Efficient Volatile Fatty Acids Production from Waste Activated Sludge

Oct 18, 2018 - The microbial community analysis elucidated that the abundance of phyla Firmicutes and Bacteroidetes increased after the combined ...
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Efficient Volatile Fatty Acids Production from Waste Activated Sludge after Ferrate Pretreatment with Alkaline Environment and the Responding Microbial Community Shift Lin Li,† Junguo He,*,† Mengfei Wang,† Xiaodong Xin,‡ Jie Xu,† and Jie Zhang*,† †

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State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, No. 73 Huanghe Road, Harbin 150090, China ‡ School of Chemical Engineering, Huaqiao University, No. 668 Jimei Avenue, Xiamen 361021, China S Supporting Information *

ABSTRACT: In this article, a promising pretreatment method (ferrate pretreatment with alkaline conditions) is reported to enhance the anaerobic fermentation performance of WAS. Experimental results revealed that the maximal VFA production of 322.6 mg COD/g VSS (volatile suspended solids) was achieved at day 5 in the combined test (ferrate of 0.5 g/g VSS with pH10) while that of the blank test was 135.1 mg COD/g VSS at day 6. Acetic acid was significantly enriched in the combined test (57%) compared with the blank (35.9%). A mechanism study showed that the ferrate with an alkaline condition accelerated all the solubilization, hydrolysis, and acidification processes, which could be concluded from the improved performance and enhanced activities of key enzymes involved. The microbial community analysis elucidated that the abundance of phyla Firmicutes and Bacteroidetes increased after the combined pretreatment, which might account for the improvement of hydrolysis and acidification. It was further supported by the fact that the most abundant genera, Macellibacteroides, Petrimonas, Proteiniclasticum, and Proteocatella, were all capable of acid production or hydrolase secretion. In addition, the sole ferrate test exhibited better performance and more similarity with the combined test than soley the pH10 test, which indicated that ferrate contributed more to the enhanced VFA production in the combined test. KEYWORDS: Waste activated sludge, Chemical pretreatment, VFAs, Anaerobic fermentation, Resource recovery

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efficiently operated.2 However, hydrolysis of WAS was generally identified as the rate-limiting step in the anaerobic treatment process owing to the compact structure of sludge flocs, leading to high retention times, low conversion rate, and unsatisfactory product output.2 Therefore, physical, chemical and biological pretreatment methods have been carried out to facilitate WAS solubilization and hydrolysis.4−6 Among them, ferrate pretreatment has recently attracted increasing attention.5−9 Ferrate(VI) is a green oxidant in water and wastewater treatment, which possesses a strong oxidizing property especially in acidic environments. It could also be adopted as a green disinfectant without concerns of disinfection byproducts given that ferrate has no reactivity with Br ions.10 Thus, ferrate could be used as a multifunctional agent for effluent disinfection and WAS pretreatment, which would get the utmost out of the ferrate generation system within WWTPs. Moreover, it was reported that methane produced from anaerobic fermentation is a low value product that may

INTRODUCTION A considerable amount of waste activated sludge (WAS) is produced during wastewater biological treatment process, which needs to be properly treated.1 It was reported that the treatment and disposal of WAS accounted for up to 14% of the total energy consumption within a wastewater treatment plant (WWTP).2 As the Water-Energy Nexus has been coming into focus in recent years, WAS has been considered as a sustainable resource owing to a high organic matter content (>40%).2,3 According to previous studies, around 60% of the initial energy of wastewater is concentrated in sewage sludge with an average calorific value of around 6094 kcal/kg (25 497 kJ/kg).3 Hence, harnessing bioenergy from WAS could simultaneously address sustainable issues and environmental concerns imposed by the increasing amount of WAS. Anaerobic fermentation (or anaerobic digestion) has been commonly regarded as a preferable technology for WAS treatment and resource recovery. The biodegradable organic fractions could be converted to methane-rich biogas while WAS is stabilized and reduced. It was estimated that the energy recovered from WAS anaerobic fermentation could offset 70− 80% of the total energy consumption within a WWTP if © XXXX American Chemical Society

