011), which can act as electron donors for Cr(VI) reduction, have been prepared to treat Cr(VI)-containing wastewater. In addition to the use of reductive materials, anaerobic biological treatment has been considered as an alternative to the reduction of Cr(VI) to Cr(III) (Fang et al. 2012). In an anaerobic process, organic substances are degraded to volatile fatty acids (VFAs) by bacteria, thereby generating electrons (Demirel & Scherer, 2008; Guyot et al., 1993). These electrons reduce H+ and produce H2 gas, which acts as an important electron carrier for hydrogenotrophic methanogens to produce methane. H2 generated in an anaerobic system has a substantial reductive ability; thus, anaerobic processes show potential for the reduction of Cr(VI) to Cr(III) (Lovley & Coates, 1997; Nerenberg & Rittmann, 2004). Cr(VI) can be reduced to Cr (III) by anaerobic sludge (Qian et al., 2016). Complex organic substances are used as initial electron donors (Mani et al., 2016; Wang
et al., 2017). Anaerobic sludge has a fast Cr(VI) reduction rate, and 1 mg∙L−1 Cr(VI) can be completely reduced within 24 min (Chung et al., 2006). Moreover, this anaerobic sludge has achieved 104 mg∙g−1 MLVSS of Cr(VI) reduction ability (Chung et al., 2006). Cr(VI) can be easily reduced to Cr(III) by anaerobic bacteria. However, HCrO4− and H2CrO4 are the dominant forms when pH is lower than 6.8, while CrO42− is stable when pH is above 6.8 (Melitas et al., 2001; Olad & Nabavi, 2007; Sun et al., 2010). These Cr(VI) anions are harmful to microorganisms because of their high toxicity. Cr(VI) ions exhibit high redox potential (Sun et al., 2010), which increases the oxidation–reduction potential and provides extreme living conditions for anaerobic microorganisms. To solve this problem, researchers sieved some special bacterial strains, which can thrive under extreme conditions, from Cr(VI)-contaminated environments and demonstrated that these strains can efficiently reduce Cr(VI). For example, Chlorella miniata efficiently removes Cr(VI) at an initial concentration as high as 100 mg∙L−1 (Han et al., 2007) because of its substantial tolerance against a high redox environment. The reduction of Cr(VI) competes electrons with hydrogenotrophic methanosarinales methanogens for producing methane. Anaerobic sludge has a loose structure. As such, Cr(VI) ions can easily diffuse to the inner anaerobic sludge. Thus, Cr(VI) significantly inhibits the activity of archaea living inside anaerobic sludge and decreases methane production. To simultaneously achieve Cr(VI) reduction and methane production under anaerobic conditions, we should develop sludge with a stable structure. Anaerobic granule is a particulate-shaped biofilm formed spontaneously by immobilizing anaerobic bacteria in the absence of a support material
(Lettinga, 1995). It has been widely used to treat organic wastewater (Liu & Tay, 2002), and it has a stable structure and protective extracellular polymeric substance (EPS) layer (Sheng et al., 2010). EPS, which is excreted by microbial population, is surrounded by granular sludge, thereby forming a protective layer for microbes in the inner layer (D’Abzac et al., 2010). Competitive and cooperative associations between the component microbes of anaerobic granules create a unique microbial ecosystem (McHugh et al., 2003a; Sekiguchi et al., 1998a). A layered structure of anaerobic granular sludge with a central archaeal core surrounded by a bacterial layer has also been proposed (Guiot et al., 1992; Lens et al., 1993; Visser et al., 1991). The unique aggregation of anaerobic granular sludge has a dense and strong microstructure, which contributes to good tolerance against toxic substances in wastewater (Adav et al., 2008; McHugh et al., 2003b). Thus, anaerobic granular sludge possibly reduces Cr(VI) to Cr(III) and simultaneously produces methane gas. In this study, anaerobic granular sludge was used to reduce toxic Cr(VI) in wastewater. (i) Sucrose as an initial electron donor for Cr(VI) reduction was added to wastewater. (ii) The effects of the initial concentrations of Cr(VI) and sucrose on Cr(VI) reduction and methane production in the granular sludge were investigated. (iii) The mechanisms involved in the efficient simultaneous Cr(VI) removal and methane production by granular sludge were revealed. 2. Materials and methods 2.1 Materials
Potassium dichromate (K2Cr2O7, ≥99.8 wt%), sucrose, phosphoric acid (H3PO4, 85 wt%), ethanol, acetic acid, propionic acid, and butyric acid were purchased from Beijing Chemical Works, China. 1,5-Dphenylcarbazide was procured from Alfa Aesar Company. All of the chemicals were used without any further treatment. Anaerobic granular sludge (VSS: 8.5 g∙L−1) was collected from a lab-scale up-flow anaerobic sludge bed reactor, which was used to treat incineration leachate for ~1 year. 2.2 Batch experiment In this experiment, 0.2, 0.5, 0.8, 1.0, and 2.0 mL of Cr(VI) stock solutions (1000 mg∙L−1) were added to 1.0 L of nutrient solution in serum bottles. Nitrogen gas was used to remove oxygen from the nutrient solution. Afterward, 300.0 mL of anaerobic granular sludge was added to the Cr(VI) nutrient solution composed of the following: 2000 mg∙L−1 sucrose, 400 mg∙L−1 NH4Cl, 80 mg∙L−1 NaHPO4, 40 mg∙L−1 KH2PO4, 39 mg∙L−1 FeCl2·4H2O, 5 mg∙L−1 MnCl2·4H2O, 5 mg∙L−1 CoCl2·6H2O, 4.5 mg∙L−1 AlCl3·6H2O, 5 mg∙L−1 (NH4)6Mo7O24·4H2O, 5 mg∙L−1 NiCl2·6H2O, 5 mg∙L−1 ZnCl2, and 5 mg∙L−1 CuSO4·5H2O. All of the bottles were incubated in a water bath at 35 ± 1 °C. Biogas produced from the bottles was collected using an aluminum foil bag. A parallel control was prepared in the same manner but without additional Cr(VI). Water and gas were sampled at different time intervals. 2.3 Analytic method The Cr(VI) concentration in the solution was determined with a colorimetric method (Qian et al., 2014). COD was measured in accordance with the procedures described in Standard Methods
