Polycyclic Aromatic Hydrocarbon Affects Acetic Acid Production during

Jun 6, 2016 - In fact, due to the wide application of combined wastewater collection systems in the developing countries, large proportions of industr...
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Polycyclic Aromatic Hydrocarbon Affects Acetic Acid Production during Anaerobic Fermentation of Waste Activated Sludge by Altering Activity and Viability of Acetogen Jingyang Luo, Yinguang Chen,* and Leiyu Feng* State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China S Supporting Information *

ABSTRACT: Till now, almost all the studies on anaerobic fermentation of waste activated sludge (WAS) for bioproducts generation focused on the influences of operating conditions, pretreatment methods and sludge characteristics, and few considered those of widespread persistent organic pollutants (POPs) in sludge, for example, polycyclic aromatic hydrocarbons (PAHs). Herein, phenanthrene, which was a typical PAH and widespread in WAS, was selected as a model compound to investigate its effect on WAS anaerobic fermentation for short-chain fatty acids (SCFAs) accumulation. Experimental results showed that the concentration of SCFAs derived from WAS was increased in the presence of phenanthrene during anaerobic fermentation. The yield of acetic acid which was the predominant SCFA in the fermentation reactor with the concentration of 100 mg/kg dry sludge was 1.8 fold of that in the control. Mechanism exploration revealed that the present phenanthrene mainly affected the acidification process of anaerobic fermentation and caused the shift of the microbial community to benefit the accumulation of acetic acid. Further investigation showed that both the activities of key enzymes (phosphotransacetylase and acetate kinase) involved in acetic acid production and the quantities of their corresponding encoding genes were enhanced in the presence of phenanthrene. Viability tests by determining the adenosine 5′-triphosphate content and membrane potential confirmed that the acetogens were more viable in anaerobic fermentation systems with phenanthrene, which resulted in the increased production of acetic acid.

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INTRODUCTION Waste activated sludge (WAS) is the main byproduct of biological wastewater treatment and has been considered as an important source of secondary pollution in aquatic environments. In 2015, the amount of WAS in China is expected to rise to 34 million metric tonnes (at a moisture content of 80%), and approximately 80% of them were disposed unsafely which put great threats to the environment.1 The effective and environment-friendly treatment and disposal of WAS are of vital importance and emergency. In recent years, anaerobic fermentation has been widely recommended as a preferred method for the treatment of sludge, considering its cost-effectiveness and energy recovery. By anaerobic fermentation large proportions of organic matters in WAS could be bioconverted into valuable bioproducts, such as short-chain fatty acids (SCFAs), hydrogen, methane, etc.2,3 © XXXX American Chemical Society

To date, most of the reported studies on WAS anaerobic fermentation focused on the influences of operating conditions, such as, pH, temperature, etc.,4,5 and pretreatment methods, such as thermal, ultrasonic and microwave pretreatments.6−9 For example, both laboratory and pilot scale tests have reported that the alkaline fermentation condition, especially pH 10, was effective in improving the accumulation of SCFAs, especially acetic acid, by showing simultaneous impacts on the solubilization, hydrolysis and methanogenesis of particulate organics in WAS.5,10 Besides, the characteristics of WAS, such as the contents of protein, carbohydrate and intracellular Received: January 1, 2016 Revised: June 2, 2016 Accepted: June 6, 2016

