How Do Biocides Occurred in Waste Activated Sludge Affect the

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How Do Biocides Occurred in Waste Activated Sludge Affect the Resource Recovery for Short-Chain Fatty Acids Production Jingyang Luo, Lijuan Wu, Qin Zhang, Fang Fang, Qian Feng, Zhaoxia Xue, Miao Cao, Zhaoqi Peng, Chao Li, and Jiashun Cao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05420 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 20, 2018

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ACS Sustainable Chemistry & Engineering

How Do Biocides Occurred in Waste Activated Sludge Affect the Resource Recovery for Short-Chain Fatty Acids Production Jingyang Luo†,‡,*, Lijuan Wu§, Qin Zhang†,‡,*, Fang Fang †,‡, Qian Feng †,‡, Zhaoxia Xue †,‡, Miao Cao †,‡, Zhaoqi Peng †,‡, Chao Li †,‡, Jiashun Cao †,‡,*

†

Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry

of Education, Hohai University, 1 Xikang Road, Nanjing210098, China ‡

College of Environment, Hohai University, 1 Xikang Road, Nanjing210098, China

§

Jiangsu Provincial Academy of Environmental Science, 176 Jiangdong North Road, Nanjing

210036, China

*Corresponding author：Jingyang Luo, Jiashun Cao, Qin Zhang E-mail: [email protected], [email protected], [email protected] Tel: 86-25-83786701 Fax: 86-25-83786701

1

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ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Anaerobic technology is an efficient and sustainable approach to dispose waste activated sludge (WAS) but the performance is easily affected by environmental conditions. In this study, the potential impacts of chlorhexidine (CHD) and hexadecyltrimethylammonium bromide (HTAB), two highly detected biocides in WAS, on the short-chain fatty acids (SCFAs) generation during anaerobic fermentation were explored. The experimental results indicated that the biocides promoted SCFAs generation unexpectedly, particularly the acetic acid. The accumulation of SCFAs increased from 567.3 mg COD/L in control to 2144.5 and 3400.0 mg COD/L with low CHD and HTAB exposure (50 mg/L). However, the enhancing effects were weakened in reactors with high loads of biocides or CHD & HTAB coexistence. Mechanisms exploration revealed that the CHD and HTAB firstly enhanced the extracellular polymeric substances disruption, and then simultaneously accelerated both the hydrolysis and acidification processes but inhibited the methanogensis. The relative activities of hydrolases, acidogenic enzymes and bioenergy involved in SCFAs production were significantly enhanced with low biocides dosage. Moreover, the abundances of active microorganisms that participated in the biological hydrolysis and acidification processes were highly enriched in biocides reactors while the acetoclastic methanogens were reduced. This work might be of significance to regulate the WAS fermentation for better resource recovery. KEYWORDS: Chlorhexidine (CHD); Hexadecyltrimethylammonium bromide (HTAB); Waste activated sludge (WAS); Short-chain fatty acids (SCFAs); Hydrolysis; Active microorganisms

INTRODUCTION As is well-known, anaerobic fermentation is currently considered as an effective and 2


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promising strategy to dispose the increasing amounts of generated waste activated sludge (WAS) in wastewater treatment plants (WWTPs)1-3. It can not only achieve WAS stabilization but also recover large amounts of valuable products from organics-enriched WAS, such as short-chain fatty acids (SCFAs), which could serves as the preferred external carbon sources for biological nutrient removal in WWTPs and potential materials for biodegradable plastics synthesis

4-6.