Received: August 19, 2018 Revised: October 1, 2018 Published: October 18, 2018 A

DOI: 10.1021/acssuschemeng.8b04115 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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make this technology economically unattractive when governmental subsidies or policies are absent.11,12 And methane upgrading and valorization is needed prior to its utilization for power generation, which would result in increased investment cost.13 Hence, VFAs produced before methane synthesis have attracted increasing interest recently owing to their wide range of biotechnology applications for products with higher monetary value, such as electricity generation by microbial fuel cells (MFC), biodiesel production by oleaginous yeast, hydrogen production via photofermentation, and synthesis of other valuable commodity chemicals.14−16 Therefore, this study was mainly focused on VFA production other than methane yield. There is little research exploring the enhancement of ferrate pretreatment on VFA production from WAS.5−9,17,18 Most of them were mainly focused on WAS disintegration and dewaterability rather than the performance of anaerobic fermentation.5,7−9,17 In Ye’s study, up to 30% of the total chemical oxygen demand (TCOD) could be solubilized after the ferrate pretreatment.7 The total volatile fatty acids (VFAs) production increased 5-fold at a high dosage of ferrate as compared to the control in another study.6 It is noteworthy that ferrate is unstable in aqueous solutions at pH below 9, which remained an impediment in its environmental applications.10 Hence, most pilot-scale studies employed the electrochemical methods to generated ferrate on site in a strong alkaline environment to improve the effective concentration of ferrate.19 However, previous studies relating to ferrate pretreatment simply added ferrate into raw WAS, without taking alkaline environment into account. The conclusions acquired might not be able to reflect the actual performance in engineering applications. In addition, the protagonists (microbes) in the anaerobic system for VFA production after ferrate pretreatment were seldom studied. Therefore, this study investigated the ferrate pretreatment with an alkaline condition pursuing the practical situation. Detailed investigation on microbial community and the specific functional activities (e.g., hydrolysis and acidification activities) were explored to understand how ferrate pretreatment with alkaline conditions improved VFA production from WAS in different stages (i.e., solubilization, hydrolysis, acidification) and the bacterial community shifts behind it. To the best of our knowledge, this is the first study about ferrate pretreatment on VFA production that takes alkaline conditions into consideration. The findings obtained herein are supposed to serve as a reference for chemical engineers to develop a promising strategy for resource recovery from WAS in fullscale. This literature aimed to investigate the effects of ferrate pretreatment combined with an alkaline condition on anaerobic WAS fermentation for VFA production. Specifically, the general effects on WAS solubilization, hydrolysis, and acidification were first investigated. Then, spectral characteristics of released organics and the activities of key enzymes relevant to hydrolysis and acidification were measured. Finally, the microbial community shift and the relationship between performance and the microbial community were explored. In addition, to understand the underlying mechanism of this process, experimental tests with sole ferrate and sole alkaline conditions were also conducted as a comparison.