A

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phenanthrene, anthracene, fluorene, and chrysene, with the concentration of 22.4, 17.6, 95.3, 6.1, 3.3, and 12.5 mg/kg dry sludge, respectively. Obviously, phenanthrene was the dominant PAH in the WAS. Hence, it was selected as the model PAH to investigate the influences on WAS anaerobic fermentation. Phenanthrene (purity 98%, Sigma-Aldrich) was dissolved in methanol (HPLC grade, Sigma-Aldrich) with a concentration of 1.0 g/L as the stock solution. Impact of Phenanthrene on SCFAs Accumulation During WAS Anaerobic Fermentation. The fermentation experiments were conducted in a series of semicontinuous-flow reactors, which were made of plexiglass with 5.0 L volume. Original WAS (4.0 L) and external phenanthrene were mixed and fermented at 20 ± 1 °C with a stirring speed of 100 rpm (rpm). External phenanthrene was added with the dosage of 0, 50, 100, 200, and 500 mg/kg dry sludge in each reactor to investigate its effects on SCFAs production under different concentrations. The fermentation pH was controlled at 10 by adding 4 M sodium hydroxide (NaOH) or 4 M hydrochloric acid (HCl). The sludge retention time (SRT) in all fermentation reactors was controlled at 8 days by withdrawing 500 mL fermentation mixture from the reactors each day and then replenishing the same volume of fresh WAS and corresponding concentration of phenanthrene. The procedures for the replenishing of fresh WAS and phenanthrene were as follows: phenanthrene stock solution was first spiked into the container and placed in a fume hood for methanol vaporization. After the vaporization of methanol, the fresh WAS was added to mix with the residual phenanthrene to obtain PAH contaminated WAS and then supplemented to the semicontinuous-flow reactors.18 The data were collected and analyzed when all the fermentation reactors ran for more than 80 days and the concentration and composition of SCFAs reached stable. After operation for 5 months, the analysis of the microbial community in the control and phenanthrene reactors (100 mg/kg) was conducted. In order to monitor the conversion of main organic substances and illustrate their fates during WAS fermentation, carbon balance study was conducted in the current fermentation system by observing the carbon contents of proteins, carbohydrates, SCFAs, lactic acid, ethanol, carbon dioxide, methane, et al. Mechanism exploration was focused on the processes during anaerobic fermentation and the activity and viability of anaerobic microorganism responsible for SCFAs production. Four steps including solubilization, hydrolysis, acidification and methanogenesis were involved in WAS anaerobic fermentation and the investigation of phenanthrene effects on these processes were described in the Supporting Information (SI). Influence of Phenanthrene on the Pure Acetogen during Anaerobic Fermentation. Proteiniphilum acetatigenes, which is isolated from anaerobic sludge and is known to produce acetate from the proteinaceous materials,19 was purchased from China General Microbiological Culture Collection Center and used as a model acetogen to further explore the phenanthrene impact on anaerobic fermentation for acetate production. It was cultured in tryptone-yeast extractglucose medium at 35 ± 1 °C for 24 h, and then inoculated to the substrates for acetic acid production, which were composed of (mg/L of tap water): tryptone 20 000, glucose 5000, NaHCO3 10 000, yeast extract 10 000, L-cysterine-HCl-H2O 500, NaCl 80, NaHCO3 400, CaCl2 8, MgSO4 8, K2HPO4 80, vitamin K1 5 and chlorohemin 12.5. The volume of substrates and inoculum was respectively 100 and 2 mL. The reactors with

polyhydroxyalkanoates (PHA), have also been documented to affect the generation of SCFAs, hydrogen and methane from WAS during anaerobic fermentation.3,11,12 Normally, WAS is a collection of various compounds, including not only the biodegradable proteins, carbohydrates and PHA but also lots of pollutants, such as heavy metals and persistent organic pollutants (POPs). In fact, due to the wide application of combined wastewater collection systems in the developing countries, large proportions of industrial wastewater enters wastewater treatment plants (WWTPs) (in China, it is approximately 35.0%), bringing in huge amounts of POPs, such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), etc.13 Commonly, the pollutants mentioned above were absorbed and concentrated in WAS after wastewater treatment.13,14 Anaerobic fermentation of sludge is a well-known biological process, which is controlled by numerous microorganisms, such as acetogens, hydrogenogens and methanogens,12,15 and is likely to be affected by these pollutants, especially in the continuous-flow mode as the WWTPs are commonly operated continuously. To our best knowledge, however, no information is currently available on how the POPs present in WAS influence anaerobic fermentation for the accumulation of fermentative products, especially SCFAs, which are the key intermediate products during anaerobic conversion of sludge organics. Among the POPs in sludge, PAHs, which are a large class of organic compounds containing two or more aromatic fused rings and derive from the incomplete combustion or pyrolysis of organic matters, have drawn much attention due to their global environmental occurrence.16,17 Therefore, the main aim of this study was to reveal the impact of absorbed PAH on anaerobic production of SCFAs from WAS at pH 10, which has been proven to be an efficient strategy for SCFAs accumulation during sludge fermentation.5,10 Phenanthrene was selected as a typical PAH considering its existence in WAS with high concentration, and the facts of what happened in each step involved in WAS anaerobic fermentation with phenanthrene were first explored. Then, the diversities of anaerobic microorganisms responsible for SCFAs production in the presence of phenanthrene were examined. Finally, the activity and viability of pure acetogens with phenanthrene during anaerobic fermentation were studied to deeply disclose the mechanism for the promotion of acetic acid in the presence of phenanthrene.