(Apha, 1998). A heating high-speed centrifugation method was used to extract EPS (Morgan et al., 1990), and the Cr concentration in the EPS solutions was identified with inductively coupled plasma method (Optima 8X00, USA). Methane concentration was detected with a glass chromatograph (Agilent 7890A, USA) equipped with a flame ionization detector and a Porapak Q column with a detector and injection port temperatures of 260 °C and 60 °C, respectively. Nitrogen was used as a carrier gas. The concentrations of VFAs, namely, acetate, propionate, and butyrate, were detected with a high-performance liquid chromatograph (Waters e2695, USA) with an eluent of 8 mM H2SO4 in accordance with previously described methods (Nevin et al., 2008). 2.4 Characterizations The morphological characteristics of anaerobic sludge after Cr(VI)-containing wastewater treatment were observed with a JEOL field emission scanning electron microscope (SEM, JSM-6700F system). X-ray photoelectron spectroscopy (XPS) measurements were performed in a Kratos AXIS 165 XPS/AES instrument by applying monochromatic Al K radiation to determine the elemental compositions and valence state of chromium. Cr 2p peaks were deconvoluted into the components consisting of a Gaussian line shape Lorentzian function (Gaussian = 80%, Lorentzian = 20%) on a Shirley background. The microorganism community was determined by using the high-throughput sequencing method (Sun et al., 2018). The live and dead cells in the anaerobic granular sludge before and after Cr(VI)-containing wastewater treatment were detected through confocal laser scanning microscopy (CLSM) by using
LIVE/DEAD® BacLightTM (Invitrogen, USA). The anaerobic granular sludge was stained with SYTO® 9 and propidium iodide in accordance with the manufacturer’s instructions (LIVE/DEAD® BacLightTM, Invitrogen, USA). Auto-PHLIP-ML and Image J were utilized to analyze the proportion of live or dead bacteria in the anaerobic granular sludge. 3. Results and Discussion 3.1 Cr(VI) reduction and methane production 3.1.1 Effect of initial Cr(VI) concentration In Fig. 1A, Cr(VI) reduction induced by granular sludge was rapid. The time needed for complete reduction increased as the initial Cr(VI) concentration increased. Furthermore, 200 µg∙L−1 Cr(VI) was completely reduced within 2 min. Moreover, only 6 min was needed to reduce 2000 µg∙L−1 Cr(VI), and this reduction is much faster than that by artificial materials, such as polyaniline(PANI) (15 min) (Qiu et al., 2014d). Granular sludge was also faster than activated sludge and bacteria. Thus, Cr(VI) was reduced to Cr(III) by the electrons generated from the additional carbon source by anaerobic bacteria. The reduction of Cr(VI) with a high initial Cr(VI) concentration was faster than that with a low initial Cr(VI) concentration. Furthermore, 2000 mg∙L−1 sucrose added as the carbon source can generate enough electrons. Thus, a fast kinetic rate was obtained at a high initial Cr(VI) concentration. Methane is an important product of anaerobic wastewater treatment. Thus, the effect of additional Cr(VI) on methane production induced by anaerobic granular sludge was investigated as shown in Fig. 1B. Our results showed that 430 mL of methane was produced by the granular sludge within 48 h. When 200 and 500
µg∙L−1 Cr(VI) were added to the reaction system, the amounts of produced methane increased to 549 and 487 mL, respectively. By contrast, the amounts of produced methane decreased when the initial Cr(VI) concentration increased from 500 µg∙L−1 to 2000 µg∙L−1. Moreover, 379.6, 306.6, 285.0, and 243.5 mL of methane were produced when 500, 1000, 1500, and 2000 µg∙L−1 Cr(VI) were added to the anaerobic system, respectively. Therefore, the additional Cr(VI) with a low concentration positively affected the anaerobic degradation of organic substances in wastewater. Sucrose was degraded to VFAs, thereby generating electrons. The effect of Cr(VI) on VFA generation was detected in this study. In Fig. 3, acetic acid was detected as the main VFA in the anaerobic system without any additional Cr(VI). Acetic acid accumulated to 3.8 mM in the first 8 h and then degraded to a stable concentration at ~0.5 mM. Approximately 4.7 mM acetic acid accumulated in the anaerobic system with an additional of 200 µg∙L−1 Cr(VI) in the first 20 h, and this value was higher than that in the control system. However, the concentration of the accumulated acetic acid increased when the initial Cr(VI) concentration increased. This result indicated that the low Cr(VI) concentration accelerated substrate degradation and led to high methane production. In Fig. 3, the redox potential of the system increased as the Cr(VI) concentration increased. The redox potential increased to −160 mV when 2000 µg∙L−1 Cr(VI) was added. The redox potential decreased during incubation, thereby facilitating methane production. A high Cr(VI) concentration could inhibit the activity of microorganisms because of the high redox potential and toxicity of Cr(VI) in wastewater. The amount of accumulated acetic acid increased after the bacteria were incubated for more than 20 h, indicating that bacteria
recovered and degraded the organic substances. Propionic acid and butyric acid were also detected in this study. In Fig. 3, the amount of propionic acid increased as the initial Cr(VI) concentration increased. With additional 2000 µg∙L−1 Cr(VI), ~4.5 mM propionic acid and 0.27 mM butyrate acid accumulated in the anaerobic system. Increased amounts of VFAs accumulated in the anaerobic system led to acidification and inhibited methanogen activity. However, the accumulated VFAs could be degraded after Cr(VI) toxicity was restored. Therefore, anaerobic granular sludge showed resistance against toxic Cr(VI). 3.1.2 Effect of carbon source Under anaerobic conditions Cr(VI) reducing bacteria may utilize a wide variety of organic sources as electron donors (Narayani & Shetty, 2013). As show in Fig. 2, the Cr(VI) removal was operated on different initial concentration of sucrose. The sucrose added was enough for complete Cr(VI) reduction. The experimental data clearly shows that a higher concentration of substrate could lead a faster Cr(VI) removal rate. According to the results of these experiments Cr(VI) removal rates were 150.0, 128.6, 90.0 and 60.0 μg∙h-1 with the initial concentration of organic carbon source were 2000, 1500, 1000, 500 mg∙L-1, respectively. These results demonstrate that higher concentration of substrate could offer more electron to reduce the Cr(VI) to Cr(III). 3.2 Cr(VI) bio-reduction mechanism 3.2.1 Cr(VI) reduction