However, the anaerobic performance was easily influenced by different environment conditions, such as the pH and temperature4, 7. The biocides, which is a diverse group of compounds that widely used in both household and industrial sectors as preservatives, disinfectants and etc., have recently raised great concerns due to their immense consumption and the potential risks to environmental organisms with low concentrations 8-10. The significant quantities of these consumed chemicals ultimately entered into WWTPs, which were the convergences for various micro-pollutants derived from industrial and sewage wastewater. In fact, numerous kinds of biocides were frequently detected in WWTPs 8, 9, 11. Moreover, they were found to show quite low degradation potential by conventional WWTPs treatment processes but adsorbed strongly to materials such as sludge and sediments 12. As a result, most of the biocides in WWTPs were concentrated in WAS. For example, the chlorhexidine (CHD) and hexadecyltrimethylammonium bromide (HTAB), which were two typical biocides with large consumption, have been estimated to range from 20 to 370 mg/kg dry sludge 9. In addition, they were believed to be still active against bacteria even when adsorbed on the WAS 13. Commonly, solubilization, hydrolysis, acidogenesis and methanogenesis are the four major processes involved in the WAS anaerobic fermentation3, 5. They were accomplished and mediated by a variety of functional anaerobic microorganisms (i.e. hydrolytic bacteria, acetogens and methanogens), and were affected by various factors 14-16. Thus, the largely existed biocides (CHD 3



and HTAB) in WAS were speculated to result in the increasing instability of SCFAs production and disturb the efficient resource recovery from WAS during anaerobic fermentation since they are persistent and still active against bacteria in WAS 12, 13, 17. However, till now, little is known on how the presence of biocides in WAS affecting the anaerobic performance for SCFAs generation, which are key intermediates during the bioconversion of sludge organics. Considering the wide occurrence of biocides with high loads in WAS and the large application of anaerobic technology for WAS disposal, the scientific attempts to investigate the potential impacts of biocides on the performance of anaerobic fermentation are significant and imperative. Besides, various microorganisms are present in WAS but only those active ones function during the anaerobic bioconversion of substrates into SCFAs. The traditional DNA-based high-throughput sequencing provided significant insights of the overall microbial community but failed to reveal the truly active microbial community in fermentation systems. Recently, the active community evaluated on RNA level via 16S rRNA amplicon sequencing was highly recommended

18.

It was supposed to reflect the interaction between biocides and fermentative

bacteria more accurately. Therefore, this study mainly aimed to disclose the potential impacts of two representative biocides (CHD and HTAB with high concentration and detection frequency) on the anaerobic fermentation for SCFAs generation from WAS. Six different SCFAs (i.e. acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids) were determined in this study. The variations of SCFAs generation under different biocides dosages were firstly investigated. Then, the influences of different biocides on the solubilization, hydrolysis, acidogenesis, and methanogenesis processes, which are the basic steps involved in sludge anaerobic fermentation were explored in details. The microbial activities correlated with SCFAs generation were also 4


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evaluated based on the key enzymes and biological energy. Finally, the active anaerobic microorganisms that involved in the SCFAs production were analyzed via amplicon sequencing of 16S rRNA transcripts. The study achieved to fill the research gap of the biocides effects on WAS anaerobic fermentation and disclose the underlying mechanisms from macro and micro perspectives, which might be beneficial to regulate the WAS anaerobic fermentation for resource recovery. EXPERIMENTAL SECTION

Characteristics of WAS and typical biocides

The WAS used in this study was obtained from the secondary sedimentation tank of a municipal WWTP in Nanjing, China.

Prior to use, the collected WAS was preconcentrated by

o

settling at 4 C for 24 h, and filtrated the large particulate solids by using a stainless steel mesh (2.0 mm). The main characteristics of WAS were concluded in Table 1. Table 1 Basic characteristics of WAS Index

Values (mg/L)

Index

Values (mg/L)

Total suspended solids (TSS)

21790 ± 455

Total proteins

10045 ± 275

Volatile suspended solids (VSS)

10150 ± 250

Total carbohydrates 2540 ± 205

Total chemical oxygen demand (TCOD)

22070 ± 550

pH

6.7 ± 0.2

Soluble chemical oxygen demand (SCOD) 445 ± 30 Chlorhexidine (purity, 97%) and hexadecyltrimethylammonium bromide (purity, 99%) were purchased from Aladdin reagent, Shanghai, China.