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MATERIALS AND METHODS WAS and Phenanthrene. WAS was collected from a secondary sedimentation tank of a municipal WWTP in Shanghai, China, in which the proportion of industrial wastewater was approximate 30%. The withdrawn WAS was first concentrated by settling at 4 °C for 24 h. The supernatant was discarded and then the concentrated WAS was filtrated by the stainless steel mesh (2.0 mm) to remove large particulates for later use. Its main characteristics (average data plus standard deviation of three tests) were as follows: pH 6.8 ± 0.1, total suspended solids (TSS) 13285 ± 255 mg/L, volatile suspended solids (VSS) 9055 ± 165 mg/L, soluble chemical oxygen demand (SCOD) 275 ± 30 mg/L, total chemical oxygen demand (TCOD) 12865 ± 245 mg/L, total carbohydrate 845 ± 125 mg COD/L, total protein 7305 ± 185 mg COD/L and lipid and oil 145 ± 15 mg COD/L. In the withdrawn WAS, several typical U.S.-EPA priority PAHs were detected, including naphthalene, acenaphthene, B

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Figure 1. Effects of phenanthrene on the concentration of total SCFAs (A) and individual SCFA (B). Error bars represent standard deviations of triplicate tests.

microbial analysis (SI). The membrane potential of pure acetogen cells was determined by flow cytometry using the anionic lipophilic potential-sensitive fluorescence dye, that is, bis(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (Dojindo, China) (SI).25 Statistical Analysis. All tests in this study were carried out in triplicate, and the results were expressed as mean ± standard deviation. An analysis of variance (ANOVA, p < 0.05) was used to test the significance of results.

the pure strain were operated in the same way with those WAS semicontinuous flow reactors, and part of the mixed fermentation substrates was discarded and replenished each day to control the retention time of fermentation substrates at 8 days. The dosage of external phenanthrene was 1.3 mg/L which was equal to the concentration of 100 mg/kg in WAS fermentation system, and the fermentation pH was controlled at 10. All the operation and inoculation were conducted under the anaerobic condition with nitrogen as protecting gas. After being operated for more than 3 months, the production of acetic acid varied little indicating that the operation of fermentation reactors went stable. The analysis of activities of key enzymes and their encoding genes closely related with acetic acid production were carried out. The adenosine 5′triphosphate (ATP) content and membrane potential of Proteiniphilum acetatigenes were also detected to reflect the effect of phenanthrene on the viability of acetogens. Analytical Methods. The determinations of SCFAs, protein, carbohydrate, lipid, COD, ammonia nitrogen (NH4+N), TSS, VSS, lactic acid, ethanol, gas component (CO2, CH4 and H2), ATP and activities of key enzymes related with acetic acid production (phosphotransacetylase (PTA) and acetate kinase (AK)) were the same as described in the previous publications.12,20,21 In this study, the result of enzyme activity was expressed in the way of relative one, that is, the enzyme activity in the control was set as 100%, while in other reactors, it was the result of comparison with that in the control. Total organic carbon (TOC) content of sludge was measured by a dry combustion method at 540 °C in an electrical furnace lasting 24 h according to Nelson,et al.22 The EPS of pure culture were first extracted by using oscillation-ultrasound method for loose-EPS and then a harsh method of cation exchange resin technique for tight-EPS.23 The PAH in WAS was extracted according to the method of Cai et al. (2007) with minor modification by sonicating for 1 h instead of Soxhletextraction,24 and analyzed via high performance liquid chromatography (Agilent 1260). The quantification of acetate-related genes, which encode PTA and AK, was conducted via real-time quantification PCR (SI). The analysis of the anaerobic microbial community responsible for SCFAs production in the presence of phenanthrene in WAS fermentation systems was conducted by Illumina MiSeq sequencing technique. The samples for sequencing were withdrawn from triplicate fermentation reactors and mixed completely to homogenize the samples for DNA extraction and

