Article pubs.acs.org/est
Effect of Humic Acids with Different Characteristics on Fermentative Short-Chain Fatty Acids Production from Waste Activated Sludge Kun Liu, Yinguang Chen,* Naidong Xiao, Xiong Zheng,* and Mu Li State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China S Supporting Information *
ABSTRACT: Recently, the use of waste activated sludge to bioproduce short-chain fatty acids (SCFA) has attracted much attention as the sludge-derived SCFA can be used as a preferred carbon source to drive biological nutrient removal or biopolymer (polyhydroxyalkanoates) synthesis. Although large number of humic acid (HA) has been reported in sludge, the influence of HA on SCFA production has never been documented. This study investigated the effects on sludgederived SCFA production of two commercially available humic acids (referred to as SHHA and SAHA purchased respectively from Shanghai Reagent Company and Sigma-Aldrich) that differ in chemical structure, hydrophobicity, surfactant properties, and degree of aromaticity. It was found that SHHA remarkably enhanced SCFA production (1.7-3.5 folds), while SAHA had no obvious effect. Mechanisms study revealed that all four steps (solubilization, hydrolysis, acidification, and methanogenesis) involved in sludge fermentation were unaffected by SAHA. However, SHHA remarkably improved the solubilization of sludge protein and carbohydrate and the activity of hydrolysis enzymes (protease and α-glucosidase) owing to its greater hydrophobicity and protection of enzyme activity. SHHA also enhanced the acidification step by accelerating the bioreactions of glyceradehyde-3P → D-glycerate 1,3-diphosphate, and pyruvate → acetyl-CoA due to its abundant quinone groups which served as electron acceptor. Further investigation showed that SHHA negatively influenced the activity of acetoclastic methanogens for its competition for electrons and inhibition on the reaction of acetyl-CoA → 5-methyl-THMPT, which caused less SCFA being consumed. All these observations were in correspondence with SHHA significantly enhancing the production of sludge derived SCFA.
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INTRODUCTION A large quantity of waste activated sludge (WAS) is generated in biological municipal wastewater treatment plants. It contains significant amounts of organic matters, such as protein and carbohydrate. Reutilization of sludge as a useful resource is considered to be a preferable method for WAS management, by which both the reduction of WAS and the production of valuable products (such as short-chain fatty acid, SCFA) are achieved simultaneously. Using WAS to produce SCFA has attracted growing interest recently owing to the fact that SCFA has been proven to be a preferred carbon source for biological nutrient removal and polyhydroxyalkanoates synthesis. Several steps are involved in the production of SCFA from sludge under anaerobic conditions. The particulate organic materials in sludge are first solubilized before they are hydrolyzed by anaerobic microbes. Then the hydrolyzed products are bioconverted to SCFA by acid-producing bacteria. The generated SCFA (especially acetic acid) can be consumed by methanogens when the anaerobic conditions are favorable. During the reuse of sludge to produce SCFA, it has been reported that polyhydroxyalkanoates and gram-staining bacteria in sludge give significant influence on SCFA production.1 Except for © XXXX American Chemical Society
protein and carbohydrate, HA has also been reported to be an important fraction in WAS.2 After sludge being fermented to produce SCFA, it was observed that HA was transferred to SCFA-containing fermentation liquid, and the use of sludgederived SCFA could improve wastewater short-cut nitrificationdenitrification and denitrifying phosphorus removal via nitrite.3 Nevertheless, the influence of HA on biological SCFA production from sludge has never been investigated. It is well-known that HA is ubiquitous in the environment. The conformational nature of HA is supposed to be supramolecular associations of many relatively small and heterogeneous organic molecules, and the major functional groups of HA are carboxylic acid, phenolic and alcoholic hydroxyls, ketone, quinone, and aldehyde,4,5 which vary significantly with different HA. In the environment HA can interact with organic or inorganic contaminants or wastes due to its diverse chemical and physical Received: January 13, 2015 Revised: March 26, 2015 Accepted: March 31, 2015
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DOI: 10.1021/acs.est.5b00200 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology properties,6 which might affect the biodegradability or bioavailability of the pollutants. In the literature HA from Sigma-Aldrich (referred to as SAHA in this study) has bee chosen as a modal HA and its properties have been studied extensively by researchers.7 As it was observed in our current study that the content of main elemental component of HA from Shanghai Reagent Company (SHHA) was more similar to the HA extracted from sludge, this paper therefore investigated the effects on sludge-derived SCFA production of two commercially available humic acids (SHHA and SAHA) that differ in chemical structure, hydrophobicity, surfactant properties, and degree of aromaticity. First, the main characteristics of SHHA and SAHA were assayed by Fourier transform infrared, fluorescence excitation emission matrix, and solid-state 13C NMR. Then, the comparison of two humic acids (HAs) affecting SCFA production from sludge was made. Finally, the mechanisms for SHHA significantly increasing SCFA production were investigated from the following aspects: (1) the influences of two HAs on the four steps involved in sludge anaerobic fermentation, (2) the role of SHHA acting as surfactant and affecting microbial enzyme activity, and (3) the function of SHHA serving as electron acceptor and affecting key biological reactions.
the influence of HA on the solubilization of particulate organic matters of sludge was obtained by analyzing the concentrations of soluble protein and carbohydrate in fermentation liquor. The HA dosage of 0.5 g/g-TCOD was chose as an example and other experiment parameters were the same as those described above except that the fermentation time was 1 day. The relative hydrophobicity of WAS was measured after the WAS contacted with HA for 1 day, and determined by microbial adhesion to nhexadecane according to the reference.8 To understand the effect of HA on the step of sludge hydrolysis, the activities of two enzymes (protease and α-glucosidase), which are respectively relevant to the hydrolysis of protein and carbohydrate, were measured at fermentation time of 2 days. Experiments of the Influence of HA on Acidification of Hydrolysis Products. The effect of HA on the acidification of hydrolyzed products was investigated with synthetic wastewater of amino acids and monosaccharide (glucose), respectively. The synthetic amino acids wastewater consisted of L-leucine, L-lysine, L-glutamic, L-alanine, and L-cysteine with concentration of 0.2 g/ L each. The concentration of glucose in the synthetic monosaccharide wastewater was 1000 mg/L. In all tests, sludge was inoculated to the synthetic wastewaters with a final concentration of 2000 mg/L, and HA was added at a dosage of 0.5 g/g-TCOD. The operation was the same as described above, and the produced SCFA was measured at time of 2 days. Experiments of the Influence of HA on Acidogens. As acetic acid was observed to be an important fraction during sludge anaerobic fermentation for SCFA production, the impact of HA on acidogens was explored as a representative for its influence on acid-forming bacteria. Saccharofermentans acetigenes, which was purchased from China General Microbiological Culture Collection Center (CGMCC) and was isolated from waste sludge, was used as a model microbe of acetic acid production bacteria in the current study. The S. acetigenes was cultured in peptone-yeast extract-glucose medium at 30 °C and 200 rpm for 12 h. Then, the culture of S. acetigenes was inoculated to acid-production substrate. The acid-production substrate was composed of (mg/L of tap water): 5000 D-glucose, 10 000 NaHCO3, 1000 K2HPO4, 1000 KH2PO4, 400 MgSO4·7H2O, and 200 CaCl2. The culture of S. acetigenes in acid-production substrate was inoculated with OD600 of 0.02. The dosage of SHHA or SAHA was 0.5 g/g-TCOD, the pH was 7.0 ± 0.2, and the fermentation time was 2 day. Experiments of the Influence of HA on Acetotrophic and Hydrogenotrophic Methanogens. It is well-known that methane is mainly synthesized from acetate or H2/CO2, and the corresponding methane production bacteria are the acetotrophic and hydrogenotrophic methanogens. Thus, the effect of HA on methane production was studied by exploring its influence on acetotrophic and hydrogenotrophic methanogens, respectively. The experiments were conducted with synthetic wastewater consisting of (mg/L of tap water) 1000 KH2PO4, 1000 NH4Cl, 100 CaCl2, 100 MgCl2·6H2O, 1.0 FeCl3, 0.5 ZnSO4·7H2O, 0.5 CoCl2· 6H2O, 1.0 NiCl2· 6H2O, 0.5 CuSO4· 5H2O, and 0.5 MnCl2·4H2O. The inoculum concentration of methanogenic sludge, which was collected from a laboratory anaerobic sludge digestion reactor and washed three times with 0.1 M PBS before inoculation, was 2000 mg/L, and the dosage of HA was 0.5 g/gTCOD. In the acetotrophic test, 3000 mg/L of CH3COONa was added as the organic substrate, and the fermentation time was 7 days. While in the hydrogenotrophic test, the mixture of hydrogen and carbon dioxide (80%:20%) was used as the substrate. The working volume was 300 mL each, and the