5



SCFAs production from WAS with different biocides exposure

The influences of CHD and HTAB at different levels on SCFAs accumulation from WAS were conducted in identical serum bottles with a working volume of 600 mL. Firstly, the fermentation reactors were fed with 350 mL concentrated WAS and spiked with different dose of biocides, which were 0, 50, 100 and 200 mg/L for CHD and 50, 100 and 200 mg/L for HTAB. Also, the simultaneous dosage of CHD (50 mg/L) & HTAB (50 mg/L) was operated to investigate their combined effects on the performance of anaerobic fermentation. Then the reactors were flushed with pure nitrogen for 3 min to eliminate oxygen and were capped with rubber stoppers and sealed to keep anaerobic environment. Finally, they were placed in an air-bath shaker at a o

stirring speed of 200 rpm and temperature 35 C.

The determination of biocides effects on WAS

anaerobic fermentation was obtained by analyzing the variations of SCFAs concentration in different reactors.

Underlying mechanisms for SCFAs promotion stimulated by biocides exposure

Solubilization, hydrolysis, acidogenesis, and methanogenesis were the four main steps involved in WAS fermentation and the influences of biocides on each step was investigated separately in order to disclose the mechanisms more accurately. The particulate organics in WAS should firstly transform into soluble ones for later utilization during solubilization process. The main organics found in WAS were carbohydrates and proteins. Therefore, the effects of CHD and HTAB on the solubilization of particulate compounds in WAS were obtained via the measurement of soluble proteins and carbohydrates concentrations from above batch tests at the fermentation time of 2 d. The surface tension, which could reflect the solubilizing performance of organic matters from WAS, was also determined 2. 6


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The influences of CHD and HTAB on the hydrolysis and acidification of solubilized organics were performed with the synthetic wastewater. Based on the analysis of main components in WAS (i.e proteins and carbohydrates), the synthetic wastewater was consisted of 2.5 g/L bovine serum albumin (BSA, model protein compound) and 1.0 g/L dextran (model polysaccharide compound) with 10% concentrated WAS from WWTPs (v/v) as the inoculum. The concentrations of biocides were controlled respectively at 0, 50 and 200 mg/L for CHD, 50 and 200 mg/L for HTAB, and 50 mg/L for CHD & HTAB. All the above reactors were also kept anaerobic by nitrogen flush and then sealed with rubber stoppers with the same operating conditions mentioned above.

The

effects of CHD and HTAB on the acidification of hydrolyzed products were obtained from the varying SCFAs concentration in anaerobic reactors. The influence of biocides (CHD and HTAB) on the methanogenesis step was investigated by determining the amounts of accumulative methane during the whole anaerobic process. The methane was sampled by gastight syringe from anaerobic reactors and injected into gas chromatography immediately for final analysis. The amount of methane was calculated by multiplying its occupied content in mixed gases and the volume at atmospheric pressure. The impacts of CHD and HTAB on the activities of hydrolases, acid-forming enzymes and coenzyme F420, and active community evolution of anaerobic microorganisms during anaerobic fermentation were explored in semi-continuous-flow reactors.

The reactors were fed with 350

mL WAS and the concentrations of biocides were controlled respectively at 0, 50 and 200 mg/L for CHD, 50 and 200 mg/L for HTAB, and CHD (50 mg/L) & HTAB (50 mg/L). The fermentation conditions were the same as above (placed in an air-bath shaker with a stirring speed o

of 200 rpm and temperature 35 C).

During the semi-continuous-flow operation, the sludge

retention time in the reactors was set as 10 d based on the results obtained from batch tests of 7



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WAS fermentation. Thus, 35 mL of WAS fermentation mixture was discharged from each reactor daily and simultaneously replenished with the same amount of raw WAS and corresponding CHD or HTAB dose. The reactors were flushed with nitrogen and re-sealed before placing them back to the shakers. The key enzyme activities and microbial community was determined after 3 months when the operation of reactors reached stable.