Fig. 4 shows the Cr 2p XPS spectra of the granular sludge for the treatment of 500 µg∙L−1 Cr(VI) solution for 30 days. The Cr 2p binding energy peaks were located at 576.0 and 585.2 eV, indicating the formation of Cr(III) (Park et al., 2008). No Cr(VI) was detected in the Cr 2p XPS spectra. These results suggested that the added Cr(VI) in the anaerobic system was completely reduced to Cr(III). In the anaerobic process, sucrose added to Cr(VI)-containing wastewater was degraded by the bacteria, generating VFAs and electrons (Demirel & Scherer, 2008; Guyot et al., 1993). These generated electrons reduce H+ and produce H2, which has a considerable reducing ability. Thus, H2 generated from the organic substances was considered as a dominant and direct electron donor to reduce Cr(VI) to Cr(III). In a neutral solution, Cr(VI) mainly exists as CrO42− and Cr2O72− (Sun et al., 2010). Cr(VI) reduction by the electrons donated by the degradation of organic substances was proposed in the following reaction (Eq. 1 & 2): 2CrO42− + 10H+ + 3H2 → 2Cr3+ + 8H2O
E0=+1.33 V
(1)
Cr2O72− + 8H+ + 3H2 → 2Cr3+ + 7H2O
E0=+1.33 V
(2)
3.2.2 Effect of VFAs In addition to H2 generated by anaerobic bacteria, the generated VFAs might be considered as a direct electron donor for Cr(VI) reduction. To determine the contribution of the generated VFAs, we investigated the Cr(VI) reduction by VFAs as the initial electron donors with and without anaerobic granular sludge. No Cr(VI) was reduced to Cr(III) by VFAs without the additional anaerobic granular sludge (Fig. S1A in Supplementary Materials), indicating that acetate, propionate, and butyrate could not act as the direct electron donor to reduce Cr(VI). However,
Cr(VI) was completely reduced to Cr(III) by VFAs within 5 min when the anaerobic granular sludge was added to this reaction system (Fig. S1B in Supplementary Materials). Acetate, propionate, and butyrate could be used as the initial electron donors and were utilized by anaerobic bacteria. In this anaerobic process, the generated electrons acted as direct electron donors to further reduce Cr(VI) to Cr(III). A fast reduction rate of Cr(VI) was achieved when acetate was used as the initial electron donor because acetate was easily utilized by anaerobic bacteria. Thus, H2 generated from organic substance degradation by bacteria was concluded as the main electron donor for Cr(VI) reduction rather than reductive VFAs. Fig. S2A shows the SEM images of the anaerobic granular sludge used to treat Cr(VI)-containing wastewater. The anaerobic granular sludge had a uniform diameter of ~1 mm. Chromium was also mapped on the granular sludge surface by using an EDX probe. Cr was evenly dispersed on the anaerobic sludge surface (Fig. S2B in Supplementary Materials). No Cr(III) ions were detected in the treated wastewater, suggesting that almost all of the obtained Cr(III) ions were adsorbed on the anaerobic granular sludge surface. The Cr(III)-attached anaerobic granular sludge was discharged from the reactors and could be treated by incineration and safe disposal. Bacteria were negatively charged in wastewater, thereby facilitating the adsorption of positively charged Cr(III) ions via electrostatic attraction. This result indicated that the anaerobic granular sludge could be effectively used to recover Cr from wastewater. The anaerobic granular sludge also had a dense structure (Fig. S2C in Supplementary Materials), which provides a good living condition for microorganisms (Mccarty, 2001; McHugh et al., 2003b; Sekiguchi et al., 1998b).
Moreover, the EPS layer of anaerobic granular sludge can improve the tolerance of microorganisms against Cr(IV) (Ozturk et al., 2009). Cr(III) distribution in granular sludge was also detected. Almost all of the adsorbed Cr(III) was distributed on the outer layer (Fig. S2D in Supplementary Materials). Almost no Cr(III) was found in the inner layer of the granular sludge, where archaea lived. This result indicated that Cr did not affect archaea. EPS layer, secreted by microorganisms, surrounded the outer layer of the anaerobic granular sludge. This EPS layer played an important role in providing protection against toxic Cr(VI) and adsorbing Cr(III). The Cr adsorbed in the EPS layer accounted for ~65% of the total Cr. Cr(VI) was adsorbed on the EPS layer by active groups, such as–NH2 and –OH, and reduced to Cr(III) by the electrons in the EPS layer. When the pH of wastewater is around 7, Cr(III) exists in the form of Cr(OH)2+ (Sun et al., 2010). Cr(OH)2+ can be chelated by carboxyl groups in the EPS layer (Doshi et al., 2007). Fig. S3 further reveals the FT-IR spectra of the EPS layer of the anaerobic granular sludge before and after Cr(VI) treatment. The strong peaks at 3284, 2923, 1635, 1537, 1404, and 1036 cm−1 were attributed to –OH, C–H, C=O, N–H, –COOH, and –CO stretching vibrations, respectively. The characterization peaks shifted to 3286, 2923, 1637, 1529, 1402, and 1031 cm−1 after Cr(VI) treatment, indicating the possibility of interactions between Cr(III) and active groups of the EPS layer. Moreover, the –C=O group had the most evident change in the reduction of Cr(VI). Thus, the functional group –C=O acted as the main group for the adsorption of Cr(III). Cr was also detected in the inner EPS layer, suggesting that Cr(VI) was diffused across the EPS layer with a high Cr(VI) concentration.
3.2.3 Microorganism activity The effect of additional Cr(VI) on bacterial activity was investigated by CLSM. In CLSM, green represented the live bacteria, while red corresponded to the dead cells in the anaerobic granular sludge. In CLSM images, the green area in the of the anaerobic granular sludge with additional 200 and 500 µg∙L−1 Cr(VI) (Fig. S4B~C) was almost the same as that without additional Cr(VI) (Fig. S4A), indicating that Cr(VI) with initial concentrations of 200 and 500 µg∙L−1 had no effect on bacterial activity. Under this condition, the EPS layer provided protection against Cr(VI). Thus, Cr(VI) did not influence the living condition of archaea in the inner layer of the granular sludge, thereby leading to stable methane production. However, Cr(VI) ions could also be diffused across the EPS layer when the Cr(VI) concentration increased. Diffused Cr(VI) ions created an extreme condition with high redox potential for bacteria, resulting in the increased number of dead cells (Fig. S4D~F). However, ~55% of the dead cells were detected in the anaerobic sludge with additional 2000 µg∙L−1 Cr(VI) (Fig. S4G), and this value was much higher than those under treating the solutions with a lower Cr(VI) concentration. 3.2.4 Microbial communities present in the granular sludge Microbial communities were detected to further investigate the reaction mechanism involved in Cr(VI) reduction and methane production. Figs. 5A and B show the bacterial and archaeal communities of the anaerobic granular sludge before and after Cr(VI)-containing wastewater treatment, respectively.