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RESULTS AND DISCUSSION Production of Acetic Acid-Enriched SCFAs from WAS in the Presence of Phenanthrene. In the current study, a constant accumulation of total SCFAs from WAS was obtained in each fermentation reactor during the whole operation time (shown in Figure 1(A)). The production of SCFAs was observed to be influenced obviously by the different concentration of phenanthrene. The average concentration of SCFAs in the reactors with phenanthrene addition of 0, 50, 100, 200, and 500 mg/kg dry sludge was 2226, 2635, 3103, 2842, and 2684 mg COD/L, respectively. The accumulation of SCFAs in phenanthrene-added reactors was enhanced compared with that in the control. When the dosage of phenanthrene was 100 mg/kg dry sludge, the concentration of SCFAs from WAS was 1.4-fold of that in the control. However, further increase of the phenanthrene dosage caused the decrease of SCFAs concentration. The reason could be attributed to the toxicity intolerance of acidogenic bacteria. Phenanthrene is a well-known organic pollutant with high toxicity to living creatures. Although the acidogenic bacteria could excrete extra EPS to weaken the toxic effect and protect themselves,26 their activities and functions would be negatively affected when the concentration of pollutants exceeded their tolerance limit. Further exploration found that among those detected SCFA in WAS fermentation reactors, the average concentrations of propionic, iso-butyric, n-butyric, iso-valeric, and n-valeric acids from WAS were almost the same in the presence or absence of phenanthrene (Figure 1(B)). However, the average concentration of acetic acid was significantly changed in the presence of phenanthrene, and was promoted respectively by 1.3-, 1.8-, 1.5-, and 1.3-fold with 50, 100, 200, and 500 mg/kg phenanthrene addition compared with that in the control. By calculating the concentration of total SCFAs, it was observed C

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solubilization, hydrolysis and acidification of WAS plus the homoacetogenesis of carbon dioxide and hydrogen are involved. In present study, the methanogenesis of acidification products was inhibited at pH 10 and thus was ignored (SI Figure S2). Therefore, it was necessary to investigate the effect of phenanthrene on each step involved in the accumulation of acetic acid during sludge fermentation. WAS is cemented and flocculated together by EPS which are mainly composed of microbiologically produced solid-state proteins and carbohydrates.29 During WAS anaerobic fermentation, the solid-state substrates are first transformed into the soluble and bioavailable small-molecule matters via solubilization and hydrolysis, which are usually considered as the ratelimiting steps and directly connected with the final concentration of hydrolyzed substrates for acetic acid formation.30 In this study, the soluble proteins and carbohydrates in the fermentation reactors, which were the reasonable indicators to reflect the efficiency of WAS solubilization,5,31 showed no significant variances in the control and phenanthrene-added reactors. The average concentration of soluble proteins was respectively 1390, 1362, 1397, 1344, and 1347 mg/L in reactors with 0, 50, 100, 200, and 500 mg/kg phenanthrene addition and the corresponding concentration of soluble carbohydrates was 268, 274, 270, 266, and 265 mg/L (SI Figure S3 (A)). Also, the hydrolysis step of WAS was not obviously affected by phenanthrene as the hydrolysis efficiencies of BSA and dextran (as model soluble protein and carbohydrate used in this study) were respectively 76.3% and 92.8%, 75.8% and 93.4% in the control and phenanthrene-added reactors (100 mg/kg) (SI Figure S3 (B)). The solubilization and hydrolysis of fermentative substrates in the presence of phenanthrene gave no contribution to the enhancement of acetic acid production during WAS fermentation. It is well-known that acidification is the main pathway for the formation of acetic acid via utilizing the hydrolyzed substrates by acidogenic bacteria during anaerobic fermentation. In this study, the effect of phenanthrene on the acidification involved in anaerobic fermentation was explored by fermenting the synthetic wastewater with L-glutamate and glucose (as model amino acid and monosaccharide compounds), and determined by the yield of acetic acid. It has found that the generation of acetic acid was enhanced remarkably due to the presence of phenanthrene (shown in SI Figure S3 (C)). With operation time of 8 days, the concentration of acetic acid was respectively 395 and 585 mg COD/L in the control and phenanthreneadded reactors, demonstrating that the presence of phenanthrene benefitted the accumulation of acetic acid during the acidification of hydrolyzed products. Moreover, during sludge anaerobic fermentation acetic acid can also be synthesized by homoacetogenic bacteria with H2 and CO2 as substrates.20 Thus, the investigation on the contribution of homoacetogenic bacteria to the enhancement of acetic acid production in the presence of phenanthrene is also necessary. It was explored by examining the H2 consumption and acetic acid production via providing sufficient H2 and CO2 for anaerobic microorganisms. However, little differences of H2 consumption and acetic acid production were observed in the control and phenanthrene-added reactors (shown in Table 1), revealing that the contribution of homoacetogenic bacteria to acetic acid promotion was negligible when the phenanthrene presented in the fermentation systems. The improved production of acetic acid from WAS with phenanthrene was