Analytical methods

The TSS, VSS and COD of WAS were measured according to the APHA standard methods19. The determination of proteins, carbohydrates, SCFAs and activities of hydrolytic and acid-forming enzymes (including the protease, α-amylase, acetate kinase (AK), phosphotransacetylase (PTA), coenzymes F420) in WAS fermentation systems were the same as previous publications 2, 20, 21. The units of SCFAs were unified and expressed as mg COD/L in the present study. The COD conversion factors for SCFAs are 1.07 mg COD/mg acetic, 1.51 mg COD/ mg propionic, 1.82 mg COD/ mg butyric, and 2.04 mg COD/ mg valeric acid, respectively. The extracellular polymeric substances (EPS) in WAS were extracted via cation exchange resin (CER) technique 22. The adenosine 5’-triphosphate (ATP) concentration was determined with ATP assay kit (Beyotime, China) following the manufacturing specific procedures.

The lactate dehydrogenase

(LDH) release assay was determined with cytotoxicity detection kit (Roche Applied Science). 10 mL WAS was withdrawn from the reactors and centrifuged at 12000 rpm for 3 min to obtain supernatants. Then the supernatants were seeded in a 96-well plate, and incubated at 30 °C for 30 min with the addition of 50 μL reaction mixture and finally analyzed at A490 via microplate readers (BioTek, USA). The surface tension of WAS was analyzed by contact angle measurement (JC2000D 8


，

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Powereach ，Shanghai), and zeta potentials were determined by Zetasizer Nano ZS 90 (Malvern, UK). For a complete understanding of active anaerobic microorganisms in WAS fermentation systems, the microbial community evaluated on RNA (active community) level via 16S rRNA amplicon sequencing was applied. The RNA was extracted with E.Z.N.A.® Soil RNA Mini Kit. The purified RNA was subsequently converted to cDNA using the cDNA Synthesis Kit and finally sent for sequencing on the Illumina Miseq platform (Biozeron, China).

The detailed

procedures were according to the literature 18 .

Statistical analysis

All the experiments in this study were conducted in triplicates. An analysis of variance was used to evaluate the significance of results, and p < 0.05 was considered to be statistically significant. RESULTS AND DISCUSSION

Effects of typical biocides on the SCFAs production from WAS

The production of SCFAs from WAS anaerobic reactors with different CHD and HTAB dosages was shown in Figure 1(A). It can be found that the SCFAs generation showed different levels of promotion when exposed to the CHD and HTAB with various dosages. The maximal accumulation of SCFAs was merely 567.3 mg COD/L (within 10 d) in the control reactor. It increased significantly to 2144.5 mg COD/L (within 10 d) at the CHD concentration of 50 mg/L, which was approximate 3.8 fold of that in control. However, the promoting effects were weakened in high concentration of CHD. The acidification rates were slowed down and the highest SCFAs concentration was reduced to 1374 and 848 mg COD/L (within 14 d) at the CHD concentration of 9



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100 and 200 mg/L, respectively. Similar trends were found in the HTAB exposure reactors but with more remarkable SCFAs promotion. The SCFAs accumulation was up to 3400 and 3615.7 mg COD/L (within 12 d) at the dosage of 50 and 100 mg/L, respectively. The SCFAs concentration also decreased evidently with further increase of HTAB dosage which was reduced to 2010.2 mg COD/L at the dosage of 200 mg/L. Clearly, both the CHD and HTAB exhibited positive effects on the anaerobic fermentation for SCFAs production, especially in low concentrations. However, the co-existence of CHD and HTAB in WAS fermentation reactors seemed to show an antagonistic effect for SCFAs generation. The SCFAs concentration in reactors with simultaneous CHD & HTAB exposure (50 mg/L) was observed to be much lower than that of individual CHD and HTAB occurred with similar concentration (Figure 1(A)).