Bacterial communities associated with the anaerobic granular sludge after Cr(VI)-containing wastewater treatment were primarily composed of four phyla, namely, Firmicutes, Chloroflexi, Proteobacteria, and Bacteroidetes, accounting for 67.5%, 11.9%, 5.9%, and 5.6% of the total communities, respectively (Fig. 5A). By contrast, the main phyla in the anaerobic granular sludge before wastewater treatment accounted for 27.9%, 32.1%, 2.1%, and 12.5% of the total communities, respectively (Fig. 5B). Thus, the abundance of Firmicutes increased significantly after Cr(VI)-containing wastewater was treated. At the genus level, Trichoccus, which is dominant in the reactor and belonging to Firmicutes, increased from 27.9% to 67.5% in the granular sludge after Cr(VI)-containing wastewater was treated. This significant increase in the bacterial communities suggested that Trichoccus might have played an important role in Cr(VI) reduction by the anaerobic granular sludge. Trichoccus can degrade complex organics substrates, such as glucose, to simple organics (Savant et al., 2002). Thus, microorganisms can be promoted to hydrolyze organic substrates to acetic acid and H2, which can be further utilized by methanogens to produce methane (Lu et al., 2017). In Fig. 5B, the archaeal communities in the anaerobic granular sludge were significantly diverse before and after Cr(VI)-containing wastewater was treated. The proportion of Methanobacterium, a type of hydrogenotrophic Methanosarinales (Lv et al., 2017), decreased from 79.1% to 12.6% after the Cr(VI)-containing wastewater was treated. The reduction of Cr(VI) competed electron donors to the methanogens. Cr(VI) can acquire electrons much more easily than hydrogenotrophic methanosarinales does, thereby decreasing methane production. This result was consistent with the result that methane
production decreased when Cr(VI) concentration was rised to 500 μg∙L−1 (Fig. 1B). However, the proportion of Methanosaeta, an aceticlastic methanosarcinales, increased from 17.3% to 68.2% in the anaerobic granular sludge, indicating that aceticlastic methanogens were the dominant microbes in the Cr(VI) reduction system. Moreover, methane production was significantly improved when the added Cr(VI) was lower than 500 μg∙L−1 (Fig. 1B). Therefore, the added low Cr(VI) concentration accelerated sucrose degradation and acetate production. Acetate accumulation improves methane production by aceticlastic methanogens (Feng et al., 2017), and this observation is consistent with the increased proportion of aceticlastic methanogens in the anaerobic granular sludge after Cr(VI)-containing wastewater is treated (Angulo et al., 2018). Cr(III) is also a trace element for microorganisms. These processes simultaneously facilitated methane production by methanosarinales and the reduction of Cr(VI). The mechanisms involved in Cr(VI) and methane production were proposed. In Fig. 6, the addition of a low concentration of Cr(VI) was adsorbed on the surface of the EPS layer, and the attached Cr(VI) ions were reduced by the electrons generated from sucrose. Thus, Cr(VI) reduction accelerated sucrose degradation, leading to rapid VFA generation and methane production. When Cr(VI) was higher than 500 μg∙L−1, Cr(VI) could be diffused across the EPS layer into the bacterial layer of the anaerobic granular sludge. This phenomenon resulted in the provision of an extreme living condition for anaerobic bacteria and a decrease in the bacterial activity. Sucrose degradation decreased because of the inhibition of Cr(VI), thereby leading to slow methane production. Cr(VI) reduction surpassed in the competition of the electron with the
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Figure list: Fig. 1 Effect of the initial Cr(VI) concentration on (A) Cr(VI) reduction and (B) methane production by the anaerobic granular sludge. Fig. 2 Cr(VI) concentration in the solution treated by the anaerobic granular sludge with different initial sucrose concentration. Fig. 3 Concentrations of (A) acetic acid, (B) propionic acid, (C) butyrate acid, and (D) ORP in the anaerobic systems with additional Cr(VI). Fig. 4 Cr2p XPS spectra of the anaerobic granular sludge after treating Cr(VI) wastewater. Fig. 5 (A) bacterial and (B) archaeal communities of the anaerobic granular sludge before and after treating Cr(VI) wastewater. Fig. 6 Proposed pathway of simultaneous Cr(VI) reduction and methane production by the granular sludge.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Highlights
Anaerobic granule sludge has a good performance on Cr(VI) removal.
Anaerobic granule sludge produced methane with high efficiency under low Cr(VI) concentration.
Trichoccus and aceticlastic methanogens were identified as the dominant microorganisms present in the Cr(VI) bio-reduction process.
Graphical abstract
S0960-8524(18)30573-X https://doi.org/10.1016/j.biortech.2018.04.060 BITE 19840
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 February 2018 12 April 2018 14 April 2018
Please cite this article as: Hu, Q., Sun, J., Sun, D., Tian, L., Ji, Y., Qiu, B., Simultaneous Cr(VI) Bio-reduction and Methane Production by Anaerobic Granular sludge, Bioresource Technology (2018), doi: https://doi.org/10.1016/ j.biortech.2018.04.060
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Simultaneous Cr(VI) Bio-reduction and Methane Production by Anaerobic Granular sludge Qian Hua, Jiaji Sunb, Dezhi Suna, Lan Tiana, Yanan Jia, Bin Qiua* a
Beijing Key Laboratory for Source Control Technology of Water Pollution, College of Environmental
Science and Engineering, Beijing Forestry University, Beijing, 100083 China b
School of Environment, Beijing Normal University, Beijing 100875, China
* Corresponding author. E-mail address: [email protected]
Abstract Wastewater containing toxic hexavalent Cr (Cr(VI)) were treated with well-organized anaerobic granular sludge in this study. Results showed that the anaerobic granular sludge rapidly removed Cr(VI), and 2000 µg∙L−1 Cr(VI) was completely eliminated within 6 min, which was much faster than the reported duration of removal by reported artificial materials. Sucrose added as a carbon source acted as an initial electron donor to reduce Cr(VI) to Cr(III). This process was considered as the main mechanism of Cr(VI) removal. Methane production by anaerobic granular sludge was improved by the addition of Cr(VI) at a concentration lower than 500 µg∙L−1. Anaerobic granular sludge had a well-organized structure, which presented good resistance against toxic Cr(VI). Trichoccus accelerated the degradation of organic substances to generate acetates with a low Cr(VI) concentration, thereby enhancing methane production by acetotrophic methanogens.