Research Article

MATERIAL AND METHODS

Source and Characteristics of WAS. The waste activated sludge (WAS) was collected from the secondary sedimentation tank of a full scale wastewater treatment plant located in Harbin, China. The WAS was concentrated by settling for 12 h and stored at 4 °C before use. The main characteristics are listed as follows: total suspended solid (TSS), 14 807 ± 103 mg/L; volatile suspended solid (VSS), 9035 ± 61 mg/L; pH, 6.7 ± 0.1; total chemical oxygen demand (TCOD), 12 700 ± 92 mg/L; soluble COD, 170 ± 11 mg/L; total protein, 8113 ± 237 mg COD/L; total carbohydrate, 1153 ± 84 mg/L (error bars represent standard deviations). Pretreatment Experiments. The pretreatment was conducted in a beaker with a working volume of 1000 mL. A total of 800 mL of WAS was transferred into the beaker. The pH was adjusted to 10 with 2.0 M NaOH, and 0.5 g/g VSS K2FeO4 (the combined test) was added into the beaker thereafter. To better understand the effects of this method, a sole pH10 test (pH adjustment to 10), sole ferrate test (addition of 0.5 g/g VSS K2FeO4), and a blank test (without any pretreatment) were also conducted. All four tests were stirred at a speed of 120 rpm under ambient temperature for 60 min. A previous pilot-scale study showed that ferrate was stable in pH10 buffer for 24 h when kept cold.19,20 Obviously, a pH of 10 is enough to maintain a high concentration of ferrate from self-decay. Alkaline pretreatment always takes several days to reach a good performance on WAS pretreatment. Considering that pH was not adjusted further after the initial adjustment, therefore, the initial alkaline environment was mainly applied to stabilize the dissolved ferrate other than pretreat the WAS directly in this study. The dosage of ferrate was selected according to a previous study,18 which reported that further increasing the ferrate dosage did not produce obvious promotion on system performance when the dosage exceeded 0.5 g/g VSS. Hence, a pH of 10 and a dosage of 0.5 g/g VSS were selected in this study. Explore the Effects of Pretreatment on VFAs Production. The VFA production tests were performed in four identical serum bottles (corresponding to the former pretreatment tests) with a working volume of 500 mL each. A total of 400 mL of pretreated WAS was fed into each bottle; then 40 mL of seed sludge was added as an inoculum.21 All four bottles were flushed with nitrogen for 10 min to eliminate oxygen and capped with rubber stoppers afterward. After that, the four serum bottles were maintained in an air-bath shaker at a speed of 160 rpm under 35 ± 1 °C. The VFA production test lasted for 12 days. The seed sludge was collected from an acidogenic reactor, which produced VFAs steadily for a couple of months. Before the inoculation, the seed sludge was centrifuged (6000 rpm, 5 min) first. The supernatant was discarded to avoid any potential interferences, and the remaining pellets were resuspended to their original volume with deionized water. The main characteristics of the seed sludge are listed as follows: TSS, 9650 ± 231 mg/L; VSS, 7835 ± 159 mg/L; pH, 6.2 ± 0.1; TCOD, 9200 ± 106 mg/L; soluble COD, 4730 ± 75 mg/L (error bars represent standard deviations). The detailed bacterial community information is shown in Supporting Information S3. Activities of Key Enzymes Involved in Anaerobic WAS Fermentation. To study the effects of pretreatment on different steps, the activities of protease, α-glucosidase, acetate kinase (AK), phosphotransacetylase (PTA), and CoA transferase were measured at a fermentation time of 2 days.22−24 Briefly, protease was extracted on ice bath by Tris-HCl buffer (pH = 8.0) and Triton X-100 with a final concentration of 10 mM and 0.5%. Then, the supernatant after 5 min of centrifugation at 8000g and 4 °C was used for a protease test. A total of 1 mL of 0.5% azocasein and 3 mL of supernatant prepared beforehand was incubated at 37 °C for 90 min, then 2 mL of 10% trichloroacetic acid (TCA) was added to terminate the reaction. After centrifuging at 8000g and 4 °C, 2 mL of supernatant was mixed with 2 mL of 2 M NaOH. One unit of enzyme (EU) activity was defined as an absorbance increase of 0.01 at 440 nm. The α-glucosidase activity was tested with 0.1% p-nitrophenyl α-D-glucopyranoside as the substrate. A total of 2 mL of 0.2 M Tris-HCl (pH = 7.6), 1 mL of substrate, and 1 mL of sludge were mixed together. After 60 min of B