Figure 1 Effects of CHD and HTAB on the SCFAs production (A) and composition (B) with different dosage Moreover, the CHD and HTAB also affected the composition of SCFAs during WAS anaerobic fermentation. Six kinds of SCFAs were determined in the present study. As shown in Figure 1(B), the SCFAs found in control reactors were composed of acetic, propionic and butyric acid with the proportion of 14.1, 27.8 and 58.1%, respectively. However, the SCFAs composition 10


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was varied greatly with the exposure of CHD and HTAB. The butyric acid ratio was evidently reduced in all reactors investigated, which ranged from approximate 10 (CHD exposure) to 18% (HTAB exposure). On the contrary, the proportions of acetic and valeric acid were remarkably promoted to the ranges of 34.1-51.1% and 16.5-24.4%, respectively. Particularly, the acetic acid was reported to be the most preferred carbon source for biological nutrient removal in WWTPs as well as the important precursor for methanogenesis 5. Therefore, the increase of acetic acid ratio in WAS fermentation reactors is supposed to further promote the economic and practical values of fermentation products. Overall, the CHD and HTAB present in WAS exhibited unexpectedly positive effects on the anaerobic fermentation for SCFAs production in view of SCFAs concentration and composition. The reasons for SCFAs promotion, especially acetic acid, that caused by biocides exposure were explored below.

Effects of biocides on the solubilization during WAS fermentation

Normally, WAS solubilization and hydrolysis are considered as the rate-limiting steps during anaerobic fermentation as the organic matters in raw WAS are mainly cemented and flocculated by EPS which have to be firstly solubilized

23, 24.

The solubilization efficiency with CHD and

HTAB exposure in WAS fermentation reactors could be determined by the concentrations of soluble proteins and carbohydrates in the fermentation liquids.

11



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Figure 2 Effects of typical CHD and HTAB on the solubilization process (A) and EPS disruption (B) in WAS fermentation reactors with different dosage at 2 d As shown in Figure 2 (A), the concentrations of soluble carbohydrates and proteins were merely 37 and 241 mg/L in the control reactor, respectively. However, they were remarkably improved with the exposure of both CHD and HTAB and directly correlated with the biocides dosage, especially the HTAB. For instance, the concentrations of carbohydrates and proteins in the reactors with 50 mg/L CHD were respectively increased to 540 and 162 mg /L and further incremented to 630 and 223 mg /L at the dosage of 100 mg/L. Similar results were found in the HTAB reactors. The concentrations of carbohydrates and proteins were respectively 672 and 195 mg /L with 50 mg/L HTAB and they were further promoted to 792 and 287 mg /L at the exposure of 100 mg/L. Obviously, the WAS solubilization process was promoted when CHD and HTAB occurred. The EPS, which were mainly composed of proteins and carbohydrates, accounted for approximate 80% of the total activated sludge mass23, 25. The disruption of EPS was therefore highly correlated with the sludge solubilization process during WAS anaerobic fermentation. As 12


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shown in Figure 2(B), the contents of proteins and carbohydrates in both loosely and tightly bound EPS were all decreased with different levels of CHD and HTAB dosage. The results demonstrated that the CHD and HTAB promoted the disruption of EPS in WAS and therefore resulted in the evident increase of soluble substrates during solubilization step. The findings can be attributed to the hydrophobic/hydrophilic properties of CHD and HTAB

12.

They were able to reduce the

surface tension between hydrophobic molecules and water due to their amphipathic molecules with both hydrophobic and hydrophilic properties

26.

In fact, the surface tension in WAS

fermentation systems were evidently decreased due to the biocides addition. It was decreased from 89 mN/m in control to 62 and 58 mN/m with 50 mg/L CHD and HTAB, respectively. The reduction of surface tension was expected to enhance the transfer and solubility of organic matters which would result in the improvement of soluble substrates2.

Effects of biocides on the hydrolysis and acidification during WAS fermentation

During WAS anaerobic fermentation, the solubilized organic substrates should be further hydrolyzed into micro-molecule products and then acidified by acid-forming bacteria.