Keywords:
Bio-reduction; Cr(VI) removal; anaerobic granular sludge; methane production
1. Introduction Hexavalent Cr (Cr(VI)) has been widely used in different industries (Gu et al., 2013) but has been detected as the main toxic substance in wastewater. Cr(VI) in wastewater has been recognized as a serious environmental problem because of its high toxicity and mobility in aqueous environments (Qiu et al., 2014b; Zhu et al., 2012; Liang et al., 2017). By contrast, Cr(III) is relatively nontoxic and immobile in aqueous solutions. Thus, the reduction of Cr(VI) to Cr(III) has been considered as an effective way to treat Cr(VI) in wastewater. Various materials, including activated carbon and magnetic carbon (Qiu et al., 2014a; Qiu et al., 2015), zero valent iron (Mitra et al., 2011), polymers (Qiu et al., 2014b; Qiu et al., 2014c; Qiu et al., 2014d), and biomass (Lin & Wang, 2012; Wang & Lee, 2011), which can act as electron donors for Cr(VI) reduction, have been prepared to treat Cr(VI)-containing wastewater. In addition to the use of reductive materials, anaerobic biological treatment has been considered as an alternative to the reduction of Cr(VI) to Cr(III) (Fang et al. 2012). In an anaerobic process, organic substances are degraded to volatile fatty acids (VFAs) by bacteria, thereby generating electrons (Demirel & Scherer, 2008; Guyot et al., 1993). These electrons reduce H+ and produce H2 gas, which acts as an important electron carrier for hydrogenotrophic methanogens to produce methane. H2 generated in an anaerobic system has a substantial reductive ability; thus, anaerobic processes show potential for the reduction of Cr(VI) to Cr(III) (Lovley & Coates, 1997; Nerenberg & Rittmann, 2004). Cr(VI) can be reduced to Cr (III) by anaerobic sludge (Qian et al., 2016). Complex organic substances are used as initial electron donors (Mani et al., 2016; Wang
et al., 2017). Anaerobic sludge has a fast Cr(VI) reduction rate, and 1 mg∙L−1 Cr(VI) can be completely reduced within 24 min (Chung et al., 2006). Moreover, this anaerobic sludge has achieved 104 mg∙g−1 MLVSS of Cr(VI) reduction ability (Chung et al., 2006). Cr(VI) can be easily reduced to Cr(III) by anaerobic bacteria. However, HCrO4− and H2CrO4 are the dominant forms when pH is lower than 6.8, while CrO42− is stable when pH is above 6.8 (Melitas et al., 2001; Olad & Nabavi, 2007; Sun et al., 2010). These Cr(VI) anions are harmful to microorganisms because of their high toxicity. Cr(VI) ions exhibit high redox potential (Sun et al., 2010), which increases the oxidation–reduction potential and provides extreme living conditions for anaerobic microorganisms. To solve this problem, researchers sieved some special bacterial strains, which can thrive under extreme conditions, from Cr(VI)-contaminated environments and demonstrated that these strains can efficiently reduce Cr(VI). For example, Chlorella miniata efficiently removes Cr(VI) at an initial concentration as high as 100 mg∙L−1 (Han et al., 2007) because of its substantial tolerance against a high redox environment. The reduction of Cr(VI) competes electrons with hydrogenotrophic methanosarinales methanogens for producing methane. Anaerobic sludge has a loose structure. As such, Cr(VI) ions can easily diffuse to the inner anaerobic sludge. Thus, Cr(VI) significantly inhibits the activity of archaea living inside anaerobic sludge and decreases methane production. To simultaneously achieve Cr(VI) reduction and methane production under anaerobic conditions, we should develop sludge with a stable structure. Anaerobic granule is a particulate-shaped biofilm formed spontaneously by immobilizing anaerobic bacteria in the absence of a support material
(Lettinga, 1995). It has been widely used to treat organic wastewater (Liu & Tay, 2002), and it has a stable structure and protective extracellular polymeric substance (EPS) layer (Sheng et al., 2010). EPS, which is excreted by microbial population, is surrounded by granular sludge, thereby forming a protective layer for microbes in the inner layer (D’Abzac et al., 2010). Competitive and cooperative associations between the component microbes of anaerobic granules create a unique microbial ecosystem (McHugh et al., 2003a; Sekiguchi et al., 1998a). A layered structure of anaerobic granular sludge with a central archaeal core surrounded by a bacterial layer has also been proposed (Guiot et al., 1992; Lens et al., 1993; Visser et al., 1991). The unique aggregation of anaerobic granular sludge has a dense and strong microstructure, which contributes to good tolerance against toxic substances in wastewater (Adav et al., 2008; McHugh et al., 2003b). Thus, anaerobic granular sludge possibly reduces Cr(VI) to Cr(III) and simultaneously produces methane gas. In this study, anaerobic granular sludge was used to reduce toxic Cr(VI) in wastewater. (i) Sucrose as an initial electron donor for Cr(VI) reduction was added to wastewater. (ii) The effects of the initial concentrations of Cr(VI) and sucrose on Cr(VI) reduction and methane production in the granular sludge were investigated. (iii) The mechanisms involved in the efficient simultaneous Cr(VI) removal and methane production by granular sludge were revealed. 2. Materials and methods 2.1 Materials
Potassium dichromate (K2Cr2O7, ≥99.8 wt%), sucrose, phosphoric acid (H3PO4, 85 wt%), ethanol, acetic acid, propionic acid, and butyric acid were purchased from Beijing Chemical Works, China. 1,5-Dphenylcarbazide was procured from Alfa Aesar Company. All of the chemicals were used without any further treatment. Anaerobic granular sludge (VSS: 8.5 g∙L−1) was collected from a lab-scale up-flow anaerobic sludge bed reactor, which was used to treat incineration leachate for ~1 year. 2.2 Batch experiment In this experiment, 0.2, 0.5, 0.8, 1.0, and 2.0 mL of Cr(VI) stock solutions (1000 mg∙L−1) were added to 1.0 L of nutrient solution in serum bottles. Nitrogen gas was used to remove oxygen from the nutrient solution. Afterward, 300.0 mL of anaerobic granular sludge was added to the Cr(VI) nutrient solution composed of the following: 2000 mg∙L−1 sucrose, 400 mg∙L−1 NH4Cl, 80 mg∙L−1 NaHPO4, 40 mg∙L−1 KH2PO4, 39 mg∙L−1 FeCl2·4H2O, 5 mg∙L−1 MnCl2·4H2O, 5 mg∙L−1 CoCl2·6H2O, 4.5 mg∙L−1 AlCl3·6H2O, 5 mg∙L−1 (NH4)6Mo7O24·4H2O, 5 mg∙L−1 NiCl2·6H2O, 5 mg∙L−1 ZnCl2, and 5 mg∙L−1 CuSO4·5H2O. All of the bottles were incubated in a water bath at 35 ± 1 °C. Biogas produced from the bottles was collected using an aluminum foil bag. A parallel control was prepared in the same manner but without additional Cr(VI). Water and gas were sampled at different time intervals. 2.3 Analytic method The Cr(VI) concentration in the solution was determined with a colorimetric method (Qian et al., 2014). COD was measured in accordance with the procedures described in Standard Methods