that the promotion of acetic acid and total SCFAs was respectively 292 and 409, 725 and 877, 469 and 616, 300, and 458 mg COD/L in the corresponding phenanthrene-added reactor. The effect of phenanthrene on SCFAs production from WAS during anaerobic fermentation mainly focused on the increased accumulation of acetic acid. Contribution of Phenanthrene Degradation to Acetic Acid Production. The phenanthrene has been demonstrated to be biodegraded under anaerobic condition.27,28 Indeed, in the current fermentation systems, phenanthrene was partly degraded and its degradation efficiency was 47.5% at the dosage of 100 mg/kg dry sludge. Meanwhile, the abiotic loss of phenanthrene was measured to be below 5% by running the same reactor under the sterile conditions, and thus it could be neglected. The transformation of phenanthrene during WAS anaerobic fermentation was completed via biodegradation. Theoretic calculation indicated that even if all the degraded phenanthrene was converted into acetic acid, the maximum yield of acetic acid was 1.6 mg COD/L (SI Table S4), which accounted for only 0.2% of the promotion of acetic acid production (725 mg COD/L). Thus, the contribution of phenanthrene degradation to acetic acid accumulation could be ignored. Carbon Mass Balance of the WAS Fermentation Systems. The study of carbon mass balance enables to monitor the conversion of main organic substances and illustrate their fates during WAS fermentation. Generally, various matters are involved in sludge anaerobic fermentation, such as proteins, carbohydrates, SCFAs, CO2 and other organic carbons (such as lactic acid, ethanol, et al). It can be seen from SI Figure S1 that the main carbon substrates in the initial sludge were proteins and carbohydrates, which were respectively 10.34 and 1.71 g (accounting for about 64% of the total carbon in WAS). During WAS anaerobic fermentation the contents of proteins and carbohydrates were remarkably decreased. The carbon contents of residual proteins and carbohydrates were respectively 8.03 and 0.93 g in the control reactor. The corresponding contents were further decreased in the phenanthrene-added reactors. For example, the carbon contents of residual proteins and carbohydrates were 6.88 and 0.74 g in the phenanthrene-added reactor (100 mg/kg), much lower than those in the control. Meanwhile, the carbon contents of SCFAs, especially acetic acid, were found to be greatly increased. The carbon of acetic acid in the initial sludge was merely 0.03 g whereas it was respectively 1.44 and 2.53 g in the control and phenanthrene-added fermentation reactors. Obviously, huge amounts of acetic acid were produced in the WAS anaerobic fermentation reactors, especially in the reactors with phenanthrene addition. Based on the results of organic carbon variations in the WAS fermentation reactors, it can be concluded that during WAS fermentation, SCFAs, especially acetic acid, were generated from proteins and carbohydrates, and more importantly the enhanced production of acetic acid in the presence of phenanthrene compared with that in the control was mainly attributed to more bioconversion of proteins and carbohydrates. Effect of Phenanthrene on Each Step Involved in Acetic Acid Accumulation during Sludge Anaerobic Fermentation. It has been found above that the effect of phenanthrene on SCFAs accumulation from WAS focused on the variation of acetic acid, which is one of the most important intermediates during anaerobic fermentation of organic matters. Normally, during the accumulation of acetic acid, the D