In this

study, the exploration of biocides effects on the hydrolysis and acidification of solubilized substrates was achieved by utilizing synthetic wastewater (containing BSA and dextran) to simulate the processes which has been reported in previous studies27, 28. As shown in Figure 3, the SCFAs production was evidently stimulated with low dosage of biocides. The concentration of SCFAs was respectively 3403 and 3697 mg COD/L at the CHD and HTAB dosage of 50 mg/L while it was merely 2389 mg COD/L in the control. However, excessive dosage of biocides (200 mg/L) and the co-existence of CHD and HTAB exhibited little effects on the SCFAs promotion. The results obtained from synthetic wastewater showed good agreement with that in WAS 13



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anaerobic fermentation reactors. Also, it demonstrated that the hydrolysis and acidification processes for SCFAs production were positively affected by the low biocides exposure.

Figure 3 Effects of CHD and HTAB on the solubilization and acidification processes with synthetic wastewater

Effects of biocides on the metabolic activity responsible for SCFAs generation

The roles of hydrolases are significant in the hydrolysis process of solubilized products during anaerobic treatment and the protease and α-amylase were the most frequently reported hydrolases involved in the proteins and carbohydrates hydrolysis 15, 29.

As can be seen in Figure

4(A), the activities of both protease and α-amylase in reactors with low CHD and HTAB were respectively 1.37- and 1.18-fold, 1.42- and 1.25-fold of those in the control. Moreover, the CHD and HTAB, which were characteristics of both hydrophilic and hydrophobic ends, could show positive influences on the interaction between enzymes and substrates by promoting the availability of reaction sites

30, 31.

Therefore, the hydrolysis rates of organic matters were 14


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accelerated.

Figure 4 Effects of CHD and HTAB on the hydrolases (A) , acid-forming enzymes (B), ATP concentrations (C), and LDH release (D) in WAS fermentation reactors with different dosage Also, there were numerous acidogenic enzymes participated in the metabolic pathway for SCFAs generation during acidification process, and their activities determined the rate of SCFAs production largely7, 20. The activities of PTA and AK, which were two key enzymes involved in the acetic acid generation (the dominant SCFA in this study), were found to be respectively enhanced by 22% and 25%, 41% and 36% in reactors with low CHD and HTAB (shown in Figure 4(B)). It meant that more substrates could be converted into acetic acids with the aid of corresponding enzymes, resulting in the high yield of acetic acid. On one hand, the improvement of enzymatic activities might attribute to the increase of bioavailable organic matters in 15



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fermentation reactors which would stimulate the microbial activities by causing selective pressures2, 15. On the other hand, the bioaccessibility of fermenting bacteria to substrates could be improved in CHD and HTAB reactors. The charge density of WAS in control was -20.8 mV, while it increased to -15.9 and -14.2 mV in the presence of 50 mg/L CHD and HTAB, respectively. The increase of electric charge in the biocides reactors could weaken the mutual repulsion and provide better adsorptions and interactions between the anaerobic bacteria and substrates32, 33. The concentration of ATP, which was widely applied to estimate the general microbial activity 34, was observed to increase from 0.85 μM/L in WAS reactor to 1.25, and 1.32 μM/L at the CHD and HTAB dosage of 50 mg/L, respectively (shown in Figure 4(C)). Clearly, the activities of anaerobic microorganisms were promoted in low biocides exposure and it was one of the main reasons for the increased accumulation of SCFAs during hydrolysis and acidification processes. However, the excessive exposure of CHD and HTAB would cause detrimental effects to the fermentative bacteria. Generally, the anaerobic microorganisms were negative charged 35, and thus the cationic CHD and HTAB were easily to bind onto fermentative bacteria. In low biocides dosage, the microbes can relieve the side-effects by forming a protective barrier with the release of substrates from EPS to entrap the toxic matters. But the biocides would directly sorb on the cell membranes and disrupt the membranes structure with increasing concentrations and ultimately result in the reduction of microbial activities. The release of LDH, which reflect the extent of cell membrane disruption36, was found to be respectively 195 and 168% of control at the dosage of 200 mg/L CHD and HTAB while they were only 125% and 118% at low dosage (50 mg/L) (Figure 4(D)). The results demonstrated the negative impacts of CHD and HTAB on cell membranes disruption and the damaging effects were linearly correlated with the biocides concentration. It could be one of the main causes for the reduction of enzymatic activities in 16