DOI: 10.1021/acssuschemeng.8b04115 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering incubation at 37 °C, the mixture was terminated in a boiled water bath for 3 min. The supernatant absorbance at 410 nm (p-nitrophenol production) was determined after a final centrifugation (8000g, 4 °C and 5 min). One EU was defined as every 1 μM p-nitrophenol produced during the incubation. Detailed determination procedures of AK, PTA, and CoA transferase are available in the Supporting Information. Analytical Methods. The sample was first centrifuged at 8000 rpm and then filtered by a cellulose membrane with a pore size of 0.45 μm; the supernatant was used for analysis.21 The TSS, VSS, TCOD, SCOD, NH4+-N, and PO43−-P were measured according to standard methods.25 Soluble protein was determined with a modified BCA kit (sangon, China). Soluble carbohydrate was measured using a phenolsulfuric acid method with glucose as a standard.26 A gas chromatograph (Agilent 6890 GC, USA) equipped with a Stabilwax-DA column (30 m × 0.32 mm × 0.5 mm) and a flame ionization detector was utilized to determine the composition of VFAs as described in a previous study.27 Extracellular polymeric substance (EPS) extraction from a sludge matrix complied with the research of Mu et al.28 In brief, 50 mL of sludge was centrifuged at 4000g for 5 min. Then it was resuspended in 0.05% NaCl solution to the original volume. After centrifuging at 4000g for another 10 min, the supernatant was regarded as loosely bound EPS (LB-EPS). The remaining pellets were resuspended again and maintained in a water bath at 60 °C for 30 min. The supernatant after centrifugation (4000g, 15 min) was regarded as tightly bound EPS (TB-EPS). Excitation emission matrix (EEM) fluorescence spectroscopy (FP6500, JASCO, Japan) was performed to determine the characteristics of soluble organics in the supernatant after pretreatment. The emission scans were performed from 220 to 550 nm in increments of 5 nm with excitation wavelengths from 220 to 450 nm at intervals of 5 nm. And the spectrum of deionized water was referred to as the blank. Fourier transform infrared (FTIR) spectroscopy was obtained in the range of 400−4000 cm−1 using a Spectrum One spectrometer (PerkinElmer, USA). After being dried, sludge samples were prepared using the potassium (KBr) pellets method.29 DNA extraction and high-throughput sequencing are described in the Supporting Information. Statistical Analysis. All measurements were performed in triplicate. An analysis of variance was processed, and p < 0.05 was referred to as statistically significant.

Figure 1. Effects of the pretreatment on the disintegration of EPS (a) and soluble protein and carbohydrate (b). Error bars represent standard deviations of triplicate tests.

78.9 mg COD/L to 234.0 mg COD/L and 1634.7 mg COD/ L, respectively. The concentration of soluble protein was much higher than that of soluble carbohydrate in the supernatant, which was in accord with the concentrations of total protein (8113 ± 237 mg COD/L) and total carbohydrate (1153 ± 84 mg COD/L). Moreover, the nutrient element NH4+-N (Figure S1) displayed a similar tendency to that of SCOD and soluble protein. It was reported that the elevated level of ammonium nitrogen could be attributed to the oxidation of protein, which was decomposed into peptides, amino acids, and eventually into carboxylate and ammonium.26 Interestingly, as we discussed above, all the SCOD, soluble protein, and carbohydrate in the sole ferrate test were higher than those in the sole pH10 test, indicating that ferrate was more favorable to WAS disintegration compared to pH10. To further explore the variations of functional groups in the solid phase, the FTIR spectra of dry sludge before and after pretreatment are illustrated in Figure S2. The adsorption band around 3300 cm−1 was reported to be the tensile vibration of O−H in the carboxyl group and N−H in amine, which represented fatty acids, sugars, proteins, and peptides.29 Both the sole ferrate and the combined tests showed weaker absorption bands compared to the blank, which indicated that plenty of macromolecules like carbohydrate and proteins were decomposed into smaller ones and thereafter released into the liquid phase. It explained the increase of SCOD, soluble carbohydrate, and proteins in the supernatant. The bands of amine groups and P−O, which represented amino acids and phospholipids that constitute microbial cell walls, were also observed at 1658 cm−1 (CO stretching amides), 1548 cm−1

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RESULTS AND DISCUSSION Effect of the Pretreatment on WAS Solubilization. EPS, which was considered a major component of the activated sludge matrix, bound cells together to form sludge aggregates.30 EPS disruption and WAS solubilization were studied first to uncover how the pretreatment improved the performance of the anaerobic system. Figure 1a showed the EPS variations after the pretreatment. TB-EPS took the dominant place of the total EPS (around 70%) without pretreatment, which was similar to previous studies.31,32 It implied a compact EPS structure which impeded the hydrolysis of WAS. SCOD increased incredibly after the pretreatment, especially in the combined test (3010 mg/L versus 170 mg/L in the blank), while no significant augmentation was observed in LB-EPS and TB-EPS. The high SCOD value of 3010 mg/L indicated an excellent performance on WAS solubilization even compared to other pretreatment methods (around 3500 mg/L and 1400 mg/L).20,33 Obviously, the EPS structure was disrupted to a large degree, and more EPS became soluble or extractable. It further indicated that the organic matter in EPS became more readily accessible to anaerobic bacteria, which would undoubtedly accelerate the hydrolysis process. Similar to SCOD, soluble carbohydrate and protein both increased remarkably in the combined test (Figure 1b). The soluble carbohydrate and protein increased from 11.5 mg COD/L and C