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MATERIALS AND METHODS Waste Activated Sludge and HA. The WAS used in this study was withdrawn from the secondary sedimentation tank of a municipal wastewater treatment plant in Shanghai, China, and was concentrated by settling at 4 °C for 24 h. The main characteristics of concentrated sludge are as follows: pH 6.7 ± 0.2, total suspended solids (TSS) 13360 ± 714 mg/L, volatile suspended solids (VSS) 9331 ± 351 mg/L, total chemical oxygen demand (TCOD) 13110 ± 602 mg/L, soluble chemical oxygen demand (SCOD) 92 ± 4 mg/L, total protein 7027 ± 115 mg COD/L, total carbohydrate 1001 ± 43 mg COD/L, and HA 1693 ± 38 mg/L. Two model HAs were purchased from Shanghai Reagent Company and Sigma-Aldrich, referred respectively as SHHA and SAHA, were used without further purification to study their influences on the anaerobic fermentation of WAS for SCFA production. The above two HAs were observed to be nonbiodegradable in the current anaerobic fermentation process (Table S1 and Figure S1, Supporting Information). It was observed that SHHA contained a higher amount of carbon and nitrogen but a lower amount of oxygen and hydrogen, and the O/C and H/C atom ratios of SAHA were much higher than SHHA (Table S2, Supporting Information). Experiments of HA Affecting the Production of SCFA from Sludge. The batch experiments were carried out in 10 identical serum bottles. These 10 bottles were divided equally into two groups to study the effect of SAHA and SHHA, respectively. For each group, 300 mL of sludge and HA with a predetermined dosage were added. The dosages of HA were 0 (control), 0.3, 0.5, 0.8, and 1.0 g/g-TCOD. The pH value in each serum bottle was adjusted to 7.0 ± 0.2 by sodium hydroxide (3 M) or hydrochloric acid (3 M). After flushed with nitrogen gas to remove oxygen, all bottles were capped with rubber stoppers, sealed, and placed in an air-bath shaker (150 rpm) with medium temperature of 35 ± 2 °C. The fermentation time was 9 days. Experiments of the Influence of HA on Sludge Solubilization and Hydrolysis. From the main characteristics of WAS, it is easy to found that the sum of protein and carbohydrate is approximately 60% of the TCOD of sludge. So B
DOI: 10.1021/acs.est.5b00200 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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nitrophenol was prepared. Enzyme activity for extracted samples was expressed as UN/mgVSS. The elemental composition of HA was determined by Elemental Analyzer Vatio EL III (Elementar, German). Before analysis, the samples were freeze-dried. The assay of HA by Fourier Transform Infrared (FTIR), fluorescence, solid-state 13C NMR and the determination of sludge HA content were detailed in Supporting Information. All other analyses, including polysaccharide, protein, total nitrogen (TN), SCFA, TCOD, SCOD, TSS, VSS, and gas component, were the same as described in our previous publications.15−17 The total SCFA was calculated as the sum of COD concentrations converted from measured acetic, propionic, n-butyric, isobutyric, n-valeric, and iso-valeric acids using appropriate conversion factors (i.e., 1 mg/ L of acetic, propionic, butyric and valeric acids equal to 1.07, 1.51, 1.81, and 2.04 mg COD/L, respectively). Statistical Analysis. All tests were performed in triplicate, and the results were expressed as mean ± standard deviation. An analysis of variance (ANOVA) was used to test the significance of results and p < 0.05 was considered to be statistically significant.
fermentation time was 2 days. All other operations were the same as described above. Experiments of HA Affecting the Intermediate Metabolites during Bioconversion of Acetate to Methane. Acetyl phosphate, acetyl-CoA, 5-methyl-THMPT, and methyl-CoM are the main intermediate metabolites when acetate is anaerobically converted to methane.9 The effect of HA on the bioconversion of acetate to acetyl phosphate was investigated by the following test. The reaction mixture was composed of 145 mM Tris-HCl buffer (pH 7.4), 200 mM CH3COOK, 10 mM MgCl2, 10 mM ATP, and 705 mM hydroxylamine hydrochloride (neutralized with KOH before addition), and 0 (control) or 100 μg/UN of HA. The reaction was started with the addition of 0.1 UN (the enzyme activity unit) of acetate kinase. The reaction time was 20 min and the temperature was controlled at 37 °C. The termination and detailed analytical procedures were referred to the literature.10 The influence of HA on the bioconversion of acetate to acetylCoA was investigated in the mixture of 136 mM potassium phosphate buffer (pH 7.5), 4 mM MgCl2, 9.1 mM ATP, 45 mM KF, 9.1 mM CH3COOK, 9.1 mM reduced glutathione, 0.35 mM coenzyme A, 182 mM hydroxylamine hydrochloride, and 0 (control) or 100 μg/UN of HA. The reaction was initiated by the addition of 0.1 UN acetyl-CoA synthase, and the reaction time was 20 min. The investigation of HA affecting the bioconversion of acetyl phosphate to acetyl-CoA was carried out with the mixture of 98.5 mM Tris-HCl buffer (pH 7.4), 1.6 mM reduced glutathione, 0.43 mM coenzyme A, 7.23 mM acetyl phosphate, 13.3 mM (NH4)2SO4, 0.1 UN phosphotransacetylase, and 0 (control) or 100 μg/UN of HA. The reaction time was 5 min. The specified experimental procedures of these two experiments were according to the references.11,12 The influence of HA on the conversion of acetyl-CoA to 5methyl-THMPT was explored by using synthetic wastewater with acetyl-CoA lithium (50 μM), sodium 2-bromoethanesulfonate (BES, 10 mM) and SHHA (0 or 500 mg/L). 500 mg/L of methanogenic sludge was used as inoculums. Other experiment parameters were the same as above. The remaining acetyl-CoA in synthetic wastewater was measured by acetyl-CoA assay kit (Sigma-Aldrich) after cultivation for 10 min. Other Analytic Methods. The assay of protease was modified based on the methods of Gessesse et al.13 Protease was extracted first by mixing the sludge sample with Tris-HCl buffer (pH 8) and Triton X-100, to give the final concentrations of 10 mM Tris-HCl and 0.5% Triton X-100. The mixed liquids were stirred for 1 h and kept on ice bath through the operation. After centrifugation at 15 120g and 4 °C for 5 min, the supernatant was used as the enzyme source. Protease activity was then determined using azocasein as the substrate, and one enzyme unit (EU) of protease activity was defined as an absorbance increase of 0.01 at 440 nm. The α-glucosidase activity was analyzed with p-nitrophenyl α-D glucopyranoside as the substrate according to the method of Goel et al.14 After termination, 10% ZnSO4 and 2 M NaOH were added to flocculate HA and this flocculation method was observed to give no effect on the absorbance of hydrolyzate (p-nitrophenol). After centrifugation (15 120g, 4 °C and 5 min) the absorbance of the clear supernatant was measured at 410 nm, and the production of p-nitrophenol was calculated according to its calibration curve. Since the hydrolysis product of α-glucosidase with the substrate used in the enzyme assay was p-nitrophenol, 1 UN was defined as the amount of enzyme which released 1 μM of p-nitrophenol per hour. To quantify the amount of p-nitrophenol released in enzyme reaction, a calibration curve using known amount of p-
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RESULTS AND DISCUSSION Chemical and Spectroscopic Characteristics of SHHA and SAHA. FTIR, fluorescence and 13C NMR are three spectroscopic measurements being used widely in the literature to study the characteristics of HA.18,19 FTIR analysis is a qualitative tool to provide valuable information about specific molecular structures and chemical groups of HA. Excitation emission matrix (EEM) fluorescence is usually used to obtain the information on molecular weight and polycondensation of aromatic compounds. 13C NMR can be served as a quantitatively technique to explore the carbon distribution and chemical environment among the chemical groups. As seen in Figure 1,
Figure 1. Comparison of the FTIR spectra of SHHA and SAHA. The main absorbance peaks and their assignments are shown in Table S3 (Supporting Information).
both spectra had bands of 3400 cm−1 (H-bonded OH), 2920 cm−1 (aliphatic C−H stretching), and 1600 cm−1 (aromatic C C and carbonyl CO). Nevertheless, there were some differences in the spectra of two HAs. For example, the band of 1706 cm−1 appeared in SHHA, but 1380 cm−1 was observed in SAHA. These bands were reported to be relevant with the CO stretching of COOH and ketonic carbonyls and the OH of phenols, COO−, −CH3, and amide II, respectively.19,20 Also, in the spectra of SHHA there was a shoulder band at 3060 cm−1, which was generally attributed to the stretching of aromatic C− H.21−23 C
DOI: 10.1021/acs.est.5b00200 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 2. Fluorescence excitation emission matrix spectra of two HAs. Experiment conditions: pH 7.0 ± 0.1, DOC = 50 mg/L, ionic strength 0.01 M KCl.