(Apha, 1998). A heating high-speed centrifugation method was used to extract EPS (Morgan et al., 1990), and the Cr concentration in the EPS solutions was identified with inductively coupled plasma method (Optima 8X00, USA). Methane concentration was detected with a glass chromatograph (Agilent 7890A, USA) equipped with a flame ionization detector and a Porapak Q column with a detector and injection port temperatures of 260 °C and 60 °C, respectively. Nitrogen was used as a carrier gas. The concentrations of VFAs, namely, acetate, propionate, and butyrate, were detected with a high-performance liquid chromatograph (Waters e2695, USA) with an eluent of 8 mM H2SO4 in accordance with previously described methods (Nevin et al., 2008). 2.4 Characterizations The morphological characteristics of anaerobic sludge after Cr(VI)-containing wastewater treatment were observed with a JEOL field emission scanning electron microscope (SEM, JSM-6700F system). X-ray photoelectron spectroscopy (XPS) measurements were performed in a Kratos AXIS 165 XPS/AES instrument by applying monochromatic Al K radiation to determine the elemental compositions and valence state of chromium. Cr 2p peaks were deconvoluted into the components consisting of a Gaussian line shape Lorentzian function (Gaussian = 80%, Lorentzian = 20%) on a Shirley background. The microorganism community was determined by using the high-throughput sequencing method (Sun et al., 2018). The live and dead cells in the anaerobic granular sludge before and after Cr(VI)-containing wastewater treatment were detected through confocal laser scanning microscopy (CLSM) by using
LIVE/DEAD® BacLightTM (Invitrogen, USA). The anaerobic granular sludge was stained with SYTO® 9 and propidium iodide in accordance with the manufacturer’s instructions (LIVE/DEAD® BacLightTM, Invitrogen, USA). Auto-PHLIP-ML and Image J were utilized to analyze the proportion of live or dead bacteria in the anaerobic granular sludge. 3. Results and Discussion 3.1 Cr(VI) reduction and methane production 3.1.1 Effect of initial Cr(VI) concentration In Fig. 1A, Cr(VI) reduction induced by granular sludge was rapid. The time needed for complete reduction increased as the initial Cr(VI) concentration increased. Furthermore, 200 µg∙L−1 Cr(VI) was completely reduced within 2 min. Moreover, only 6 min was needed to reduce 2000 µg∙L−1 Cr(VI), and this reduction is much faster than that by artificial materials, such as polyaniline(PANI) (15 min) (Qiu et al., 2014d). Granular sludge was also faster than activated sludge and bacteria. Thus, Cr(VI) was reduced to Cr(III) by the electrons generated from the additional carbon source by anaerobic bacteria. The reduction of Cr(VI) with a high initial Cr(VI) concentration was faster than that with a low initial Cr(VI) concentration. Furthermore, 2000 mg∙L−1 sucrose added as the carbon source can generate enough electrons. Thus, a fast kinetic rate was obtained at a high initial Cr(VI) concentration. Methane is an important product of anaerobic wastewater treatment. Thus, the effect of additional Cr(VI) on methane production induced by anaerobic granular sludge was investigated as shown in Fig. 1B. Our results showed that 430 mL of methane was produced by the granular sludge within 48 h. When 200 and 500
µg∙L−1 Cr(VI) were added to the reaction system, the amounts of produced methane increased to 549 and 487 mL, respectively. By contrast, the amounts of produced methane decreased when the initial Cr(VI) concentration increased from 500 µg∙L−1 to 2000 µg∙L−1. Moreover, 379.6, 306.6, 285.0, and 243.5 mL of methane were produced when 500, 1000, 1500, and 2000 µg∙L−1 Cr(VI) were added to the anaerobic system, respectively. Therefore, the additional Cr(VI) with a low concentration positively affected the anaerobic degradation of organic substances in wastewater. Sucrose was degraded to VFAs, thereby generating electrons. The effect of Cr(VI) on VFA generation was detected in this study. In Fig. 3, acetic acid was detected as the main VFA in the anaerobic system without any additional Cr(VI). Acetic acid accumulated to 3.8 mM in the first 8 h and then degraded to a stable concentration at ~0.5 mM. Approximately 4.7 mM acetic acid accumulated in the anaerobic system with an additional of 200 µg∙L−1 Cr(VI) in the first 20 h, and this value was higher than that in the control system. However, the concentration of the accumulated acetic acid increased when the initial Cr(VI) concentration increased. This result indicated that the low Cr(VI) concentration accelerated substrate degradation and led to high methane production. In Fig. 3, the redox potential of the system increased as the Cr(VI) concentration increased. The redox potential increased to −160 mV when 2000 µg∙L−1 Cr(VI) was added. The redox potential decreased during incubation, thereby facilitating methane production. A high Cr(VI) concentration could inhibit the activity of microorganisms because of the high redox potential and toxicity of Cr(VI) in wastewater. The amount of accumulated acetic acid increased after the bacteria were incubated for more than 20 h, indicating that bacteria
recovered and degraded the organic substances. Propionic acid and butyric acid were also detected in this study. In Fig. 3, the amount of propionic acid increased as the initial Cr(VI) concentration increased. With additional 2000 µg∙L−1 Cr(VI), ~4.5 mM propionic acid and 0.27 mM butyrate acid accumulated in the anaerobic system. Increased amounts of VFAs accumulated in the anaerobic system led to acidification and inhibited methanogen activity. However, the accumulated VFAs could be degraded after Cr(VI) toxicity was restored. Therefore, anaerobic granular sludge showed resistance against toxic Cr(VI). 3.1.2 Effect of carbon source Under anaerobic conditions Cr(VI) reducing bacteria may utilize a wide variety of organic sources as electron donors (Narayani & Shetty, 2013). As show in Fig. 2, the Cr(VI) removal was operated on different initial concentration of sucrose. The sucrose added was enough for complete Cr(VI) reduction. The experimental data clearly shows that a higher concentration of substrate could lead a faster Cr(VI) removal rate. According to the results of these experiments Cr(VI) removal rates were 150.0, 128.6, 90.0 and 60.0 μg∙h-1 with the initial concentration of organic carbon source were 2000, 1500, 1000, 500 mg∙L-1, respectively. These results demonstrate that higher concentration of substrate could offer more electron to reduce the Cr(VI) to Cr(III). 3.2 Cr(VI) bio-reduction mechanism 3.2.1 Cr(VI) reduction