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enzymes and acetic acid during anaerobic degradation of organic matters,38,39 was also increased in the phenanthreneadded fermentation systems. Further exploration at genus level found that the abundances of microbes related with acetic acid production, that is, Lactobacillus, Proteiniclasticum, Ruminococcus, Anaerovorax, Bacteroides, Paludibacte, Parabacteroides, Caldilinea, and Collinsella, were all increased remarkably in the phenanthrene reactors (Seen in SI Table S5). For example, the Proteiniclasticum, which is a kind of anaerobic proteinutilizing bacteria with the acetic acid as the major fermentation products,40 was detected and occupied respectively 2.59% and 3.07% in the control and phenanthrene-added reactors. Obviously, the microbial community in the phenanthrene reactors was shifted to the direction that was advantageous to the accumulation of acetic acid. The change of microbial community was consistent with the higher concentration of acetic acid observed in WAS fermentation reactors with phenanthrene. Effect of Phenanthrene on the Activity of Proteiniphilum acetatigenes during Anaerobic Fermentation. Based on the results mentioned above, it can be found that the due to the presence of phenanthrene in WAS, the microbial community was changed in the semicontinuous-flow fermentation reactors. The phenanthrene mainly influenced the aceticforming bacteria which resulted in the enhancement of acetic acid accumulation. In order to disclose the interaction between phenanthrene and acetogens more deeply, the effect of phenanthrene on the acetogens was further explored via pure Proteiniphilum acetatigenes, which has the ability of converting protein-containing organic matter (the predominant organics in WAS) into acetic acid in the pH range of 6−10.19 Acetic acid is the metabolite of anaerobic acetogens, and its production is directly related with bacterial activity which is commonly reflected and determined by the corresponding enzymes. Acetic acid is mainly generated via acetyl-CoA (derived from hydrolyzed substrates) to acetyl phosphate and then to acetic acid during WAS fermentation (Figure 3(A)). Conversion of acetyl-CoA to acetyl phosphate requires PTA, and breakdown of acetyl phosphate to acetate utilizes AK.41 In this study, the activities of PTA and AK were investigated to demonstrate the activity of acetogens in the anaerobic fermentation reactors for acetic acid production. As seen from Figure 3(B), in the fermentation reactors with phenanthrene the activity of PTA was increased by 80% compared with that in the control, indicating that more acetylCoA could be transformed into acetyl phosphate. Meanwhile, the activity of AK was found to be increased by 47.1%,

Table 1. Comparison of Acetic Acid Production and H2 Consumption with H2/CO2 Synthetic Gas Fillinga acetic acid (mg COD/L)b

H2 consumption (%)

control reactors

H2/CO2 filling No H2/CO2 filling

1163 ± 36 1105 ± 40

1.6 ± 0.2

phenanthrene reactorsc

H2/CO2 filling No H2/CO2 filling

1851 ± 38 1804 ± 30

1.4 ± 0.3

a The operation time was 2 days. bBefore synthetic gas filling, the initial concentration of acetic acid was 981 mg COD/L and 1595 mg COD/ L in the control and phenanthrene reactors, respectively. cThe dosage of phenanthrene in the fermentation reactor is 100 mg/kg dry sludge.

derived from the enhancement of bioconversion pathway of hydrolyzed substrates to acetic acid. Shift in Microbial Community in the Presence of Phenanthrene. In order to further explore the mechanism of phenanthrene effect on acetic acid production in WAS fermentation systems, the communities of anaerobic bacteria related with acetic acid accumulation were analyzed by Illumina Miseq 16S rRNA genes technique. Experimental data showed that anaerobic microorganisms in both fermentation reactors were mainly grouped into five phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Chlorof lexi (shown in Figure 2), which have all been reported to have the ability of degrading a wide range of complex organic matters, including proteins and carbohydrates, for the production of SCFAs and were widely distributed throughout various anaerobic habitats.32−36 However, the structure and composition of microbial species were changed significantly between the control and phenanthrene-added reactors. Venn analysis showed that only 2819 OUTs were shared by the two reactors and the others (nearly 4000 OTUs) were unique to the individual reactor (SI Figure S4). The abundance of Firmicutes, which was the predominant bacteria in the present fermentation systems and part of them have been reported to be acetic acid consumers,34 was reduced in the presence of phenanthrene (74.7% in the control versus 65.5% in the phenanthrene reactors). Proteobacteria, which was the second dominant bacteria and generated acetic acid as major acidification products,32,37 showed a higher abundance in the phenanthrene reactors (13.0% versus 9.3% in the control). Another dominant phylum, Bacteroidetes (mainly distributed in class Bacteroidia), which has been reported to produce lytic