EEM spectra of two HAs are shown in Figure 2. It can be seen that two apparent fluorophores (i.e., peak A at Ex = 440−470 nm and Em = 530−570 nm, and peak B at Ex = 280−340 nm and Em = 515−545 nm) were observed in SAHA, but only peak A appeared in SHHA. Also, the intensity of peak A of SAHA was less than that of SHHA. The fluorescence intensity is related with the molecular weight and polycondensation of aromatic compounds within macromolecules.24 The fluorescence peaks at longer wavelengths (such as peak A) suggest the existence of extended, linearly condensed aromatic ring networks and other unsaturated bonds, which are mainly connected with high molecular weight and humification degree; whereas peaks at shorter wavelength (such as peak B) are associated with the presence of simple structural components, that is, comparatively small molecular weight, high content of aliphatic fraction and low degree of aromatic polycondensation, conjugated chromophores and humification.24−26 The quantitative analyses of two HAs by Solid-state 13C NMR are shown in Figure 3 and the major bands are listed in Table 1.
Table 1. Distribution of different carbons in SAHA and SHHA calculated from 13C NMR spectra.a carboxylic
aromatic
anomeric
carbohydrate
alkyl
sample
220−161
161−113
113−93
93−44
44−0
HI/ HBb
SHHA SAHA
20.5 19.3
50.1 35.7
9.2 9.9
11.0 14.2
9.2 20.9
0.34 0.42
a
The percentage peak areas of individual peaks were calculated by dividing their areas by the total spectral peak area of the sample. Except HI/HB, the unit is %. bHI/HB = ((113−44) + (200−161))/ ((44−0) + (161−113)).
aromaticity degree of SHHA was higher than SAHA (50.08% against 35.78%). Effects of HA on SCFA Production. The effects of two HAs on total SCFA production at different fermentation time are shown in Figure 4A1 and A2. The data in Figure 4A1 indicated that in the blank test the SCFA concentration was increased with fermentation time from 1 to 5 days, but further increasing time to 7 days caused the decline of SCFA, which was mainly attributed to the activity of methanogens.15 At fermentation time of 5 day the maximal SCFA was 790 mg COD/L in the blank test. When SAHA was added to sludge fermentation system, almost the same observation was made (Figure 4A1). The maximal SCFA was 760, 816, 837, and 840 mg COD/L at SAHA dosage of 0.3, 0.5, 0.8, and 1.0 g/g-TCOD, respectively. Apparently, the presence of SAHA did not significantly affect the total SCFA production at any dosage investigated (p > 0.05). From Figure 4A2 it can be seen that as SHHA was added to sludge fermentation system, the maximal SCFA appeared at time of 7 days no matter what the dosage was. The concentration of SCFA was increased from 1379 to 2741 mg COD/L with the increase of SHHA from 0.3 to 0.8 g/g-TCOD, which was 1.7−3.5 folds of the blank. Apparently, the presence of SHHA greatly enhanced the production of SCFA from sludge. It is known that landfill refuse, digested sludge, peat soil, brown coal, weathered coal or compost are abundant in HA. These materials are available at low cost and might be used to accelerate the accumulation of SCFA from WAS, by which not only more SCFA will be obtained, but also the wastes can be reused. As shown in Figure 4B1, the content of individual SCFA in the presence of SAHA was similar to that in the blank. However, in
Figure 3. 13C NMR spectra of SAHA and SHHA.
According to Figure 3, the bands of alkyl carbon (44−0 ppm) and carbohydrate carbon (93−44 ppm) regions with SHHA were broader and their intensities were less than with SAHA. On the contrary, the intensity of aromatic carbon region (161−113 ppm) in the spectrum of SHHA was apparently higher than that in SAHA, and this region was reported to be attributed to the unsubstituted, carbon-substituted, oxygen or nitrogen substituted aromatic carbons, and olefinic carbons.19,27 The data in Table 1 revealed that the HI/HB ratio (an empirical index of hydrophilic) of SHHA was lower than that of SAHA, and the D
DOI: 10.1021/acs.est.5b00200 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 4. Comparison of two HAs affecting the total SCFA production at different time (A1: SAHA; A2: SHHA) and the composition of SCFA at fermentation time of 7 days (B1: SAHA; B2: SHHA). 0.3, 0.5, 0.8, and 1.0 in the graph represent the dosages of 0.3 g/g-TCOD, 0.5 g/g-TCOD, 0.8 g/gTCOD, and 1.0 g/g-TCOD, respectively. A, P, iso-B, n-B, iso-V, and n-V were respectively acetic, propionic, iso-butyric, n-butyric, iso-valeric, and nvaleric acids. Error bars represent standard deviations of triplicate tests.
Figure 5. Effects of SAHA and SHHA on the solubilization of protein and carbohydrate (A) and the activity of protease and α-glucosidase (B). Error bars represent standard deviations of triplicate tests. The control group was set as 100%.
expressed by the changes of soluble protein and carbohydrate concentrations in fermentation liquor. The data in Figure 5A showed that both soluble protein and carbohydrate were more than 150% of the control in the presence of SHHA, while they were almost same with the control in the presence of SAHA. Thus, the solubilization of sludge protein and carbohydrate were remarkably enhanced by SHHA. Some HAs have been reported to have the features of surfactant and show the ability of decreasing surface tension of water, which is mainly attributed to their amphiphilic character and water−air interface accumulation,29 and the appearance of such HA can increase the solubility of organic compounds.30 As seen from Table 1, the HI/HB ratio of SHHA was lower than that of SAHA (0.34 against 0.42), which suggested that the hydrophobicity of SHHA was stronger and the hydrophilicity of SAHA was higher. The hydrodynamic size of dissolved HA is related to the distribution of hydrophobic and hydrophilic components, which is small when the HA rich in hydrophilic
the SHHA added test the fraction of individual SCFA was different with that in the control (Figure 4B2). The presence of SHHA increased the percentages of acetic, n-butyric, and nvaleric acids, but decreased propionic, iso-butyric, and iso-valeric acids. Clearly, the influences of SHHA on total SCFA production and individual SCFA fraction were different with those of SAHA. The mechanisms for two HAs showing different influences on SCFA production were discussed in the following text. Influence of HA on Sludge Solubilization and Hydrolysis. There are four steps (solubilization, hydrolysis, acidification, and methanogenesis) involved in anaerobic fermentation of WAS, and the solubilization of sludge particulate organic matters is the prerequisite step for SCFA production. As protein and carbohydrate are the two important components of sludge, which account for respectively 54% and 8% of sludge TCOD, their degradation has been correlated with the formation of SCFA during sludge anaerobic fermentation.28 Therefore, in this study the solubilization of sludge organic matters was E
DOI: 10.1021/acs.est.5b00200 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 6. Metabolic pathways of methane production from acetate and H2/CO2. AK, acetate kinase; ACS, acetyl-coenzyme A synthase; PTA, phosphotransacetylase; ACDS, acetyl-CoA decarbonylase; THMPT, tetrahydromethanopterin; mtrA, THMPT methyltransferase; mcrA, methyl conenzyme reductase; hdrA, heterodisulfide reductase; fmdA, formyl-MFR dehydrogenase; ftr, formyl-MFR-THMPT formyltransferase; mch, methenyl- THMPT cyclohydrolase; mtd, 5,10-methylene-THMPT dehydrogenase; mer, methylene-THMPT reductase; frhA, coenzyme F420 hydrogenase.
Table 2. Comparison between Two HAs Affecting the Activities of Acetotrophic and Hydrogenotrophic Methanogensa acetotrophic methanogensb hydrogenotrophic methanogensc
acetic acid consumption (mg/L) methane generation (mL) H2 consumption (mL) methane generation (mL)
control
SHHA
SAHA
2918 ± 43 198 ± 6 218 ± 4 42 ± 2
1754 ± 25 101 ± 4 220 ± 4 42 ± 1
2889 ± 38 195 ± 7 215 ± 3 41 ± 2
a
The data reported are the averages and their standard deviations. bThe results were obtained by analyzing the residual acetate and accumulated methane from synthetic acetate wastewater at fermentation time of 7 days. cThe gas ingredient was analyzed at fermentation time of 48 h.