Fig. 4 shows the Cr 2p XPS spectra of the granular sludge for the treatment of 500 µg∙L−1 Cr(VI) solution for 30 days. The Cr 2p binding energy peaks were located at 576.0 and 585.2 eV, indicating the formation of Cr(III) (Park et al., 2008). No Cr(VI) was detected in the Cr 2p XPS spectra. These results suggested that the added Cr(VI) in the anaerobic system was completely reduced to Cr(III). In the anaerobic process, sucrose added to Cr(VI)-containing wastewater was degraded by the bacteria, generating VFAs and electrons (Demirel & Scherer, 2008; Guyot et al., 1993). These generated electrons reduce H+ and produce H2, which has a considerable reducing ability. Thus, H2 generated from the organic substances was considered as a dominant and direct electron donor to reduce Cr(VI) to Cr(III). In a neutral solution, Cr(VI) mainly exists as CrO42− and Cr2O72− (Sun et al., 2010). Cr(VI) reduction by the electrons donated by the degradation of organic substances was proposed in the following reaction (Eq. 1 & 2): 2CrO42− + 10H+ + 3H2 → 2Cr3+ + 8H2O
E0=+1.33 V
(1)
Cr2O72− + 8H+ + 3H2 → 2Cr3+ + 7H2O
E0=+1.33 V
(2)
3.2.2 Effect of VFAs In addition to H2 generated by anaerobic bacteria, the generated VFAs might be considered as a direct electron donor for Cr(VI) reduction. To determine the contribution of the generated VFAs, we investigated the Cr(VI) reduction by VFAs as the initial electron donors with and without anaerobic granular sludge. No Cr(VI) was reduced to Cr(III) by VFAs without the additional anaerobic granular sludge (Fig. S1A in Supplementary Materials), indicating that acetate, propionate, and butyrate could not act as the direct electron donor to reduce Cr(VI). However,
Cr(VI) was completely reduced to Cr(III) by VFAs within 5 min when the anaerobic granular sludge was added to this reaction system (Fig. S1B in Supplementary Materials). Acetate, propionate, and butyrate could be used as the initial electron donors and were utilized by anaerobic bacteria. In this anaerobic process, the generated electrons acted as direct electron donors to further reduce Cr(VI) to Cr(III). A fast reduction rate of Cr(VI) was achieved when acetate was used as the initial electron donor because acetate was easily utilized by anaerobic bacteria. Thus, H2 generated from organic substance degradation by bacteria was concluded as the main electron donor for Cr(VI) reduction rather than reductive VFAs. Fig. S2A shows the SEM images of the anaerobic granular sludge used to treat Cr(VI)-containing wastewater. The anaerobic granular sludge had a uniform diameter of ~1 mm. Chromium was also mapped on the granular sludge surface by using an EDX probe. Cr was evenly dispersed on the anaerobic sludge surface (Fig. S2B in Supplementary Materials). No Cr(III) ions were detected in the treated wastewater, suggesting that almost all of the obtained Cr(III) ions were adsorbed on the anaerobic granular sludge surface. The Cr(III)-attached anaerobic granular sludge was discharged from the reactors and could be treated by incineration and safe disposal. Bacteria were negatively charged in wastewater, thereby facilitating the adsorption of positively charged Cr(III) ions via electrostatic attraction. This result indicated that the anaerobic granular sludge could be effectively used to recover Cr from wastewater. The anaerobic granular sludge also had a dense structure (Fig. S2C in Supplementary Materials), which provides a good living condition for microorganisms (Mccarty, 2001; McHugh et al., 2003b; Sekiguchi et al., 1998b).
Moreover, the EPS layer of anaerobic granular sludge can improve the tolerance of microorganisms against Cr(IV) (Ozturk et al., 2009). Cr(III) distribution in granular sludge was also detected. Almost all of the adsorbed Cr(III) was distributed on the outer layer (Fig. S2D in Supplementary Materials). Almost no Cr(III) was found in the inner layer of the granular sludge, where archaea lived. This result indicated that Cr did not affect archaea. EPS layer, secreted by microorganisms, surrounded the outer layer of the anaerobic granular sludge. This EPS layer played an important role in providing protection against toxic Cr(VI) and adsorbing Cr(III). The Cr adsorbed in the EPS layer accounted for ~65% of the total Cr. Cr(VI) was adsorbed on the EPS layer by active groups, such as–NH2 and –OH, and reduced to Cr(III) by the electrons in the EPS layer. When the pH of wastewater is around 7, Cr(III) exists in the form of Cr(OH)2+ (Sun et al., 2010). Cr(OH)2+ can be chelated by carboxyl groups in the EPS layer (Doshi et al., 2007). Fig. S3 further reveals the FT-IR spectra of the EPS layer of the anaerobic granular sludge before and after Cr(VI) treatment. The strong peaks at 3284, 2923, 1635, 1537, 1404, and 1036 cm−1 were attributed to –OH, C–H, C=O, N–H, –COOH, and –CO stretching vibrations, respectively. The characterization peaks shifted to 3286, 2923, 1637, 1529, 1402, and 1031 cm−1 after Cr(VI) treatment, indicating the possibility of interactions between Cr(III) and active groups of the EPS layer. Moreover, the –C=O group had the most evident change in the reduction of Cr(VI). Thus, the functional group –C=O acted as the main group for the adsorption of Cr(III). Cr was also detected in the inner EPS layer, suggesting that Cr(VI) was diffused across the EPS layer with a high Cr(VI) concentration.
3.2.3 Microorganism activity The effect of additional Cr(VI) on bacterial activity was investigated by CLSM. In CLSM, green represented the live bacteria, while red corresponded to the dead cells in the anaerobic granular sludge. In CLSM images, the green area in the of the anaerobic granular sludge with additional 200 and 500 µg∙L−1 Cr(VI) (Fig. S4B~C) was almost the same as that without additional Cr(VI) (Fig. S4A), indicating that Cr(VI) with initial concentrations of 200 and 500 µg∙L−1 had no effect on bacterial activity. Under this condition, the EPS layer provided protection against Cr(VI). Thus, Cr(VI) did not influence the living condition of archaea in the inner layer of the granular sludge, thereby leading to stable methane production. However, Cr(VI) ions could also be diffused across the EPS layer when the Cr(VI) concentration increased. Diffused Cr(VI) ions created an extreme condition with high redox potential for bacteria, resulting in the increased number of dead cells (Fig. S4D~F). However, ~55% of the dead cells were detected in the anaerobic sludge with additional 2000 µg∙L−1 Cr(VI) (Fig. S4G), and this value was much higher than those under treating the solutions with a lower Cr(VI) concentration. 3.2.4 Microbial communities present in the granular sludge Microbial communities were detected to further investigate the reaction mechanism involved in Cr(VI) reduction and methane production. Figs. 5A and B show the bacterial and archaeal communities of the anaerobic granular sludge before and after Cr(VI)-containing wastewater treatment, respectively.