DOI: 10.1021/acssuschemeng.8b04115 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Figure 2. Fluorescence excitation emission matrix spectra of the four tests. All the samples were diluted by 80 times.

and has been reported previously to be an important component in WAS.22 Obviously, all three of these peaks were enhanced significantly in the combined test as compared to the blank, which was in agreement with the reduction of FTIR spectra bands and the SCOD augmentation. On the basis of the above observation, it could be reasonably deduced that the pretreatment by ferrate combined with pH10 caused severe disintegration of WAS (SCOD from 170 mg/L to 3010 mg/L), and sole ferrate (SCOD of 1550 mg/L) was supposed to be more beneficial than sole pH10 (SCOD of 850 mg/L) in this process. Destruction of microbial cell walls was supposed to happen during the pretreatment. What’s more, it seems that pH10 and ferrate together gave rise to a positive synergistic effect on WAS solubilization. More organic matter such as protein and carbohydrate were solubilized and thereby provided massive readily biodegradable substrates for the succeeding hydrolysis and acidification steps. Effects of the Pretreatment on Hydrolysis and Acidification. After WAS solubilization, the released organics will undergo hydrolysis and acidification before they are finally converted to VFAs. To study the influences of the pretreatment on hydrolysis and acidification processes, the activity measurements of specific key enzymes were conducted. As for the hydrolysis process, considering that protein and carboxylate are two major components of WAS, the activities of two common enzymes, namely protease and α-glucosidase, were determined here to study the influence of the pretreatment on the hydrolysis step.21,22 As shown in Figure 3a, the activities of both protease and α-glucosidase were enhanced after the pretreatment. The activities of protease increased by 30.1%, 88.5%, and 103.6% in sole pH10, sole ferrate, and the

(N−H bending and C−N stretching in amide groups) and around 1045 cm−1 (P−O−C stretch).29,34 These bands were reduced significantly in the sole ferrate and the combined tests. Considering that ferrate is able to degrade and inactivate persistent compounds and microorganisms, it implied the destruction of microbial cells caused by the pretreatment.10,29 Aromatics were also detected at 722 cm−1 (C−H bending in aromatic ring), 540 cm−1 (C−O−C bonding and deformation in aromatics), and 467 cm−1 (−COOH and alkenes or ArH).35,36 Similarly, remarkable reduction on these aromatic bands was also observed in the two tests with ferrate. The EEM fluorescence spectroscopy was widely used as a practical technology to characterize the EPS structure and soluble microbial products. Hence, EEM spectra were obtained after solubilization to further study the dissolved organics. As shown in Figure 2, a main peak (peak A) was detected at excitation/emission wavelengths (Ex/Em) of 280/345−355, and another weak peak (peak C) was observed at an Ex/Em of 225−230/345−350. It was reported that both of these peaks represented protein-like substances, which explained the large amount of protein in Figure 1b. Specifically, peak A was associated with the soluble microbial byproduct-like materials such as tryptophan protein-like substances, while peak C was related to some simple aromatic proteins such as tyrosine.37 A previous study reported that tryptophan-like compounds, which present as free molecules or as bound in proteins and peptides, could be easily used by microorganisms to meet their energy requirements. Besides the protein-like peaks, a humic acid-like peak (peak B) located at an Ex/Em around 350−360/ 435−450 was also observed except for the blank test.38 It could be explained that humic acid is ubiquitous in the environment D

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Figure 3. Effects of the pretreatment on the activities of key enzymes (a), total VFA production (b) and VFAs distribution (c). Enzyme activities of blank test were set as 100%. AK and PTA were acetate kinase and phosphotransacetylase. A, P, iso-B, n-B, iso-V, and n-V were acetic, propionic, iso-butyric, n-butyric, iso-valeric, and n-valeric acids, respectively. Error bars represent standard deviations of triplicate tests.