Influence of HA on Acidification and Acidogens. It is well-known that the dissolved protein and carbohydrate are first hydrolyzed respectively to amino acids and monosaccharide, and then bioconverted to pyruvate and SCFA in acidification step.34,35 Thus, the amino acids and monosaccharide synthetic wastewaters were used to investigate the influences of two HAs on acidification step. The data in Figure S2 (Supporting Information) showed that in the presence of SHHA the SCFA produced from either amino acids or monosaccharide synthetic wastewater was much greater than the blank. Nevertheless, when SAHA was added to the synthetic wastewaters, the produced SCFA was almost the same as the blank. These results indicated that the acidification step was enhanced by SHHA. Acidogens play a vital role in acidification. As seen in Figure 4, acetic acid was a very important SCFA fraction after SHHA was added to sludge fermentation system. The impact of SAHA and SHHA on the bacteria relevant to acetic acid production was explored by using S. acetigenes as a model acidogens, a phyletic of Firmicutes, and Firmicutes is reported to be one of the abundant phyla relevant to sludge fermentation.36 The results showed that the generation of acetic acid from glucose in the presence of SHHA was 123% of the blank and 121% of the SAHA. It is wellknown that in the pathway of acetic acid production from glucose the electrons are generated in the steps of glyceradehyde-3P → DGlycerate 1,3-diphosphate, and pyruvate → acetyl-CoA. The production of acetic acid would be enhanced if other electron acceptors were presented. HA can act as electron acceptor, and the quinone groups in HA are responsible for this property.37−39 From the above chemical characteristics of two HAs it can be
components is dissolved in aqueous solution. Also, it has been reported that materials with greater hydrophobic property are easily self-assembled in larger hydrodynamic dimensions, which shows greater decrease of total free energy of dissolved system.27 In this study the relative hydrophobicity of sludge in the test of blank, SHHA, and SAHA was 66.9%, 50.55%, and 61.8%, respectively. It seems that the presence of SHHA, compared with SAHA, made it easier for sludge contacting with water, and a greater solubilization of sludge particulate organic matters was therefore observed. Two common hydrolysis enzymes, that is, protease and αglucosidase, were analyzed to study the influence of HA on the hydrolysis of solubilized protein and carbohydrates, respectively. As seen in Figure 5B, the activity of both protease and αglucosidase in the test of SHHA was much greater than that of SAHA. It is well-known that the hydrolysis enzymes in activated sludge are associated with sludge floc matrix. These enzymes, however, are easy to leave sludge and dispersed into liquid phase during anaerobic fermentation, and their activities are easy to get lost due to dissolution in water solution or the formation of complex ingredients between enzymes and metal ions released from sludge.31 In the literature some specific surfactants (such as Tween-80) were used to maintain the activity of enzyme in aqueous bioreaction because they could form the protective micelles or facilitate the enzyme−substrate interaction.32,33 It might be that these properties with SHHA were greater than with SAHA, which caused greater activities of two sludge hydrolysis enzymes being observed in the presence of SHHA. F
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efficiency of acetyl-CoA in the presence of SHHA was only 16.1% of the control. Obviously, the step IV was remarkably inhibited by SHHA. It can be concluded therefore that the reason for SHHA decreasing methane generation was attributed to its inhibitory effect on the biotransformation of acetyl-CoA to 5methyl-THMPT. In the metabolic step IV, the C−C and C−S bonds of acetylCoA are split by the CO dehydrogenase and acetyl-CoA synthase complex, and the carbonyl group is oxidized with contributing two electrons to the small protein electron carrier ferredoxin.9,45 The suppression effect of SHHA on step IV was most likely induced by the electron accepting capacity of SHHA, which enabled it to compete for the electrons and block the way of ferredoxin regeneration. This observation is consistent with the literatures that some specific HA can be utilized as electron acceptor and low CH4 generation.40,41
found that the aromaticity of SHHA is higher than that of SAHA, indicating that SHHA has more abundance of quinone groups. Thus, SHHA showed a stronger electron accepting capacity, and more acetic acid was produced. Influence of HA on Methane Generation and Acetotrophic and Hydrogenotrophic Methanogens. It was reported that the methane production could be inhibited by HA.40−42 In the current study, however, the results in Figure S3 (Supporting Information) showed that only SHHA inhibited the methane generation when sludge was anaerobically fermented. With the increase of SHHA the amount of methane was decreased significantly (p < 0.05), and its inhibitory effect on methane was 14%, 42%, 85%, and 97% at SHHA dosage of 0.3, 0.5, 0.8, and 1.0 g/g-sludge, respectively. Usually, acetotrophic and hydrogenotrophic methanogens are the two main types of methane producers,43,44 and they can respectively bioconvert acidification products acetate and H2/ CO2 to methane (Figure 6). In this study the effects of SHHA and SAHA on acetoclastic methanogenesis were conducted in acetic acid synthetic wastewater. As seen in Table 2 the presence of SAHA did not significantly affect the consumption of acetic acid and the generation of methane in the acetoclastic methanogenesis test (p > 0.05). However, in the presence of SHHA the consumption of acetate and the generation of methane were respectively decreased to 60% and 51% of the control, which suggested that both acetic acid uptake and methane production were remarkably decreased by SHHA. Hence, SHHA rather than SAHA showed the inhibitory effect on acetoclastic methanogenesis. The results of hydrogenotrophic methanogenesis test in Table 2 showed that the presence of either SHHA or SAHA did not affect the uptake of hydrogen and the generation of methane. It can be concluded therefore that the reason for the inhibitory effect of SHHA on methane generation was due to the metabolism of acetoclastic methanogens instead of hydrogenotrophic methanogens being suppressed. As SHHA decreased the consumption of acidified products, the increased production of SCFA was observed. Several steps are involved in the generation of methane from acetate by acetoclastic methanogens (Figure 6). In order to dig out the detailed reason for SHHA inhibiting the metabolism of acetoclastic methanogens, it is necessary to investigate its influence on each step. As seen in Figure 6, both the generation of methane from acetate and H2/CO2 undergoes the following metabolic steps: 5-methyl-THMPT → methyl-CoM → CH4. The above discussion had indicated that the influence of SHHA on methane production from H2/CO2 was insignificant, which suggested that the effects of SHHA on the bioconversions of 5methyl-THMPT → methyl-CoM → CH4 might not be taken into consideration. Thus, in the aceticlastic pathway only steps I, II, III, and IV could be influenced by SHHA. According to the experiment of SHHA affecting the bioconversion of acetate to acetyl phosphate, it was found that the presence of SHHA did not significantly influence the step I (p > 0.05). The experiment of the influence of SHHA on the bioconversion of acetate to acetyl-CoA showed that the generated acetyl-CoA in the presence of SHHA was 97.2− 105.5% of the control, which indicated that the step II was not significantly affected by SHHA. Also, the produced acetyl-CoA in the test of SHHA affecting the bioconversion of acetyl phosphate to acetyl-CoA was 98.3−103.8% of the control, suggesting that the influence of SHHA on the step III was marginal. However, the experiment of the influence of HA on the conversion of acetyl-CoA to 5-methyl-THMPT showed that the utilization
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ASSOCIATED CONTENT
S Supporting Information *
Additional analytical methods, Tables S1−S3, and Figures S1− S3. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(Y.C.) Phone: 86-21-65981263; fax: 86-21-65986313; e-mail: [email protected]. *(X.Z.) E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (51278354, 51178324 and 51425802) and the Program of Shanghai Subject Chief Scientist (15XD1503400).