Bacterial communities associated with the anaerobic granular sludge after Cr(VI)-containing wastewater treatment were primarily composed of four phyla, namely, Firmicutes, Chloroflexi, Proteobacteria, and Bacteroidetes, accounting for 67.5%, 11.9%, 5.9%, and 5.6% of the total communities, respectively (Fig. 5A). By contrast, the main phyla in the anaerobic granular sludge before wastewater treatment accounted for 27.9%, 32.1%, 2.1%, and 12.5% of the total communities, respectively (Fig. 5B). Thus, the abundance of Firmicutes increased significantly after Cr(VI)-containing wastewater was treated. At the genus level, Trichoccus, which is dominant in the reactor and belonging to Firmicutes, increased from 27.9% to 67.5% in the granular sludge after Cr(VI)-containing wastewater was treated. This significant increase in the bacterial communities suggested that Trichoccus might have played an important role in Cr(VI) reduction by the anaerobic granular sludge. Trichoccus can degrade complex organics substrates, such as glucose, to simple organics (Savant et al., 2002). Thus, microorganisms can be promoted to hydrolyze organic substrates to acetic acid and H2, which can be further utilized by methanogens to produce methane (Lu et al., 2017). In Fig. 5B, the archaeal communities in the anaerobic granular sludge were significantly diverse before and after Cr(VI)-containing wastewater was treated. The proportion of Methanobacterium, a type of hydrogenotrophic Methanosarinales (Lv et al., 2017), decreased from 79.1% to 12.6% after the Cr(VI)-containing wastewater was treated. The reduction of Cr(VI) competed electron donors to the methanogens. Cr(VI) can acquire electrons much more easily than hydrogenotrophic methanosarinales does, thereby decreasing methane production. This result was consistent with the result that methane
production decreased when Cr(VI) concentration was rised to 500 μg∙L−1 (Fig. 1B). However, the proportion of Methanosaeta, an aceticlastic methanosarcinales, increased from 17.3% to 68.2% in the anaerobic granular sludge, indicating that aceticlastic methanogens were the dominant microbes in the Cr(VI) reduction system. Moreover, methane production was significantly improved when the added Cr(VI) was lower than 500 μg∙L−1 (Fig. 1B). Therefore, the added low Cr(VI) concentration accelerated sucrose degradation and acetate production. Acetate accumulation improves methane production by aceticlastic methanogens (Feng et al., 2017), and this observation is consistent with the increased proportion of aceticlastic methanogens in the anaerobic granular sludge after Cr(VI)-containing wastewater is treated (Angulo et al., 2018). Cr(III) is also a trace element for microorganisms. These processes simultaneously facilitated methane production by methanosarinales and the reduction of Cr(VI). The mechanisms involved in Cr(VI) and methane production were proposed. In Fig. 6, the addition of a low concentration of Cr(VI) was adsorbed on the surface of the EPS layer, and the attached Cr(VI) ions were reduced by the electrons generated from sucrose. Thus, Cr(VI) reduction accelerated sucrose degradation, leading to rapid VFA generation and methane production. When Cr(VI) was higher than 500 μg∙L−1, Cr(VI) could be diffused across the EPS layer into the bacterial layer of the anaerobic granular sludge. This phenomenon resulted in the provision of an extreme living condition for anaerobic bacteria and a decrease in the bacterial activity. Sucrose degradation decreased because of the inhibition of Cr(VI), thereby leading to slow methane production. Cr(VI) reduction surpassed in the competition of the electron with the
hydrogenotrophic methanogens. This process was also considered as the main mechanism leading to the decreased methane production when high-concentration Cr(VI) was added to the anaerobic granular sludge system. 4. Conclusions Anaerobic sludge could be used to efficiently detoxify and recover Cr(VI) from wastewater. Organic substrates acted as initial electron donors for Cr(VI) reduction. Active groups in an EPS layer played an important role in the adsorption of Cr(III). The EPS layer acted as a protective layer against toxic Cr(VI), and low Cr(VI) concentrations improved methane production by accelerating the degradation of organic substances by Trichoccus. Conversely, high Cr(VI) concentrations inhibited methane production because of the toxicity of Cr(VI) and the competition of hydrogen between Cr(VI) reduction and hydrogenotrophic methanogens. Acknowledgement This project is financially supported by the Fundamental Research Funds for the Central Universities (2015ZCQ-HJ-01) and the National Natural Science Foundation of China (NO.51608037). References 1. Adav, S.S., Lee, D.J., Show, K.Y., Tay, J.H, 2008. Aerobic granular sludge: recent advances. Biotechnol. Adv. 26(5), 411-423. 2. Angulo, E., Bula, L., Mercado, I, 2018. Bioremediation of Cephalexin with Non-Living Chlorella sp., Biomass after Lipid Extraction. Bioresource Technol. 257, 17-22. 3. APHA, AWWA, WPCF, Standard methods for the examination of water and wastewater,
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Figure list: Fig. 1 Effect of the initial Cr(VI) concentration on (A) Cr(VI) reduction and (B) methane production by the anaerobic granular sludge. Fig. 2 Cr(VI) concentration in the solution treated by the anaerobic granular sludge with different initial sucrose concentration. Fig. 3 Concentrations of (A) acetic acid, (B) propionic acid, (C) butyrate acid, and (D) ORP in the anaerobic systems with additional Cr(VI). Fig. 4 Cr2p XPS spectra of the anaerobic granular sludge after treating Cr(VI) wastewater. Fig. 5 (A) bacterial and (B) archaeal communities of the anaerobic granular sludge before and after treating Cr(VI) wastewater. Fig. 6 Proposed pathway of simultaneous Cr(VI) reduction and methane production by the granular sludge.
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Highlights
Anaerobic granule sludge has a good performance on Cr(VI) removal.
Anaerobic granule sludge produced methane with high efficiency under low Cr(VI) concentration.
Trichoccus and aceticlastic methanogens were identified as the dominant microorganisms present in the Cr(VI) bio-reduction process.
Graphical abstract