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anaerobic fermentation reactors with high biocides dosage (Figure 4(A) and 4(B)). The hydrolysis and acidification rates were therefore inhibited which resulted in the low efficiency of SCFAs generation though more soluble substrates were provided. In addition, the LDH release and microbial activity reduction in reactor with CHD & HTAB co-existence (50 mg/L) were observed to be comparable to that of individual CHD or HTAB with high loads (200 mg/L). It seemed to form a synergistic disrupting effect between the CHD and HTAB which may require further investigation. But the aggravating detrimental effects of CHD and HTAB highlighted the potential risks of the various coexisted pollutants in environments.

Effects of biocides on the methanogenesis during WAS fermentation

The accumulation of SCFAs during anaerobic treatment was also dependent on the methanogenesis which was a SCFAs consumption process. As shown in Figure 5, the contents of accumulative methane was 11.2 mL/g VSS during the whole fermentation process in the control reactor while it was gradually decreased to 8.5 and 7.2 mL/g VSS at the CHD and HTAB dosage of 50 mg/L, respectively. It was further reduced to 4.5 and 4.3 mL/g VSS at the CHD and HTAB dosage of 200 mg/L, respectively. Moreover, the activity of coenzyme F420, which was responsible for the transformative activity of electron donors of redox-driven proton translocation in methanogenic Archaea 21, was significantly restrained at different level of biocides. The obtained results indicated that the CHD and HTAB exhibited inhibitory effects on the methanogenic activity, especially at high loads. Therefore, the SCFAs consumption for methane production would be reduced which was in accordance with the high SCFAs accumulation in anaerobic reactors with biocides exposure.

17



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Figure 5 Effects of CHD and HTAB on the methanogenesis process in WAS fermentation reactors with different dosage

Effects of biocides on the shifts of active microorganisms during WAS fermentation Fermentative bacteria responsible for SCFAs production The abundances of active fermentative microorganisms responsible for SCFAs production in reactors with different biocides exposure were performed via amplicon sequencing of 16S rRNA transcripts. As shown in Table 2, the richness and diversity of anaerobic microorganisms in reactors with biocides exposure were reduced evidently. The OUTs was found to be 2020 in the control while it was only 1547 and 1417 in the CHD and HTAB reactors. The reason may be attributed to the toxic effects of biocides on those sensitive and fragile microorganisms. The main active phyla included Proteobacteria, Bacteroidetes, Firmicutes, Patescibacteria, Chloroflexi, Acidobacteria, Actinobacteria and Euryarchaeota (Figure 6 (A)), which accounted for approximate 90% of the total abundances in all the investigated reactors of this study. However, the abundance of particular phylum was varied with CHD and HTAB exposure. Firmicutes, which had the ability of excreting hydrolases to degrade those complex organic 18


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macromolecules (including both proteins and carbohydrates) with the metabolites of propionic and acetic acids 7, was increased from 2.3% to 12.0 and 25.1% in reactors with CHD and HTAB, respectively. The abundances of Patescibacteria, which was an typical anaerobic fermenter

37, 38,

was also observed to increment from 9.0% to 24.9 and 20.5% with CHD and HTAB exposure, respectively. Besides, the proportions of Acidobacteria that consisted of various anaerobic species with the ability of decomposing solid wastes and producing organic acids, were respectively improved from 3.3% to 7.7 and 7.2%. The increase of these fermentative bacterial would certainly be advantageous to the hydrolysis of WAS and SCFAs generation which were observed in the WAS fermentation reactors. However, Proteobacteria was reduced from 38.8 % in the control to 18.6-19.6% with the biocides exposure. They were considered as the main acetate consumers during anaerobic simultaneous denitrification and methanogenesis

2, 39,

and therefore its decrease

was actually beneficial to the SCFAs accumulation. Table 2 Summary of pyrosequencing data in control and biocides exposing reactors α = 0.03 Sample ID