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REFERENCES
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(10) Aceti, D. J.; Ferry, J. G. Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis. J. Biol. Chem. 1988, 263 (30), 15444−15448. (11) Enzymatic Assay of S-Acetyl Coenzyme a Synthetase. Assay library of Sigma-Aldrich Life Science Website. http://www. sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_ Assay/sacetylcoenzyasynth.pdf. (12) Enzymatic Assay of Phosphotransacetylase. Assay library of Sigma-Aldrich Life Science Website. http://www.sigmaaldrich.com/ content/da m/sigma-aldrich/docs/Sigma /Enzyme_Assa y/ phosphotransacetylase.pdf. (13) Gessesse, A.; Dueholm, T.; Petersen, S. B.; Nielsen, P. H. Lipase and protease extraction from activated sludge. Water Res. 2003, 37 (15), 3652−3657. (14) Goel, R.; Mino, T.; Satoh, H.; Matsuo, T. Enzyme activities under anaerobic and aerobic conditions inactivated sludge sequencing batch reactor. Water Res. 1998, 32 (7), 2081−2088. (15) Chen, Y.; Jiang, S.; Yuan, H.; Zhou, Q.; Gu, G. Hydrolysis and acidification of waste activated sludge at different pHs. Water Res. 2007, 41 (3), 683−9. (16) Yuan, H.; Chen, Y.; Zhang, H.; Jiang, S.; Zhou, Q.; Gu, G. Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline conditions. Environ. Sci. Technol. 2006, 40 (6), 2025−2029. (17) Zhao, Y.; Chen, Y.; Zhang, D.; Zhu, X. Waste activated sludge fermentation for hydrogen production enhanced by anaerobic process improvement and acetobacteria inhibition: The Role of Fermentation pH. Environ. Sci. Technol. 2010, 44 (9), 3317−3323. (18) Kang, K. H.; Shin, H. S.; Park, H. Characterization of humic substances present in landfill leachates with different landfill ages and its implications. Water Res. 2002, 36 (16), 4023−4032. (19) Amir, S.; Jouraiphy, A.; Meddich, A.; El Gharous, M.; Winterton, P.; Hafidi, M. Structural study of humic acids during composting of activated sludge-green waste: Elemental analysis, FTIR and 13C NMR. J. Hazard. Mater. 2010, 177 (1−3), 524−529. (20) Smidt, E.; Schwanninger, M. Characterization of waste materials using FTIR spectroscopy: Process monitoring and quality assessment. Spectrosc. Lett. 2005, 38 (3), 247−270. (21) Cunha, T. J. F.; Novotny, E. H.; Madari, B. E.; Martin-Neto, L.; Rezende, M. O.; Canelas, L. P.; Benites, V. Spectroscopy Characterization of humic acids isolated from amazonian dark earth soils (Terra ́ In Amazonian Dark Earths: Wim Sombroek’s Vision; Preta De Indio). Woods, W., Teixeira, W., Lehmann, J., Steiner, C., WinklerPrins, A., Rebellato, L., Eds.; Springer: Netherlands, 2009; pp 363−372. (22) Kang, S. H.; Xing, B. S. Phenanthrene sorption to sequentially extracted soil humic acids and humins. Environ. Sci. Technol. 2005, 39 (1), 134−140. (23) Tatzber, M.; Stemmer, M.; Splegel, H.; Katziberger, C.; Haberhauer, G.; Mentler, A.; Gerzabek, M. H. FTIR-spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH, Na4P2O7, and Na2CO3 extraction procedures. J. Plant Nutr. Soil Sci. 2007, 170 (4), 522−529. (24) Chen, J.; Gu, B. H.; LeBoeuf, E. J.; Pan, H. J.; Dai, S. Spectroscopic characterization of the structural and functional properties of natural organic matter fractions. Chemosphere 2002, 48 (1), 59−68. (25) Chai, X. L.; Liu, G. X.; Zhao, X.; Hao, Y. X.; Zhao, Y. C. Fluorescence excitation-emission matrix combined with regional integration analysis to characterize the composition and transformation of humic and fulvic acids from landfill at different stabilization stages. Waste Manage. 2012, 32 (3), 438−447. (26) Fukushima, M.; Kikuchi, A.; Tatsumi, K.; Tanaka, F. Separation of fulvic acid from soil extracts based on ion-pair formation with a cationic surfactant. Anal. Sci. 2006, 22 (2), 229−233. (27) Šmejkalová, D.; Piccolo, A. Aggregation and disaggregation of humic supramolecular assemblies by NMR diffusion ordered spectroscopy (DOSY-NMR). Environ. Sci. Technol. 2007, 42 (3), 699−706. (28) Jiang, S.; Chen, Y.; Zhou, Q.; Gu, G. Biological short-chain fatty acids (SCFAs) production from waste-activated sludge affected by surfactant. Water Res. 2007, 41 (14), 3112−20. H
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Effect of Humic Acids with Different Characteristics on Fermentative Short-Chain Fatty Acids Production from Waste Activated Sludge Kun Liu, Yinguang Chen,* Naidong Xiao, Xiong Zheng,* and Mu Li State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China S Supporting Information *
ABSTRACT: Recently, the use of waste activated sludge to bioproduce short-chain fatty acids (SCFA) has attracted much attention as the sludge-derived SCFA can be used as a preferred carbon source to drive biological nutrient removal or biopolymer (polyhydroxyalkanoates) synthesis. Although large number of humic acid (HA) has been reported in sludge, the influence of HA on SCFA production has never been documented. This study investigated the effects on sludgederived SCFA production of two commercially available humic acids (referred to as SHHA and SAHA purchased respectively from Shanghai Reagent Company and Sigma-Aldrich) that differ in chemical structure, hydrophobicity, surfactant properties, and degree of aromaticity. It was found that SHHA remarkably enhanced SCFA production (1.7-3.5 folds), while SAHA had no obvious effect. Mechanisms study revealed that all four steps (solubilization, hydrolysis, acidification, and methanogenesis) involved in sludge fermentation were unaffected by SAHA. However, SHHA remarkably improved the solubilization of sludge protein and carbohydrate and the activity of hydrolysis enzymes (protease and α-glucosidase) owing to its greater hydrophobicity and protection of enzyme activity. SHHA also enhanced the acidification step by accelerating the bioreactions of glyceradehyde-3P → D-glycerate 1,3-diphosphate, and pyruvate → acetyl-CoA due to its abundant quinone groups which served as electron acceptor. Further investigation showed that SHHA negatively influenced the activity of acetoclastic methanogens for its competition for electrons and inhibition on the reaction of acetyl-CoA → 5-methyl-THMPT, which caused less SCFA being consumed. All these observations were in correspondence with SHHA significantly enhancing the production of sludge derived SCFA.
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INTRODUCTION A large quantity of waste activated sludge (WAS) is generated in biological municipal wastewater treatment plants. It contains significant amounts of organic matters, such as protein and carbohydrate. Reutilization of sludge as a useful resource is considered to be a preferable method for WAS management, by which both the reduction of WAS and the production of valuable products (such as short-chain fatty acid, SCFA) are achieved simultaneously. Using WAS to produce SCFA has attracted growing interest recently owing to the fact that SCFA has been proven to be a preferred carbon source for biological nutrient removal and polyhydroxyalkanoates synthesis. Several steps are involved in the production of SCFA from sludge under anaerobic conditions. The particulate organic materials in sludge are first solubilized before they are hydrolyzed by anaerobic microbes. Then the hydrolyzed products are bioconverted to SCFA by acid-producing bacteria. The generated SCFA (especially acetic acid) can be consumed by methanogens when the anaerobic conditions are favorable. During the reuse of sludge to produce SCFA, it has been reported that polyhydroxyalkanoates and gram-staining bacteria in sludge give significant influence on SCFA production.1 Except for © XXXX American Chemical Society
protein and carbohydrate, HA has also been reported to be an important fraction in WAS.2 After sludge being fermented to produce SCFA, it was observed that HA was transferred to SCFA-containing fermentation liquid, and the use of sludgederived SCFA could improve wastewater short-cut nitrificationdenitrification and denitrifying phosphorus removal via nitrite.3 Nevertheless, the influence of HA on biological SCFA production from sludge has never been investigated. It is well-known that HA is ubiquitous in the environment. The conformational nature of HA is supposed to be supramolecular associations of many relatively small and heterogeneous organic molecules, and the major functional groups of HA are carboxylic acid, phenolic and alcoholic hydroxyls, ketone, quinone, and aldehyde,4,5 which vary significantly with different HA. In the environment HA can interact with organic or inorganic contaminants or wastes due to its diverse chemical and physical Received: January 13, 2015 Revised: March 26, 2015 Accepted: March 31, 2015
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Environmental Science & Technology properties,6 which might affect the biodegradability or bioavailability of the pollutants. In the literature HA from Sigma-Aldrich (referred to as SAHA in this study) has bee chosen as a modal HA and its properties have been studied extensively by researchers.7 As it was observed in our current study that the content of main elemental component of HA from Shanghai Reagent Company (SHHA) was more similar to the HA extracted from sludge, this paper therefore investigated the effects on sludge-derived SCFA production of two commercially available humic acids (SHHA and SAHA) that differ in chemical structure, hydrophobicity, surfactant properties, and degree of aromaticity. First, the main characteristics of SHHA and SAHA were assayed by Fourier transform infrared, fluorescence excitation emission matrix, and solid-state 13C NMR. Then, the comparison of two humic acids (HAs) affecting SCFA production from sludge was made. Finally, the mechanisms for SHHA significantly increasing SCFA production were investigated from the following aspects: (1) the influences of two HAs on the four steps involved in sludge anaerobic fermentation, (2) the role of SHHA acting as surfactant and affecting microbial enzyme activity, and (3) the function of SHHA serving as electron acceptor and affecting key biological reactions.