different conditions. The sole pH10 test imposed the slightest influence on the relevant enzymes, while the combined test showed the highest relative activities in all three acid-forming enzymes. Take AK for example, the relative activities increased by 23%, 66%, and 89% relative to the blank in the sole pH10, sole ferrate, and the combined test, respectively. These results were in good agreement with the enhancement of WAS solubilization (Figure 1) and VFA production (Figure 3b). They all implied improved acidification activity. The effects of the pretreatment on VFA production are shown in Figure 3b and c. In all four tests, the total VFAs concentrations increased with the fermentation time at first and then declined rapidly, which was mainly attributed to the activity of methanogens.22 The maximal VFA production was 254.1 and 322.6 mg COD/g VSS in sole ferrate (day 6) and the combined test (day 5), respectively, which were much higher than that in the blank test (135.1 mg COD/g VSS, day 6). The high VFA production of 322.6 mg COD/g VSS in the combined test was comparable to other studies with different pretreatment methods, where around 270−370 mg COD/g VSS was achieved.20,22,33 However, only 159.2 mg COD/g VSS of VFAs, similar to the blank, was achieved at day 6 in the sole pH10 test. Obviously, the ferrate pretreatment with alkaline conditions improved the VFA production and shortened the fermentation time significantly, and ferrate contributed more to the positive effects according to the performance in the individual pretreatment test. Moreover, the combined test displayed a better performance than both sole ferrate and sole pH10 tests, which indicated a synergistic effect

combined test, respectively. However, the pretreatment imposed less influence on α-glucosidase compared to protease, which probably resulted from a much lesser quantity of carboxylate (1153 ± 84 mg COD/L) than protein (8113 ± 237 mg COD/L). The activities of α-glucosidase after pretreatment were enhanced by 3.3%, 32.2%, and 26.0% compared to the blank, respectively. It is well-known that a large proportion of exoenzymes such as hydrolase are immobilized in EPS by adsorption.39 Thus, many enzymes might be released into the supernatant after the pretreatment. Moreover, the disrupted EPS structure was supposed to facilitate the mass transfer between the substrate and the hydrolase. Therefore, the promoted mass transfer efficiency coupled with massive hydrolase released might be the main reason for which the hydrolysis step was enhanced. After WAS solubilization and hydrolysis, the produced amino acids and monosaccharide were bioconverted to VFAs. Determining the activities of key enzymes involved is an alternative method to evaluate the influences of the pretreatment on the acidification step. As shown in Figure S5, the acetyl-CoA is first converted to acetyl phosphate by PTA, then the produced acetyl phosphate is further converted to acetate by AK. Propionate is produced from propionyl-CoA by CoA transferase.40 Considering that acetate and propionate were two main VFAs produced here (Figure 3c), the activities of PTA, AK, and CoA transferase were investigated in this study to reveal the activities of acid-producing bacteria. As shown in Figure 4a, the relative activities of all the tested acid-forming enzymes were improved with the same tendency under E

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presented in Figure 4. As shown in Figure 4a, when the number of sequences exceeded 5000 in each test, the Shannon indexes approached the plateau, which indicated that the library size of over 39 000 sequences in this study was competent to characterize the overall bacterial communities. Table S2 showed that the species richness estimators (ACE and Chao1) declined after the pretreatment. Moreover, the Shannon index decreased while the Simpson index increased at the same time, indicating that the bacterial diversity was reduced by the imposed pretreatment. Principal component analysis (PCA) of these samples was carried out to investigate the difference of bacterial communities between these tests. As shown in Figure 4b, principal components 1 and 2 were supposed to explain 64% and 23% of the community variations, respectively. Obviously, five samples were clustered into three groups. The community of the sole ferrate test was more similar to that of the combined test, while the sole pH10 test was more close to the blank. It indicated that the microbial community of the inoculum was reshaped after the anaerobic fermentation, and ferrate imposed a greater influence on the bacterial structure compared to pH10, which was in accordance with the observations in the former sections. Effect of the Pretreatment on Functional Bacterial Population. Varieties of bacteria were involved in the anaerobic fermentation of WAS for VFAs. Figure 5 illustrated

Figure 4. Rarefaction curves of Shannon diversity index (a) and principal component analysis (PCA) analysis of the five samples (b).