Figure 2. Distribution of bacterial populations at phylum level in the (A) control and (B) phenanthrene reactors (100 mg/kg). E

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Figure 3. (A) Key enzymes and genes involved in acetic acid production; (B) Relative activities of key enzymes related with acetic acid production; (C) Quantities of PTA and AK genes in the control and phenanthrene-added reactors (100 mg/kg). All the values were normalized by the number of acetogens. Error bars represent standard deviations of triplicate tests.

in reactors with phenanthrene revealed that the acetogens had better viability and more bioenergy to produce acetic acid.

demonstrating that more acetyl phosphate was further converted into acetic acid. The promotion of activities of key enzymes directly related with acetic acid production exhibited that the acetogens were much more active, which showed good agreement with the enhancement of acetic acid production in the fermentation reactors with phenanthrene. It is well-known that the enzymes secreted by anaerobes were encoded and controlled by the corresponding functional genes. Whether the PTA and AK operon, which encoded the enzymes of PTA and AK involved in the production of acetic acid,42 were affected and changed due to the presence of phenanthrene was important. Based on the results investigated by real-time qPCR, it was observed that the quantities of PTA and AK genes from Proteiniphilum acetatigenes in the phenanthrene-added reactors were respectively 1.53 × 105 and 1.22 × 107 copies/mL, which was much higher than that in the control (7.36 × 104 and 6.86 × 106 copies/mL) (Figure 3(C)). The results were in accordance with the observation in the mixed WAS microbial communities. In the phenanthreneadded reactors, the quantities of PTA and AK genes were respectively 5.58 × 103 and 4.86 × 105 copies/g TSS, whereas they were 2.71 × 103 and 1.88 × 105 copies/g TSS in the control. However, the quantities of 16S rRNA genes (as an indicator of the total bacteria) were almost the same in the two reactors mentioned above (1.72 × 1010 and 1.68 × 1010 copies/ g TSS). It indicated that more acetic-forming related genes regulated the synthesis and expression of corresponding PTA and AK enzymes in the phenanthrene-added reactors, which led to the enhancement of acetic acid production during WAS anaerobic fermentation. Effect of Phenanthrene on the viability of Proteiniphilum acetatigenes during Anaerobic Fermentation. It is understandable that the activity of an organism is determined by its viability under different conditions. Thus, the viability of Proteiniphilum acetatigenes in the presence of phenanthrene was also examined and determined by measuring the content of adenosine 5′-triphosphate (ATP), which is present in all living organisms and is considered as a practical indicator for evaluating the cell viability.43,44 In the fermentation reactors with phenanthrene addition, the concentration of ATP from Proteiniphilum acetatigenes was 0.67 μg/L, whereas it was only 0.35 μg/L in the control (Figure 4). The higher content of ATP

Figure 4. Concentration of ATP and EPS from Proteiniphilum acetatigenes in the control and phenanthrene reactors (100 mg/kg). All the values were normalized by the number of acetogens. Error bars represent standard deviations of triplicate tests.