the influence of HA on the solubilization of particulate organic matters of sludge was obtained by analyzing the concentrations of soluble protein and carbohydrate in fermentation liquor. The HA dosage of 0.5 g/g-TCOD was chose as an example and other experiment parameters were the same as those described above except that the fermentation time was 1 day. The relative hydrophobicity of WAS was measured after the WAS contacted with HA for 1 day, and determined by microbial adhesion to nhexadecane according to the reference.8 To understand the effect of HA on the step of sludge hydrolysis, the activities of two enzymes (protease and α-glucosidase), which are respectively relevant to the hydrolysis of protein and carbohydrate, were measured at fermentation time of 2 days. Experiments of the Influence of HA on Acidification of Hydrolysis Products. The effect of HA on the acidification of hydrolyzed products was investigated with synthetic wastewater of amino acids and monosaccharide (glucose), respectively. The synthetic amino acids wastewater consisted of L-leucine, L-lysine, L-glutamic, L-alanine, and L-cysteine with concentration of 0.2 g/ L each. The concentration of glucose in the synthetic monosaccharide wastewater was 1000 mg/L. In all tests, sludge was inoculated to the synthetic wastewaters with a final concentration of 2000 mg/L, and HA was added at a dosage of 0.5 g/g-TCOD. The operation was the same as described above, and the produced SCFA was measured at time of 2 days. Experiments of the Influence of HA on Acidogens. As acetic acid was observed to be an important fraction during sludge anaerobic fermentation for SCFA production, the impact of HA on acidogens was explored as a representative for its influence on acid-forming bacteria. Saccharofermentans acetigenes, which was purchased from China General Microbiological Culture Collection Center (CGMCC) and was isolated from waste sludge, was used as a model microbe of acetic acid production bacteria in the current study. The S. acetigenes was cultured in peptone-yeast extract-glucose medium at 30 °C and 200 rpm for 12 h. Then, the culture of S. acetigenes was inoculated to acid-production substrate. The acid-production substrate was composed of (mg/L of tap water): 5000 D-glucose, 10 000 NaHCO3, 1000 K2HPO4, 1000 KH2PO4, 400 MgSO4·7H2O, and 200 CaCl2. The culture of S. acetigenes in acid-production substrate was inoculated with OD600 of 0.02. The dosage of SHHA or SAHA was 0.5 g/g-TCOD, the pH was 7.0 ± 0.2, and the fermentation time was 2 day. Experiments of the Influence of HA on Acetotrophic and Hydrogenotrophic Methanogens. It is well-known that methane is mainly synthesized from acetate or H2/CO2, and the corresponding methane production bacteria are the acetotrophic and hydrogenotrophic methanogens. Thus, the effect of HA on methane production was studied by exploring its influence on acetotrophic and hydrogenotrophic methanogens, respectively. The experiments were conducted with synthetic wastewater consisting of (mg/L of tap water) 1000 KH2PO4, 1000 NH4Cl, 100 CaCl2, 100 MgCl2·6H2O, 1.0 FeCl3, 0.5 ZnSO4·7H2O, 0.5 CoCl2· 6H2O, 1.0 NiCl2· 6H2O, 0.5 CuSO4· 5H2O, and 0.5 MnCl2·4H2O. The inoculum concentration of methanogenic sludge, which was collected from a laboratory anaerobic sludge digestion reactor and washed three times with 0.1 M PBS before inoculation, was 2000 mg/L, and the dosage of HA was 0.5 g/gTCOD. In the acetotrophic test, 3000 mg/L of CH3COONa was added as the organic substrate, and the fermentation time was 7 days. While in the hydrogenotrophic test, the mixture of hydrogen and carbon dioxide (80%:20%) was used as the substrate. The working volume was 300 mL each, and the
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MATERIALS AND METHODS Waste Activated Sludge and HA. The WAS used in this study was withdrawn from the secondary sedimentation tank of a municipal wastewater treatment plant in Shanghai, China, and was concentrated by settling at 4 °C for 24 h. The main characteristics of concentrated sludge are as follows: pH 6.7 ± 0.2, total suspended solids (TSS) 13360 ± 714 mg/L, volatile suspended solids (VSS) 9331 ± 351 mg/L, total chemical oxygen demand (TCOD) 13110 ± 602 mg/L, soluble chemical oxygen demand (SCOD) 92 ± 4 mg/L, total protein 7027 ± 115 mg COD/L, total carbohydrate 1001 ± 43 mg COD/L, and HA 1693 ± 38 mg/L. Two model HAs were purchased from Shanghai Reagent Company and Sigma-Aldrich, referred respectively as SHHA and SAHA, were used without further purification to study their influences on the anaerobic fermentation of WAS for SCFA production. The above two HAs were observed to be nonbiodegradable in the current anaerobic fermentation process (Table S1 and Figure S1, Supporting Information). It was observed that SHHA contained a higher amount of carbon and nitrogen but a lower amount of oxygen and hydrogen, and the O/C and H/C atom ratios of SAHA were much higher than SHHA (Table S2, Supporting Information). Experiments of HA Affecting the Production of SCFA from Sludge. The batch experiments were carried out in 10 identical serum bottles. These 10 bottles were divided equally into two groups to study the effect of SAHA and SHHA, respectively. For each group, 300 mL of sludge and HA with a predetermined dosage were added. The dosages of HA were 0 (control), 0.3, 0.5, 0.8, and 1.0 g/g-TCOD. The pH value in each serum bottle was adjusted to 7.0 ± 0.2 by sodium hydroxide (3 M) or hydrochloric acid (3 M). After flushed with nitrogen gas to remove oxygen, all bottles were capped with rubber stoppers, sealed, and placed in an air-bath shaker (150 rpm) with medium temperature of 35 ± 2 °C. The fermentation time was 9 days. Experiments of the Influence of HA on Sludge Solubilization and Hydrolysis. From the main characteristics of WAS, it is easy to found that the sum of protein and carbohydrate is approximately 60% of the TCOD of sludge. So B
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nitrophenol was prepared. Enzyme activity for extracted samples was expressed as UN/mgVSS. The elemental composition of HA was determined by Elemental Analyzer Vatio EL III (Elementar, German). Before analysis, the samples were freeze-dried. The assay of HA by Fourier Transform Infrared (FTIR), fluorescence, solid-state 13C NMR and the determination of sludge HA content were detailed in Supporting Information. All other analyses, including polysaccharide, protein, total nitrogen (TN), SCFA, TCOD, SCOD, TSS, VSS, and gas component, were the same as described in our previous publications.15−17 The total SCFA was calculated as the sum of COD concentrations converted from measured acetic, propionic, n-butyric, isobutyric, n-valeric, and iso-valeric acids using appropriate conversion factors (i.e., 1 mg/ L of acetic, propionic, butyric and valeric acids equal to 1.07, 1.51, 1.81, and 2.04 mg COD/L, respectively). Statistical Analysis. All tests were performed in triplicate, and the results were expressed as mean ± standard deviation. An analysis of variance (ANOVA) was used to test the significance of results and p < 0.05 was considered to be statistically significant.
fermentation time was 2 days. All other operations were the same as described above. Experiments of HA Affecting the Intermediate Metabolites during Bioconversion of Acetate to Methane. Acetyl phosphate, acetyl-CoA, 5-methyl-THMPT, and methyl-CoM are the main intermediate metabolites when acetate is anaerobically converted to methane.9 The effect of HA on the bioconversion of acetate to acetyl phosphate was investigated by the following test. The reaction mixture was composed of 145 mM Tris-HCl buffer (pH 7.4), 200 mM CH3COOK, 10 mM MgCl2, 10 mM ATP, and 705 mM hydroxylamine hydrochloride (neutralized with KOH before addition), and 0 (control) or 100 μg/UN of HA. The reaction was started with the addition of 0.1 UN (the enzyme activity unit) of acetate kinase. The reaction time was 20 min and the temperature was controlled at 37 °C. The termination and detailed analytical procedures were referred to the literature.10 The influence of HA on the bioconversion of acetate to acetylCoA was investigated in the mixture of 136 mM potassium phosphate buffer (pH 7.5), 4 mM MgCl2, 9.1 mM ATP, 45 mM KF, 9.1 mM CH3COOK, 9.1 mM reduced glutathione, 0.35 mM coenzyme A, 182 mM hydroxylamine hydrochloride, and 0 (control) or 100 μg/UN of HA. The reaction was initiated by the addition of 0.1 UN acetyl-CoA synthase, and the reaction time was 20 min. The investigation of HA affecting the bioconversion of acetyl phosphate to acetyl-CoA was carried out with the mixture of 98.5 mM Tris-HCl buffer (pH 7.4), 1.6 mM reduced glutathione, 0.43 mM coenzyme A, 7.23 mM acetyl phosphate, 13.3 mM (NH4)2SO4, 0.1 UN phosphotransacetylase, and 0 (control) or 100 μg/UN of HA. The reaction time was 5 min. The specified experimental procedures of these two experiments were according to the references.11,12 The influence of HA on the conversion of acetyl-CoA to 5methyl-THMPT was explored by using synthetic wastewater with acetyl-CoA lithium (50 μM), sodium 2-bromoethanesulfonate (BES, 10 mM) and SHHA (0 or 500 mg/L). 500 mg/L of methanogenic sludge was used as inoculums. Other experiment parameters were the same as above. The remaining acetyl-CoA in synthetic wastewater was measured by acetyl-CoA assay kit (Sigma-Aldrich) after cultivation for 10 min. Other Analytic Methods. The assay of protease was modified based on the methods of Gessesse et al.13 Protease was extracted first by mixing the sludge sample with Tris-HCl buffer (pH 8) and Triton X-100, to give the final concentrations of 10 mM Tris-HCl and 0.5% Triton X-100. The mixed liquids were stirred for 1 h and kept on ice bath through the operation. After centrifugation at 15 120g and 4 °C for 5 min, the supernatant was used as the enzyme source. Protease activity was then determined using azocasein as the substrate, and one enzyme unit (EU) of protease activity was defined as an absorbance increase of 0.01 at 440 nm. The α-glucosidase activity was analyzed with p-nitrophenyl α-D glucopyranoside as the substrate according to the method of Goel et al.14 After termination, 10% ZnSO4 and 2 M NaOH were added to flocculate HA and this flocculation method was observed to give no effect on the absorbance of hydrolyzate (p-nitrophenol). After centrifugation (15 120g, 4 °C and 5 min) the absorbance of the clear supernatant was measured at 410 nm, and the production of p-nitrophenol was calculated according to its calibration curve. Since the hydrolysis product of α-glucosidase with the substrate used in the enzyme assay was p-nitrophenol, 1 UN was defined as the amount of enzyme which released 1 μM of p-nitrophenol per hour. To quantify the amount of p-nitrophenol released in enzyme reaction, a calibration curve using known amount of p-
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RESULTS AND DISCUSSION Chemical and Spectroscopic Characteristics of SHHA and SAHA. FTIR, fluorescence and 13C NMR are three spectroscopic measurements being used widely in the literature to study the characteristics of HA.18,19 FTIR analysis is a qualitative tool to provide valuable information about specific molecular structures and chemical groups of HA. Excitation emission matrix (EEM) fluorescence is usually used to obtain the information on molecular weight and polycondensation of aromatic compounds. 13C NMR can be served as a quantitatively technique to explore the carbon distribution and chemical environment among the chemical groups. As seen in Figure 1,
Figure 1. Comparison of the FTIR spectra of SHHA and SAHA. The main absorbance peaks and their assignments are shown in Table S3 (Supporting Information).