Reads

OTUs

Coverage

Chao1a

Shannonb

Control

37538

2020

0.99

2561

5.95

CHD (50mg/L)

40945

1574

0.99

1874

4.93

HTAB (50mg/L)

36376

1417

0.99

1750

4.54

a

Chao1 richness estimator: the total number of OTUs estimated by infinite sampling. A higher number indicates higher richness. b Shannon diversity index: an index to characterize species diversity. A higher value represents more diversity. The functional evolution of microbial community responsible for SCFAs production can be better illustrated by analyzing the relative abundances of predominant phylogenetic groups at genus level. As shown in Figure 6(B), various anaerobic species showed lower abundance due to 19



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the presence of CHD and HTAB, such as the Acinetobacter, Sapropiraceae, Anaerolineaceae and et al. However, the dominant fermentative bacteria, which were expected to utilize various organic substrates for SCFAs generation, were highly enriched in reactors with CHD and HTAB exposure. For example, Bacteroides that contributed significantly to acetic and propionic acids production 40,

and Macellibacteroides that could metabolize carbohydrates with acetate as the main

fermentation product

41,

were both increased in the biocides reactors. Acidaminobacter, which

was reported to involve in the sludge biological hydrolysis and organics fermentation for acetic and propionic acids production

41, 42,

was found to be respectively 8.87 and 22.05% in the CHD

and HTAB reactors while it was only 0.08% in the control. Saccharibacteria, and Proteiniclasticum, which were generally linked to the proteins metabolism with acetate as the major product43,

44,

were also highly enriched in the biocide reactors. Particularly, the

Saccharibacteria was respectively 23.85 and 19.85% in the CHD and HTAB reactors and became one of the predominant groups in WAS anaerobic reactors. Obviously, the remarkable increase of these functional fermentative bacteria in the reactors of biocides exposure could accelerate the hydrolysis of WAS (enriched with protein- and carbohydrate-containing substrates) and then enhance the acidification process for SCFAs production during WAS fermentation, especially the acetic acid.

20


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Figure 6 Effects of CHD and HTAB on the variations of microbial community in WAS fermentation reactors with different dosage (A) Phylum level; (B) Genus level 21



Page 22 of 28

Typical archaea community involved in methanogenesis Generally, the lower abundance of methanogens in the WAS fermentation systems would be beneficial to the SCFAs accumulation since they may consume the SCFAs for methane formation. The abundances of Euryarchaeota, which consisted of numerous methanogenic archaeal clones5, were found to be reduced from 1.27% in the control to 0.69 and 0.46% in the CHD and HTAB reactors (Figure 6(A)). Further analysis found that the Euryarchaeota was mainly consisted of Methanosaeta (Figure 6(B)), which was the typical acetoclastic methanogen 45, 46. The decrease of acetoclastic ones in biocides reactors would reduce the acetic acid consumption for methane production which was consistent with the low methane volume but high SCFAs concentration, especially the acetic acid. The negative effects of biocides on the methanogens could further be confirmed by the decrease of non-methanogenic archaea Woesearchaeota, which was generally symbiotic with methanogens

45.

Methanogens were supposed to be quite sensitive to the

surrounding environments which required stringent conditions to maintain their high microbial viability and activity 47. Thus, it was reasonable that the abundances of methanogens were reduced in CHD and HTAB reactors considering their biocidal effects. CONCLUSION During anaerobic fermentation, the biocides existed in WAS, especially the HTAB, could positively affect the resource recovery for SCFAs production. The maximal SCFAs concentration was respectively 3.8- and 6.3-folds in the reactors with low CHD and HTAB exposure when compared with that of control. The reasons for the SCFAs promotion were mainly attributed to the simultaneous promotion of sludge solubilization, hydrolysis and acidification with the inhibition

22


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Page 28 of 28

For Table of Contents Use Only

Synopsis: The disclosure of correlations between contaminates occurred in WAS and anaerobic fermentation performance would better regulate the resource recovery from WAS.

28


How Do Biocides Occurred in Waste Activated Sludge Affect the

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