Membrane potential is another most definitive proof to determine the viability of cells,45−47 and only live cells are able to maintain membrane potential steadily. Membrane depolarization, i.e. the increase of membrane potential, often indicates the structural damage of cell membrane and thus the decrease in cell viability, resulting in the change of physiological status (cell size and cell density) and loss of cellular function.37,48 Usually, the membrane potential is determined by flow cytometry with proper probes, which enter the damaged cell and emit the fluoresecence while those cells with intact membranes are impermeable to molecular probes. As shown in Figure 5 (A) and (B), the average fluorescence intensity of acetogen cells in the control and phenanthrene reactors at pH 10 was respectively 1194.82 and 530.88, which meant that the cell membranes of numerous acetogens were destructed in both fermentation reactors and large amounts of DiBAC4 (3) probes entered the cells considering that the fluorescence intensity was merely 117.95 at pH 7 (the optimal pH for Proteiniphilum acetatigenes) in Figure 5(C). It has been reported that the F

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Figure 5. Flow cytometric analysis of membrane potential and physiological status of Proteiniphilum acetatigenes with DiBAC4 (3) probe. (A) + (D) Cells without phenanthrene at pH 10; (B) + (E) Cells with phenanthrene at pH 10; (C) + (F) Cells without phenanthrene at pH 7. FSC: forward scattering (proportional to cell size); SSC: side scattering (proportional to cell density).

extreme alkaline condition would give damaging effects on the integrity of cell membranes due to the large amounts of hydroxyl anions.49 Thus, the acetogen cells were in a depolarized state and their viability was negatively influenced at pH 10, which was also illustrated by the deviation of physiological status of cells from the normal ones (Figure 5 (D) and (F)). However, the negative impacts of pH 10 on acetogen cells were observed to be less severe in the phenanthrene reactors. The fluorescence intensity declined remarkably and the physiological status of cells (Figure 5 (B) and (E)) was more close to that at pH 7, meaning that the acetogen cells in the phenanthrene reactors were more inclined to maintain their membrane potential, and thus showed better viability than those in the control at pH 10. Previously, microorganisms have been reported to produce extra EPS to protect themselves when being exposed to the toxic compounds by making the cell wall thicker to reduce the mass transfer of toxicant into the cells.26,50 In this study, due to the presence of phenanthrene, the acetogens were stimulated to synthesize more EPS to resist the negative effect of strong alkaline condition on the viability of acetogen cells. The content of EPS from Proteiniphilum acetatigenes was found to be enhanced by 32% in the phenanthrene reactors compared with that in the control (Figure 4). Thus, the acetogens were more tolerant under the alkaline condition with phenanthrene addition, showing stronger viability with more acetic acid production. Overall Understandings of Enhanced Acetic Acid Accumulation from WAS in the Presence of Phenanthrene. Based on the above discussions, the mechanism for the enhancement of acetic acid accumulation from WAS in the presence of phenanthrene during anaerobic fermentation was concluded. When WAS was fermented at pH 10, the methanogenesis was inhibited and the phenanthrene showed little effects on the solubilization and hydrolysis of sludge organics. However, the acidification process for the formation

of SCFAs, especially acetic acid, was positively influenced by phenanthrene. The phenanthrene changed the microbial community in WAS fermentation reactors shifting to acetic acid-forming species and enhanced the activity and viability of acetogens, resulting in the enhanced accumulation of acetic acid during WAS anaerobic fermentation. Interestingly, a different sludge, which was withdrawn from another WWTP (its main characteristics were shown in SI), was tested to investigate the phenanthrene effect on acetic acid production, and the similar results were obtained. The concentration of acetic acid was respectively 1105 and 1602 mg COD/L in the control and phenanthrene-added reactors while the concentrations of other SCFAs were almost the same. It seemed that the enhanced accumulation of acetic acid in the presence of phenanthrene during sludge anaerobic fermentation was not a specific phenomenon unique to particular sludge.

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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/acs.est.6b00003. Additional material as noted in the text (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(Y.C.) Phone: 86-21-65981263; fax: 86-21-65986313; e-mail: [email protected]. *(L.F.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work is financially supported by the National Natural Science Foundation of China (51425802, 51108332 and 51278354), the Research Fund of Tianjin Key Laboratory of G

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Aquatic Science and Technology (CTJKLAST-ZD-2014-05), Fundamental Research Funds for the Central University, the Program of Shanghai Subject Chief Scientist (15XD1503400), and Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation (STGEF).

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DOI: 10.1021/acs.est.6b00003 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.est.6b00003 Environ. Sci. Technol. XXXX, XXX, XXX−XXX