both spectra had bands of 3400 cm−1 (H-bonded OH), 2920 cm−1 (aliphatic C−H stretching), and 1600 cm−1 (aromatic C C and carbonyl CO). Nevertheless, there were some differences in the spectra of two HAs. For example, the band of 1706 cm−1 appeared in SHHA, but 1380 cm−1 was observed in SAHA. These bands were reported to be relevant with the CO stretching of COOH and ketonic carbonyls and the OH of phenols, COO−, −CH3, and amide II, respectively.19,20 Also, in the spectra of SHHA there was a shoulder band at 3060 cm−1, which was generally attributed to the stretching of aromatic C− H.21−23 C
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Figure 2. Fluorescence excitation emission matrix spectra of two HAs. Experiment conditions: pH 7.0 ± 0.1, DOC = 50 mg/L, ionic strength 0.01 M KCl.
EEM spectra of two HAs are shown in Figure 2. It can be seen that two apparent fluorophores (i.e., peak A at Ex = 440−470 nm and Em = 530−570 nm, and peak B at Ex = 280−340 nm and Em = 515−545 nm) were observed in SAHA, but only peak A appeared in SHHA. Also, the intensity of peak A of SAHA was less than that of SHHA. The fluorescence intensity is related with the molecular weight and polycondensation of aromatic compounds within macromolecules.24 The fluorescence peaks at longer wavelengths (such as peak A) suggest the existence of extended, linearly condensed aromatic ring networks and other unsaturated bonds, which are mainly connected with high molecular weight and humification degree; whereas peaks at shorter wavelength (such as peak B) are associated with the presence of simple structural components, that is, comparatively small molecular weight, high content of aliphatic fraction and low degree of aromatic polycondensation, conjugated chromophores and humification.24−26 The quantitative analyses of two HAs by Solid-state 13C NMR are shown in Figure 3 and the major bands are listed in Table 1.
Table 1. Distribution of different carbons in SAHA and SHHA calculated from 13C NMR spectra.a carboxylic
aromatic
anomeric
carbohydrate
alkyl
sample
220−161
161−113
113−93
93−44
44−0
HI/ HBb
SHHA SAHA
20.5 19.3
50.1 35.7
9.2 9.9
11.0 14.2
9.2 20.9
0.34 0.42
a
The percentage peak areas of individual peaks were calculated by dividing their areas by the total spectral peak area of the sample. Except HI/HB, the unit is %. bHI/HB = ((113−44) + (200−161))/ ((44−0) + (161−113)).
aromaticity degree of SHHA was higher than SAHA (50.08% against 35.78%). Effects of HA on SCFA Production. The effects of two HAs on total SCFA production at different fermentation time are shown in Figure 4A1 and A2. The data in Figure 4A1 indicated that in the blank test the SCFA concentration was increased with fermentation time from 1 to 5 days, but further increasing time to 7 days caused the decline of SCFA, which was mainly attributed to the activity of methanogens.15 At fermentation time of 5 day the maximal SCFA was 790 mg COD/L in the blank test. When SAHA was added to sludge fermentation system, almost the same observation was made (Figure 4A1). The maximal SCFA was 760, 816, 837, and 840 mg COD/L at SAHA dosage of 0.3, 0.5, 0.8, and 1.0 g/g-TCOD, respectively. Apparently, the presence of SAHA did not significantly affect the total SCFA production at any dosage investigated (p > 0.05). From Figure 4A2 it can be seen that as SHHA was added to sludge fermentation system, the maximal SCFA appeared at time of 7 days no matter what the dosage was. The concentration of SCFA was increased from 1379 to 2741 mg COD/L with the increase of SHHA from 0.3 to 0.8 g/g-TCOD, which was 1.7−3.5 folds of the blank. Apparently, the presence of SHHA greatly enhanced the production of SCFA from sludge. It is known that landfill refuse, digested sludge, peat soil, brown coal, weathered coal or compost are abundant in HA. These materials are available at low cost and might be used to accelerate the accumulation of SCFA from WAS, by which not only more SCFA will be obtained, but also the wastes can be reused. As shown in Figure 4B1, the content of individual SCFA in the presence of SAHA was similar to that in the blank. However, in
Figure 3. 13C NMR spectra of SAHA and SHHA.
According to Figure 3, the bands of alkyl carbon (44−0 ppm) and carbohydrate carbon (93−44 ppm) regions with SHHA were broader and their intensities were less than with SAHA. On the contrary, the intensity of aromatic carbon region (161−113 ppm) in the spectrum of SHHA was apparently higher than that in SAHA, and this region was reported to be attributed to the unsubstituted, carbon-substituted, oxygen or nitrogen substituted aromatic carbons, and olefinic carbons.19,27 The data in Table 1 revealed that the HI/HB ratio (an empirical index of hydrophilic) of SHHA was lower than that of SAHA, and the D
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Figure 4. Comparison of two HAs affecting the total SCFA production at different time (A1: SAHA; A2: SHHA) and the composition of SCFA at fermentation time of 7 days (B1: SAHA; B2: SHHA). 0.3, 0.5, 0.8, and 1.0 in the graph represent the dosages of 0.3 g/g-TCOD, 0.5 g/g-TCOD, 0.8 g/gTCOD, and 1.0 g/g-TCOD, respectively. A, P, iso-B, n-B, iso-V, and n-V were respectively acetic, propionic, iso-butyric, n-butyric, iso-valeric, and nvaleric acids. Error bars represent standard deviations of triplicate tests.
Figure 5. Effects of SAHA and SHHA on the solubilization of protein and carbohydrate (A) and the activity of protease and α-glucosidase (B). Error bars represent standard deviations of triplicate tests. The control group was set as 100%.
expressed by the changes of soluble protein and carbohydrate concentrations in fermentation liquor. The data in Figure 5A showed that both soluble protein and carbohydrate were more than 150% of the control in the presence of SHHA, while they were almost same with the control in the presence of SAHA. Thus, the solubilization of sludge protein and carbohydrate were remarkably enhanced by SHHA. Some HAs have been reported to have the features of surfactant and show the ability of decreasing surface tension of water, which is mainly attributed to their amphiphilic character and water−air interface accumulation,29 and the appearance of such HA can increase the solubility of organic compounds.30 As seen from Table 1, the HI/HB ratio of SHHA was lower than that of SAHA (0.34 against 0.42), which suggested that the hydrophobicity of SHHA was stronger and the hydrophilicity of SAHA was higher. The hydrodynamic size of dissolved HA is related to the distribution of hydrophobic and hydrophilic components, which is small when the HA rich in hydrophilic
the SHHA added test the fraction of individual SCFA was different with that in the control (Figure 4B2). The presence of SHHA increased the percentages of acetic, n-butyric, and nvaleric acids, but decreased propionic, iso-butyric, and iso-valeric acids. Clearly, the influences of SHHA on total SCFA production and individual SCFA fraction were different with those of SAHA. The mechanisms for two HAs showing different influences on SCFA production were discussed in the following text. Influence of HA on Sludge Solubilization and Hydrolysis. There are four steps (solubilization, hydrolysis, acidification, and methanogenesis) involved in anaerobic fermentation of WAS, and the solubilization of sludge particulate organic matters is the prerequisite step for SCFA production. As protein and carbohydrate are the two important components of sludge, which account for respectively 54% and 8% of sludge TCOD, their degradation has been correlated with the formation of SCFA during sludge anaerobic fermentation.28 Therefore, in this study the solubilization of sludge organic matters was E
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Figure 6. Metabolic pathways of methane production from acetate and H2/CO2. AK, acetate kinase; ACS, acetyl-coenzyme A synthase; PTA, phosphotransacetylase; ACDS, acetyl-CoA decarbonylase; THMPT, tetrahydromethanopterin; mtrA, THMPT methyltransferase; mcrA, methyl conenzyme reductase; hdrA, heterodisulfide reductase; fmdA, formyl-MFR dehydrogenase; ftr, formyl-MFR-THMPT formyltransferase; mch, methenyl- THMPT cyclohydrolase; mtd, 5,10-methylene-THMPT dehydrogenase; mer, methylene-THMPT reductase; frhA, coenzyme F420 hydrogenase.
Table 2. Comparison between Two HAs Affecting the Activities of Acetotrophic and Hydrogenotrophic Methanogensa acetotrophic methanogensb hydrogenotrophic methanogensc
acetic acid consumption (mg/L) methane generation (mL) H2 consumption (mL) methane generation (mL)
control
SHHA
SAHA
2918 ± 43 198 ± 6 218 ± 4 42 ± 2
1754 ± 25 101 ± 4 220 ± 4 42 ± 1
2889 ± 38 195 ± 7 215 ± 3 41 ± 2
a
The data reported are the averages and their standard deviations. bThe results were obtained by analyzing the residual acetate and accumulated methane from synthetic acetate wastewater at fermentation time of 7 days. cThe gas ingredient was analyzed at fermentation time of 48 h.
Influence of HA on Acidification and Acidogens. It is well-known that the dissolved protein and carbohydrate are first hydrolyzed respectively to amino acids and monosaccharide, and then bioconverted to pyruvate and SCFA in acidification step.34,35 Thus, the amino acids and monosaccharide synthetic wastewaters were used to investigate the influences of two HAs on acidification step. The data in Figure S2 (Supporting Information) showed that in the presence of SHHA the SCFA produced from either amino acids or monosaccharide synthetic wastewater was much greater than the blank. Nevertheless, when SAHA was added to the synthetic wastewaters, the produced SCFA was almost the same as the blank. These results indicated that the acidification step was enhanced by SHHA. Acidogens play a vital role in acidification. As seen in Figure 4, acetic acid was a very important SCFA fraction after SHHA was added to sludge fermentation system. The impact of SAHA and SHHA on the bacteria relevant to acetic acid production was explored by using S. acetigenes as a model acidogens, a phyletic of Firmicutes, and Firmicutes is reported to be one of the abundant phyla relevant to sludge fermentation.36 The results showed that the generation of acetic acid from glucose in the presence of SHHA was 123% of the blank and 121% of the SAHA. It is wellknown that in the pathway of acetic acid production from glucose the electrons are generated in the steps of glyceradehyde-3P → DGlycerate 1,3-diphosphate, and pyruvate → acetyl-CoA. The production of acetic acid would be enhanced if other electron acceptors were presented. HA can act as electron acceptor, and the quinone groups in HA are responsible for this property.37−39 From the above chemical characteristics of two HAs it can be
components is dissolved in aqueous solution. Also, it has been reported that materials with greater hydrophobic property are easily self-assembled in larger hydrodynamic dimensions, which shows greater decrease of total free energy of dissolved system.27 In this study the relative hydrophobicity of sludge in the test of blank, SHHA, and SAHA was 66.9%, 50.55%, and 61.8%, respectively. It seems that the presence of SHHA, compared with SAHA, made it easier for sludge contacting with water, and a greater solubilization of sludge particulate organic matters was therefore observed. Two common hydrolysis enzymes, that is, protease and αglucosidase, were analyzed to study the influence of HA on the hydrolysis of solubilized protein and carbohydrates, respectively. As seen in Figure 5B, the activity of both protease and αglucosidase in the test of SHHA was much greater than that of SAHA. It is well-known that the hydrolysis enzymes in activated sludge are associated with sludge floc matrix. These enzymes, however, are easy to leave sludge and dispersed into liquid phase during anaerobic fermentation, and their activities are easy to get lost due to dissolution in water solution or the formation of complex ingredients between enzymes and metal ions released from sludge.31 In the literature some specific surfactants (such as Tween-80) were used to maintain the activity of enzyme in aqueous bioreaction because they could form the protective micelles or facilitate the enzyme−substrate interaction.32,33 It might be that these properties with SHHA were greater than with SAHA, which caused greater activities of two sludge hydrolysis enzymes being observed in the presence of SHHA. F
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efficiency of acetyl-CoA in the presence of SHHA was only 16.1% of the control. Obviously, the step IV was remarkably inhibited by SHHA. It can be concluded therefore that the reason for SHHA decreasing methane generation was attributed to its inhibitory effect on the biotransformation of acetyl-CoA to 5methyl-THMPT. In the metabolic step IV, the C−C and C−S bonds of acetylCoA are split by the CO dehydrogenase and acetyl-CoA synthase complex, and the carbonyl group is oxidized with contributing two electrons to the small protein electron carrier ferredoxin.9,45 The suppression effect of SHHA on step IV was most likely induced by the electron accepting capacity of SHHA, which enabled it to compete for the electrons and block the way of ferredoxin regeneration. This observation is consistent with the literatures that some specific HA can be utilized as electron acceptor and low CH4 generation.40,41
found that the aromaticity of SHHA is higher than that of SAHA, indicating that SHHA has more abundance of quinone groups. Thus, SHHA showed a stronger electron accepting capacity, and more acetic acid was produced. Influence of HA on Methane Generation and Acetotrophic and Hydrogenotrophic Methanogens. It was reported that the methane production could be inhibited by HA.40−42 In the current study, however, the results in Figure S3 (Supporting Information) showed that only SHHA inhibited the methane generation when sludge was anaerobically fermented. With the increase of SHHA the amount of methane was decreased significantly (p < 0.05), and its inhibitory effect on methane was 14%, 42%, 85%, and 97% at SHHA dosage of 0.3, 0.5, 0.8, and 1.0 g/g-sludge, respectively. Usually, acetotrophic and hydrogenotrophic methanogens are the two main types of methane producers,43,44 and they can respectively bioconvert acidification products acetate and H2/ CO2 to methane (Figure 6). In this study the effects of SHHA and SAHA on acetoclastic methanogenesis were conducted in acetic acid synthetic wastewater. As seen in Table 2 the presence of SAHA did not significantly affect the consumption of acetic acid and the generation of methane in the acetoclastic methanogenesis test (p > 0.05). However, in the presence of SHHA the consumption of acetate and the generation of methane were respectively decreased to 60% and 51% of the control, which suggested that both acetic acid uptake and methane production were remarkably decreased by SHHA. Hence, SHHA rather than SAHA showed the inhibitory effect on acetoclastic methanogenesis. The results of hydrogenotrophic methanogenesis test in Table 2 showed that the presence of either SHHA or SAHA did not affect the uptake of hydrogen and the generation of methane. It can be concluded therefore that the reason for the inhibitory effect of SHHA on methane generation was due to the metabolism of acetoclastic methanogens instead of hydrogenotrophic methanogens being suppressed. As SHHA decreased the consumption of acidified products, the increased production of SCFA was observed. Several steps are involved in the generation of methane from acetate by acetoclastic methanogens (Figure 6). In order to dig out the detailed reason for SHHA inhibiting the metabolism of acetoclastic methanogens, it is necessary to investigate its influence on each step. As seen in Figure 6, both the generation of methane from acetate and H2/CO2 undergoes the following metabolic steps: 5-methyl-THMPT → methyl-CoM → CH4. The above discussion had indicated that the influence of SHHA on methane production from H2/CO2 was insignificant, which suggested that the effects of SHHA on the bioconversions of 5methyl-THMPT → methyl-CoM → CH4 might not be taken into consideration. Thus, in the aceticlastic pathway only steps I, II, III, and IV could be influenced by SHHA. According to the experiment of SHHA affecting the bioconversion of acetate to acetyl phosphate, it was found that the presence of SHHA did not significantly influence the step I (p > 0.05). The experiment of the influence of SHHA on the bioconversion of acetate to acetyl-CoA showed that the generated acetyl-CoA in the presence of SHHA was 97.2− 105.5% of the control, which indicated that the step II was not significantly affected by SHHA. Also, the produced acetyl-CoA in the test of SHHA affecting the bioconversion of acetyl phosphate to acetyl-CoA was 98.3−103.8% of the control, suggesting that the influence of SHHA on the step III was marginal. However, the experiment of the influence of HA on the conversion of acetyl-CoA to 5-methyl-THMPT showed that the utilization
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ASSOCIATED CONTENT
S Supporting Information *
Additional analytical methods, Tables S1−S3, and Figures S1− S3. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*(Y.C.) Phone: 86-21-65981263; fax: 86-21-65986313; e-mail: [email protected]. *(X.Z.) E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (51278354, 51178324 and 51425802) and the Program of Shanghai Subject Chief Scientist (15XD1503400).
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REFERENCES
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