between pH10 and ferrate. Given that the initial adjustment of pH10 did not affect the total VFA production much, it could be inferred that pH10 might not pretreat the WAS directly but supply an alkaline condition which stabilized ferrate from selfdecay and therefore promoted the system performance. Figure 3c presented the distribution of individual VFAs in the four tests. The acetic acid was the most abundant VFA in all four tests, which account for 35.9%, 36.9%, 57.6%, and 57.0% of the total VFAs, respectively. The n-valeric acid took the lowest proportions in all tests. Similar observations were also reported in other studies.20,22 Apparently, the ferrate pretreatment with pH10 enhanced the acetic acid production markedly, while the production of other VFAs such as propionic and iso-valeric acids was reduced. The sole ferrate test exhibited similar VFAs distribution to that of the combined test. However, the individual VFAs distribution of the sole pH10 test bore resemblance to the blank test. Considering that the production of VFAs from WAS was mainly a biological process, based on the above results, it could be further inferred that the combined pretreatment might impose a huge impact on the microbial community, which will be discussed in the following section, and ferrate clearly played a major role in this process. Overall Analysis on the Microbial Community. It can be seen from Table S2 that over 39 000 sequences were generated for each test after quality control, which was much more than previous studies.41,42 To obtain a general evaluation of the sequencing analysis, the rarefaction curves were

Figure 5. Phylum level distribution of bacterial population of the four samples.

the phylum level distribution of bacterial OTUs. It could be seen that the majority of bacteria were grouped into the phyla of Firmicutes, Bacteroidetes, Proteobacteria, and Chlorof lesi, which all have been reported to be capable of degrading complex organic matters for VFA production previously.41,43 However, the Venn analysis (Figure S4) displayed that only 977 OTUs out of 5624 and 3798 OTUs respectively were shared by the blank and combined tests, indicating that the pretreatment changed the microbial structure significantly. The relative abundance of Firmicutes, the most predominant bacteria, was remarkably enhanced in the combined test (41.7% versus 30.2% in blank test). Many microbes belonging to Firmicutes were reported to have the ability to excrete extracellular hydrolytic enzymes such as protease and cellulose,40,44 which was in accordance with the observed enhancement of the hydrolysis process. Bacteroidetes, which was reported to play a crucial role in hydrolysis and acidification steps,45 showed a higher abundance in the combined test as well (30.1% versus 23.0% in blank test). Other dominant phyla, Proteobacteria and Chlorof lesi, were also F

DOI: 10.1021/acssuschemeng.8b04115 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering found to be important in anaerobic fermentation.45 Proteobacteria was reduced from 20.7% (blank test) to 11.2% (combined test), while Chloroflesi did not change much. In addition, the abundance of Bacteroidetes in the sole ferrate test was enhanced dramatically (44.4% versus 23.0% in blank), which was similar to the combined test. It could be inferred that the enrichment of Bacteroidetes might be attribute to the ferrate pretreatment, given that no obvious enhancement of Bacteroidetes was observed in the sole pH10 test. More detailed information about the bacterial community was illustrated by the genus level distribution of bacterial OTUs in Table S3. Macellibacteroides and Petrimonas, affiliated with phylum Bacteroidetes, were enriched significantly and became the most abundant genera in the combined test (15.0% and 9.8% versus 5.9% and 0.03% in the blank test). These two genera are typical fermenting bacteria which could produce VFAs like acetate and butyrate from carbohydrate.46,47 Furthermore, the abundances of Proteiniclasticum (9.2%), Proteocatella (7.0%), Sedimentibacter (5.1%) ,and Acetoanaerobium (5.1%), affiliated with phylum Firmicutes, were all increased remarkably in the combined test as well. Among them, Proteiniclasticum and Sedimentibacter were previously reported to utilize protein with acetate as the major fermentation product.43 Acetoanaerobium forms acetate from H2 and CO2.48 And Proteocatella was believed to have proteolytic, chitinolyti,c and amylolytic activities.49 On the basis of the above findings, all the dominant bacteria were related to acid producing and hydrolyzing bacteria. Protein, carbohydrate, and H2/CO2 could all be utilized for VFA production. Obviously, these acid producing and hydrolyzing bacteria were enriched significantly after the pretreatment, which could explain the enhanced performance on acidification and hydrolysis. What’s more, it was reported that communities with higher values of Shannon indices will have more species and even distribution of abundance than those with lower values of Shannon indices.50 And unevenness might imply an enhanced community functional organization,51 which represents VFA production and hydrolysis ability in this study. Therefore, it is reasonable to speculate that the pretreatment enhanced the system performance by enriching specific bacteria relating to VFA production and hydrolysis. In addition, the genera distribution of the sole ferrate test in Table S3 exhibited more similarity with the combined test as compared with the sole pH10 test, which accorded with the PCA analysis, and it further supported the inference that ferrate contributed more to the bacterial community shift compared to the sole pH10 environment. Implications. This study proposed a new pretreatment strategy (i.e., ferrate pretreatment with alkaline conditions) to enhance VFA production from anaerobic WAS fermentation. It confirmed that the ferrate combined with an alkaline environment enhanced the VFA production, especially acetic acid, significantly from WAS. As reported before, most pilotscale studies employed in situ generated ferrate, which contained much hydroxide, for wastewater treatment.19 Therefore, the findings obtained here were expected to provide a scientific basis for further engineering applications, where ferrate and hydroxide are provided on site simultaneously. The generation facility could be shared among wastewater treatment, effluent disinfection, and WAS pretreatment systems in a wastewater treatment plant. The VFA-containing fermentation liquid, which displayed a better mass transfer efficiency owing to a lower viscosity as compared to WAS slurry, could be

directly used for enhanced nutrient removal, synthesis of PHAs, and other commodity chemicals. Moreover, no additional chemicals are needed in this technology, which makes it more economically attractive. According to previous studies, the net energy within a WWTP increased from −0.27 to 0.14 kWh/m3 sewage treated by the free ammonia pretreatment, which displayed comparable enhancement on VFA production.33,52 The electricity consumption for ferrate production is around 0.21 kWh/m3 sewage treated under the same conditions. It indicated that around 0.2 kWh/m3 sewage treated could be saved if the proposed methods are used and more might be saved if the parameters are carefully optimized.19 Hence, it seems that the proposed ferrate pretreatment combined with an alkaline environment is practically feasible. However, a more detailed operation strategy and a corresponding performance should be further investigated in pilot and full-scale systems to estimate the economic feasibility. And the performance might differ from region to region because of different characteristics of the WAS. Despite all this, this work could still inspire engineers to develop new strategies for efficient WAS management.

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CONCLUSIONS In this study, a promising pretreatment strategy, i.e., ferrate pretreatment combined with an initial alkaline environment, was studied. Experimental results showed that plenty of macromolecules were decomposed and converted into smaller ones and hence released into the liquid phase from the suspended solids. Destruction of cell walls was supposed to happen resulting from the pretreatment. Moreover, the activities of key enzymes relating to hydrolysis and acidification were also enhanced significantly, which explained the improved performance. In the phylum level, Firmicutes and Bacteroidetes, were enriched remarkably in the combined test (41.7% and 30.1% versus 30.2% and 23.0% in the blank respectively). However, Macellibacteroides (15.0%) and Petrimonas (9.8%) became the dominant genera compared to the blank (5.9% and 0.03% respectively). The enriched bacteria were all capable of acid production or hydrolase excretion, which was in good agreement with the observation of improved enzyme activity. Thus, the proposed pretreatment method facilitates the solubilization, hydrolysis, and acidification processes, and more VFAs were produced while less fermentation time was required.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b04115.

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Additional methods, nutrient element release, FTIR spectra, bacterial distribution of inoculum, Venn analysis, brief pathway, EPS distribution, richness and diversity indexes, and genus distribution of bacterial communities (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-0451-86283001. Fax: +86-0451-86283001. Email: [email protected]. *E-mail: [email protected]. G

DOI: 10.1021/acssuschemeng.8b04115 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Research Article

ACS Sustainable Chemistry & Engineering ORCID

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Junguo He: 0000-0002-2787-4694 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Program of International S&T Cooperation (No. 2016YFE0123400) and National Natural Science Foundation of China (No. 51778179).

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DOI: 10.1021/acssuschemeng.8b04115 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX