Free Ammonia-Based Pretreatment Promotes Short-Chain Fatty Acid

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Free ammonia-based pretreatment promotes shortchain fatty acid production from waste activated sludge Chang Zhang, Yuge Qin, Qiuxiang Xu, Xuran Liu, Yiwen Liu, BingJie Ni, Qi Yang, Dongbo Wang, Xiaoming Li, and Qilin Wang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01452 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Sustainable Chemistry & Engineering

Free ammonia-based pretreatment promotes short-chain fatty acid production from waste activated sludge Chang Zhanga,b, Yuge Qina,b, Qiuxiang Xua,b, Xuran Liua,b, Yiwen Liuc, Bing-Jie Nic, Qi Yanga,b, Dongbo Wanga,b,*, Xiaoming Lia,b, Qilin Wangd,* a

College of Environmental Science and Engineering, Hunan University, Changsha 410082, P.R. China

b

Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education,

Changsha 410082, P.R. China c

Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University

of Technology Sydney, Sydney, NSW 2007, Australia d

Griffith School of Engineering & Centre for Clean Environment and Energy & Environmental Futures

Research Institute, Griffith University, QLD, Australia

Corresponding author Email: [email protected] (Dongbo Wang); Tel: +86-731-88823967 Email: [email protected] (Qilin Wang); Tel: +61 7 3735 5036

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ABSTRACT: This work reported a new WAS pretreatment method based on free ammonia (FA) for promoting the generation of SCFA. Experimental results showed that pretreatment of WAS for 3 d with FA largely improved WAS disintegration, with the highest dissolution (soluble COD 3400 ± 120 mg/L at initial FA level of 237.8 mg/L) being 4.5-fold that without FA pretreatment. The pretreatment method by FA facilitated the breakdown of extracellular polymeric substances and cell envelop of sludge cells, killed more live microbial cells, which thereby accelerated the dissolution of substances from WAS. It was also found that FA severely suppressed the SCFA consumption process, but unaffected acetogenesis process. Although FA also inhibited hydrolysis, acidogenesis, and homoacetogenesis to some extents, the inhibitions did not largely affect the biodegradation of the relevant substances at all the tested FA levels. Finally, using FA to pretreat WAS for SCFA enhancement was confirmed. When FA concentrations ranging from 53.5 to 176.5 mg/L, the maximum generation of SCFA enhanced from 196.8 to 267.2 mg COD/g VSS, which was 2.3~3.2 times of that from the blank.

Further FA level (237.8 mg/L) caused a slight decline of maximum SCFA

generation (226.9 mg COD/g VSS). The findings reported may instruct engineers to develop an economic and effective strategy to enhance SCFA production, which might support the operation of WWTPs in sustainable paradigms with low energy input in the future. KEYWORDS: Sludge pretreatment, Anaerobic fermentation, Free ammonia, Short chain fatty acid INTRODUCTION Biological nutrient removal is an effective technique to treat municipal wastewater and is widely applied worldwide [1, 2]. However, it is often upset by insufficient carbon sources in wastewater in real-world operations of wastewater treatment plants (WWTPs) [3]. In these situations, researchers and engineers generally add some additional carbon sources (e.g., acetate) to keep desirable biological nutrient removal [4-7]. Although this approach is effective, it increases not only operational costs but also carbon footprint of

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WWTPs, which makes it uneconomical and unsustainable in long-term operation. Waste activated sludge is daily generated at high amounts in wastewater treatment plants. According to our communications with industry partners, it is estimated that a WWTP (Q = 105 m3) may produce ~50 tons WAS daily. On one hand, WAS treatment and disposal is expensive, which is approximately 50% ~ 60% of the whole operational cost of a WWTP [8-10]. On the other hand, as the amount of biodegradable organic substrates are contained in sludge, WAS should be recovered and reused [11, 12]. By using WAS as the fermentation substrate, SCFA could be in-situ produced in WWTPs and utilized as supplementary carbon sources to promote wastewater biological nutrient removal [13-15], which simultaneously reduces the sludge volume and the operational cost of wastewater treatment. Therefore, the generation of SCFA from anaerobic fermentation of sludge has recently attracted much attention [16, 17]. Anaerobic fermentation of WAS generally includes six steps, i.e., solubilization, hydrolysis, etc. Solubilization and hydrolysis provide soluble substrates for production of SCFA, which are considered the major rate-limiting steps [18-20]. SCFA is generated in acidogenesis, acetogenesis, and homoacetogenes steps and readily consumed in methanogenesis step.

Clearly, the yield of SCFA production can be promoted

by enhancing the first five steps (especially sludge solubilization step) and inhibiting the last step. Based on this principle, a large number of sludge pretreatment methods including alkaline, thermal and ultrasonic have been tested and proposed to promote SCFA yield in the past years [21-24]. For example, When 1.8 mg HNO2-N/L pretreated WAS for 48h, sludge solubilization was substantially promoted, resulting in a 3.7-time increase in SCFA yield than that in blank [25, 26]. Although these approaches are effective, they are either expensive due to serious inputs of chemicals or application-difficult due to lack of nitritation reactor for HNO2 production in most of the current WWTPs, which diminish their values. It reported that free ammonia (FA) have a severe biocidal influence on microorganisms [27, 28]. FA can

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enter the cell without energy consumption, causing the proton imbalance by the difference in intracellular pH and/or potassium deficiency [29].

Previous studies showed that the accumulation of FA in anaerobic

digestion could inhibit severely methane production [30-32]. It was found that 40 mg FA/L caused 50% inhibition to methanogenesis while 150 mg FA/L completely ceased this process [33]. In addition, it has been recently indicated that FA may largely facilitated solubilization of WAS [34]. These previous outcomes enlighten us to raise an assumption that a FA-based sludge pretreatment might enhance SCFA generation from sludge. This can be readily achieved by recycling sludge fermentation liquid into FA pretreatment unit. Considering the fact that FA was able to be generated from fermentation liquid without any side-stream nitritation reactor, this FA-based technology should be economically attractive and easily implemented in field situations. Although the inhibition of anaerobic digestion by FA has been well acknowledged in the past years, the impact of FA on SCFA generation from WAS has not been documented so far. Using modeling analyses, Studies demonstrated that pretreatment technologies could significantly increase the sludge hydrolysis and biochemical methane potential in the past years [35]. To date, however, in terms of experimental evidences, mechanism of how FA-based sludge pretreatment affect the whole process of WAS fermentation (e.g., solubilization), which is a mystery. This study was therefore to assess whether and how FA-based sludge pretreatment enhance SCFA generation from the sludge.

Firstly, the pretreatment time of FA was determined by investigating the

dynamics in soluble COD with pretreatment time.

Secondly, details of how FA pretreatment enhances sludge

solubilization was elucidated by investigating the function of FA on breakdown of EPS and cell envelope, and the influence of FA on the viability of bacteria. Thirdly, the impact of FA on hydrolysis, acidogenesis, acetogenesis, homoacetogenesis, and methanogenesis was evaluated as well. Based on these investigations, the feasibility of FA pretreatment promoting SCFA production was finally confirmed. The findings reported

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gave an economic method for SCFA generation, which might support the operation of WWTPs in sustainable paradigms with low energy input in the future. MATERIALS AND METHODS Source of WAS Secondary sludge was utilized to conduct this test. The collected WAS was separated with a 1mm×1mm screen to remove impurities and stored at 4 ℃ in a refrigerator for approximately 24 h prior to utilize. The properties of the used sludge are: pH 6.8 ± 0.1, total suspended soils (TSS) 36620 ± 440 mg/L, volatile suspended soils (VSS) 15960 ± 370 mg/L, soluble COD 560 ± 20 mg/L, total COD 19300 ± 190 mg/L, total protein 9830 ± 240 mg COD/L, total carbohydrate 1680 ± 100 mg COD /L, total SCFA 9.5 ± 1.1 mg/L, and NH4+-N 36 ± 3 mg/L. Optimization of FA pretreatment time In experiment, six replicate reactors (1 L each) were conducted. Each reactor was first filled with 0.5 L WAS. NH4Cl solution (4.0 M) with a certain volumes was then added into these reactors to attain the desired NH4+-N level of 36, 36, 152, 326, 501 or 675 mg/L. Each reactor was placed in a temperature-controlled room with temperature at 20 ± 1℃ and stirring at 120 rpm. NH4Cl solution was not added into the first two reactors (set as the blank and pH 9 reactors, respectively), and the background value of NH4+-N was 36 mg/L. Except one 36 mg/L NH4+-N reactor without pH control (the blank), pH in all other reactors was constantly maintained at 9.0 ± 0.1 by adding 4.0 M HCl solution or 4.0 M NaOH solution during the entire time. This resulted in initial FA level of 0.1, 12.7, 53.5, 114.8, 176.5, or 237.8 mg/L, which was obtained by the equation (1)

FA =

S( NH − N + NH + − N ) × 10pH 3 4 （K b / K w + 10pH）

(1)

Where S(NH3-N+NH4+-N) is the concentration of NH3-N + NH4+-N, Kb is the ionization constant of the ammonia 5



equilibrium equation, and Kw is the ionization constant of water [29]. The value of Kb/Kw was determined by equation of Kb/Kw=e6.344/(273+T) . The pretreatment was lasted for 96 h, during which the contents of protein, carbohydrate, and SCOD released in fermentation liquid as well as VSS reduction in solid phase were measured periodically. Effect of FA pretreatment on breakdown of EPS and cell envelope

To investigate whether FA pretreatment affect breakdown of EPS and cell envelope, three replicate reactors(0.1 L each) (set up as pH 9 reactor, FA reactor, and blank reactor, respectively) were operated with temperature at 20℃. The blank reactor was conducted without either pH control or additional NH4+-N addition, while the pH 9 reactor was operated at pH 9.0 condition without additional NH4+-N addition. As comparison, the FA reactor was conducted at pH 9.0 condition with an l NH4+-N level of 501 NH4+-N/L (FA level of 176.5 mg/L). To judge whether the cell envelope is broken, the release of the lactate dehydrogenase (LDH, a cell membrane integrity marker) and COD mass balance analyses were employed. 0.5 L of WAS were added into each reactor. The total COD of EPS in raw sludge were determined before sludge was added into these reactors. After the adjustment of pH and NH4+-N addition, all the reactors were stirred at 120 rpm. The LDH release and COD mass balance analyses were made at different sampling times.

For COD mass balance analysis, the soluble COD was first measured after sampling, then a

heating extraction method was used to treat sludge for extracting LB-EPS and TB- EPS of sludge cells [36]. The detailed extraction method was presented in additional information. The soluble COD, heating extraction of COD, and the total COD in the raw WAS were utilized for the analysis of COD mass balance. If the sum of first two are not significantly increased compared to the total COD in the raw WAS, cell envelope will not disrupt; or else, it will disrupt. Effect of FA pretreatment on hydrolysis, acidogenesis, acetogenesis, homoacetogenesis and

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methanogenesis processes

Except for sludge disintegration, anaerobic fermentation of WAS as well contains few other bio-processes (e.g., hydrolysis). These bio-processes are closely associated with SCFAs yield. To reveal whether and how FA influences these bio-processes, a sequence of batch fermentation experiments were performed by serving model compounds as fermentation substrates according to the literature [9, 18]. In the tests, 30 replicate reactors (1 L each) were carried out, which were divided into Group-I, Group-II, Group-III, Group-IV, and Group–V with six in each. Group-I: 0.54 L synthetic medium and 0.06 L same inocula were added into each reactor. The inocula utilized in experiment were derived from an anaerobic fermenter operated in lab, and 0.5 g dextran/L was contained in the synthetic medium. Among these reactors, two of them were respectively defined as blank reactor and the sole pH 9 reactors, the other four were set as the FA reactors. pH and temperature in these FA reactors were maintained at 9.0 and 20 ℃, respectively.

The levels of FA in the four reactors were

controlled at 61.5, 101.2, 138.8 and 178.9 mg/L, respectively (these levels of FA were respectively the average FA levels obtained from the fermenters filled with 53.5, 114.8, 176.5, and 237.8 mg/L of FA treated sludge, details please see below). By comparing the dextran content in synthetic medium, the influence of FA on hydrolysis could be assessed. Group-II: The test was carried out with similar method depicted in Group-I except that 0.9 g/L glucose replaced 0.5 g/L dextran in the synthetic wastewater. The influence of FA pre-treatment on acidogenesis could be evaluated via comparing the glucose content in synthetic medium. Group-III: To order to evaluate the influence of FA pre-treatment on acetogenesis, the test was employed. The test was conducted with the same approach described in Group-I. In addition, 0.5 g/L dextran was utilized to replace 10 g/L sodium propionate.

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Group-IV: the effect of FA on homoacetogenesis was obtained in this experiment. All reactors received 0.06 L of same inoculua and 0.54 L tap water. Then, each reactor was sparged with synthetic gas (40% H2, 10% CO2, and 50% N2) for 5 min in order to ensure that the reactor was fed with the hydrogen-containing gas. Finally, each reactor was sealed and stirred at 120 rpm. All other operations were the same as those in Group-I. Group-V: the test was used to evaluate the influence of FA on methonogenesis. Except the different fermentation substrate (5 g/L sodium acetate) this experiment was performed with similar operational procedure depicted in Group-I. The effect of FA on the content of dextran, glucose, propionate, hydrogen, and acetate in synthetic medium was also evaluated by the first order kinetic model (Eq 1) and the noncompetitive inhibition model (Eq 2): Lo-Lt = Lo × {1-exp (-X × t)}

(1)

Where Lo represents the initial level of model compounds, Ct represents the level of model compounds when fermentation is over, t represents the fermentation time (d), and X represents the degradation kinetics rate (mg/(L•d)) of model compounds among 3 d fermentation. ଵ ଡ଼ୱ,୧

=(

ଵ

ଡ଼ୱ,୭×୏ୱ,୧

× Ii +

ଵ

(2)

)

ଡ଼ୱ,୭

Where, X represents the degradation rate as depicted in Eq 1, subindex "s" represents the fermentation substrate and "i" represents the inhibitor, and Ii represents inhibitor levels (mg/L). SCFA generation from WAS pretreated with different FA levels

Six replicate anaerobic fermentation reactors (1 L each) were carried out at 20 ± 2 ℃

in a

temperature-controlled room. Each reactor first received 0.5 L WAS, and certain volumes of NH4Cl solution were added into the reactors to obtain the initial NH4+-N level of 36, 36, 152, 326, 501 or 675 mg/L. The former two reactors were respectively served as the blank and sole pH 9 reactors, while the latter four were

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defined as the FA reactors.

According to the results obtained from Section “Optimization of FA pretreatment

time”, FA pretreatment in the FA reactors were 72 h, during which pH in these reactors was constantly keep at 9.0 ± 0.2. After FA pretreatment, pH in these FA reactors was not controlled. The pH in the sole pH 9 reactor without extra NH4+-N addition was constantly controlled at 9.0 in the entire fermentation time. In the blank reactor, NH4+-N was not added and pH was also not controlled in entire fermentation time. Analytical methods

based on the standard method, The measure of TCOD, SCOD, TSS and VSS were applied [37]. Protein was measured according to the modified Lowry method [38]. Carbohydrate was determined according to the phenol-sulfuric acid method [39]. The method for SCFA measurement was the same as described in the previous literatures [40], and the detailed operational procedure was placed in Additional information. A gas chromatography (GC112A, China) was used to analyze the content of hydrogen and methane in biogas according to the details documented in the literature [40]. the changes of organic substrates in fermentation liquid was characterized by Excitation emission matrix (EEM) fluorescence spectroscopy (F-7000 FL spectrophotometer, Hitachi, Japan) [41], and the specific operation was placed in Additional information. The LDH release was assessed by LDH-Cytotoxicity Assay Kit (BioVision, USA), and the details method was provided in our previous publication [42]. The Live/Dead® BaclightTM bacterial viability kit (L-7012) was utilized to quantitatively determine live and dead cells according to the literature [43]. Firstly, 25 mL of the sludge samples were centrifuged at 10000 g for 15 min to remove the supernatant, and re-suspended in 2 mL of 0.85% NaCl wash buffer. The sludge mixture was incubated at room temperature for 1 h and mixed every 15 min. Then the mixture was centrifuged (10000 g for 15 min) to remove the supernatant and re-suspended in 20 mL of 0.85% NaCl wash buffer. After centrifuging, the concentrated sludge cells were re-suspended in 10 mL of 0.85% NaCl 3 mL of

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this supernatant was monitored the optical density at 670 nm in glass or acrylic absorption cuvettes. Afterwards, 1 mL of this supernatant was trapped between a slide and an 18 mm square coverslip, and a 3 µL freshly prepared mixture of SYTO 9 green-fluorescent and propidium iodide stains was added to the sludge cells. After reacting at room temperature in darkness for 15 min, the sludge cells were examined by fluorescence microscopy (Nikon Eclipse 80i, Japan).

It is reported that bacteria with a complete cell

membranes is fluorescent green, whereas bacteria with damaged cell membranes is fluorescent red [43]. For quantitative analysis, 20 microscopic fields were investigated with Image Pro Plus 6.0. Statistical analysis

All experiments were carried out in triplicate. An analysis of variance was employed to evaluate the significance of results and p < 0.05 (p > 0.05) was considered to statistically significant (insignificant). RESULTS AND DISCUSSION Effect of FA Pretreatment on Sludge Solubilization

As shown in Figure 1a, soluble COD (SCOD) in the blank was maintained at low levels (350~750 mg/L). Compared with the blank, alkaline condition (pH 9 in this work) largely enhanced soluble COD content (the maximal soluble COD of 2040 ± 120 mg/L was achieved at 3d). The same results were reported previously [21, 44].

When WAS was pretreated by FA, more soluble COD was obtained.

For example, SCOD

increased from 350 ± 120 to 2280 ± 120 mg/L with the treatment time expanding from 0 to 3 d when WAS were treated by 53.5 mg/L of initial FA. The increase of pretreatment time caused insignificant increase of SCOD (p > 0.05). Similar observations were also made for other FA levels, suggesting that the optimal time of FA pretreatment was 3 d. It was also found that FA level affect sludge solubilization obviously.

With the

initial FA concentration at 53.5 - 176.5 mg/L, the maximal SCOD (measured on 3d) increased from 2280 ± 120 to 3240 ± 120 mg/L. when FA level was further increased to 237.8 mg/L, the maximum soluble COD was

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not insignificant increased o to 3400 ± 160 mg/L（p > 0.05(Figure 1a), suggesting that the optimal FA pretreatment level was 176.5 mg/L. The data presented in Figure 1a could be further supported by the results of VSS reduction. On 3 d treatment time, 4.0 ± 1.0% and 10.1 ± 0.8% of VSS reduction were respectively achieved in the blank and pH 9 reactors, whereas the corresponding data were 11.8 ± 0.9%, 14.7 ± 1.0%, 16.8 ± 1.2% and 17.6 ± 1.2% in the initial FA of 53.5, 114.8, 176.5, and 237.8 mg/L reactors, respectively. All these data demonstrated that FA enhanced sludge disintegration. In the next text, details of how FA enhances sludge solubilization would be investigated. Details of How FA Pretreatment Enhances Sludge Solubilization

It is known that sludge solubilization process will be enhanced by accelerating the breakdown of EPS and cell envelop. Heating method is widely employed to extract EPS of cells in the literature [18, 44]. In this test, the total value of the soluble COD and the extracted COD by heating method measured at one pretreatment time was significantly higher than the initial TCOD , indicating that the presence of FA resulted in the breakdown of cell envelop. It can be seen from Figure 2a that the breakdown of cell envelop at 1h of pretreatment in all the blank reactor, the pH 9 reactor, and the FA (176.5 mg/L) reactor did not occur, because compared with 0 h, the total value of soluble COD and the extracted COD did not significantly increase (p > 0.05, Table S1), suggesting that soluble COD measured at this time was obtained from the breakdown of EPS. This deduction could be confirmed by the data of LDH release assay (Figure 2b). Compared with either the blank reactor or the pH 9 reactor, the FA reactor had more soluble COD released.

The results indicated that

FA pretreatment accelerated the disruption of EPS. At 2 h of pretreatment time, no significant disruption of cell envelop was also observed in blank reactor and pH 9 reactor (Figure 2a and 2b).

In the FA reactor, however, both LDH release and the total value of the

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soluble COD and the extracted COD increased significantly (p < 0.05, Table S1), indicating an obvious release of intracellular substances. The results suggested that cell envelop of sludge cells in the FA reactor disrupted earlier than that in the other two reactors. Figure 3 presents the visual results of the BacLight test using the sludge from the blank, pH 9, or FA reactor after 24 h pretreatment. The ratio of live cells to total cells was 90.8 ± 7.6% in the blank reactor and 78.9 ± 7.1% in the pH 9 reactor, whereas it decreased to 66.7 ± 5.4% in the FA reactor. This suggested that the biocidal impact of FA killed more live microbial cells from sludge than pH 9, which were the same results with the reported above. Also, all the facts indicated that FA facilitated the lysis of sludge cells and then accelerated the solubilization of sludge. In other words, more EPS and/or cells became soluble substances under the condition of FA-treated WAS, as compared with those in pH 9 reactor or blank reactor (Figure 4). For example, the soluble proteins (carbohydrates) concentration at 10 h treatment was 322.5 ± 99 mg/L (44.0 ± 19 mg/L) in blank reactor and 567.5 ± 75 mg/L (92.6 ± 16 mg/L) in pH 9 reactor, whereas the corresponding datum was 920.2 ± 101 mg/L (176.0 ± 9 mg/L) in the FA reactor (Figure 4a and 4b). Similar observation was also made at other treatment time. Figure 4c shows the EEM fluorescence spectroscopy of sludge liquid after 24 pretreatment, which is often applied to depict the structure change of fermentation liquid in the literature [45]. The location and intensity of fluorescence peak were used to indicate the difference in the chemical structures. It is reported that a red-shift (or blue-shift) of emission wavelength and an strong (or weak) of fluorescence intensity are respectively associated with the variation of particular functional groups and organic substances concentration [46]. Two main peaks (Peak A and Peak B) were located from fluorescence spectroscopy in all samples. Peak A and Peak B respectively identified at the excitation/emission wavelengths of 215-240/290-310 nm and 265-290/290-335 nm in the spectroscopy, belonging to aromatic proteins and tryptophan protein-like

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substances, respectively [47]. Compared with either the blank or pH 9, FA caused red-shifts of emission wavelength and increased fluorescence intensity (Figure 4c), confirming again that compared with pH 9 or the blank, more soluble organic matters was released in the anaerobic fermentation liquid due to the presence of FA.

It can be therefore concluded that FA facilitated breakdown of EPS and cell envelop of sludge cells,

benefited the damage of cell membrane, and accelerated the organics released from WAS. Effect of FA on hydrolysis, acidogenesis, acetogenesis, homoacetogenesis and methanogenesis

After sludge solubilization process, several processes in anaerobic fermentation (e.g., hydrolysis), are as well associated with SCFA accumulation. To reveal the potential influence of FA on these processes, five batch experiments (i.e., Group-I, Group-II, Group-III, Group-IV, and Group-V) using model substrates was performed, and the results are summed up in Table 1. The FA levels utilized in these batch test, i.e., 61.5, 101.2 138.8, and 178.8 mg/L, were respectively the average FA levels determined from the fermenters filled with 53.5, 114.8, 176.5, and 237.8 mg/L of FA treated sludge performed in the Section “SCFA generation from WAS pretreated with different FA levels ” (see Figure S1). The content of dextran and glucose in synthetic medium at different concentrations of FA were respectively utilized to show the influence of FA on hydrolysis and acidogenesis.

FA was found to decrease

the content of dextran and glucose in compared with the blank (or pH 9). As the FA concentration increased, the degradation ratios decreased. For instance, on 1 d of fermentation, the degradation ratios of dextran and glucose decreased from 45.2 ± 4.4% and 47.1 ± 1.5% to 32.5 ± 2.1% and 30.7 ± 1.3% respectively by FA pretreatment at 61.5 - 178.9 mg/L. Similar trend can be as well seen on 3 d of fermentation. However, it should be noticed that with 178.9 mg/L FA (i.e. the highest level), 76.1 ± 2.3% of dextran and 73.6 ± 2.2% of glucose were degraded at 3 d fermentation, indicating that the hydrolysis and acidogenesis was inhibited by

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the presence of FA to some extents, but the inhibition have no largely influence on the degradation of these substrates. Acetogenesis is relevant to the generation of acetic acid, and the impact of FA on acetogenesis was confirmed by analyzing the content of propionate in this work. When FA level enhanced from 61.5 to 178.9 mg/L, the degradation of propionate varied between 43.4 ± 2.6% and 40.4 ± 2.0% at 1 d fermentation and between 45.6 ± 2.0% and 43.2 ± 1.7% at 3 d fermentation. Compared with the blank, all these data had no significant variations (p > 0.05), suggesting that FA did not affect the acetogenesis significantly Apart from acetogenesis, acetic acid could be also produced via homoacetogenesis process, thus we further assessed the effect of FA on the homoacetogenesis process by measuring hydrogen consumption (i.e., Group-IV).

It was found that FA decreased both the consumption of hydrogen and the production of acetic

acid. On 1 d fermentation, hydrogen consumption and acetic acid production were respectively 24.0 ± 1.2% and 56.1 ± 3.1 mg/L in the blank reactor and 19.3 ± 3.1% and 50.6 ± 3.4 mg/L in the pH 9 reactor, while the corresponding data decreased from 18.6 ± 1.2% and 43.3 ± 2.7 mg/L to 10.5 ± 1.9% and 28.1 ± 2.3 mg/L, respectively, with FA concentration range from 61.5 to 178.9 mg/L (Table 1). Similar observation was observed on 3 d of fermentation as well. The results indicated that FA suppressed the homoacetogenesis. The influence of FA on methanogenesis process was evaluated as well due to the acetic acid produced was consumed on this process. Compared with either the blank or pH 9, FA decreased the degradation of acetate. Also, as the FA concentration increased, the degradation decreased. The results indicated that FA inhibited the methanogenesis process. In order to further clarify the FA’s influence on these processes, the correlative inhibition constant (Ks,i) as well was utilized to evaluate the impact of FA on the degradations of dextran, glucose, acetate, and hydrogen (Figure S2), and Table 2 summarizes the inhibition constant (Ks,i). As FA significantly unaffected the

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acetogenesis (Table 1), the inhibition of FA on the degradation of propionate was not considered here. From Table 2, for different fermentation substrates, the higher FA level, the lower Xs,i, which proved the FA suppress these processes. Moreover, Ks,i was in the order of acetate > hydrogen > glucose > dextran, indicating that the compared with other bioprocesses, methanogenesis was most sensitive, which benefited SCFA accumulation in the fermentation systems. SCFA generation from WAS pretreated with different FA levels

Figure 5a exhibits the profiles of SCFA generation from WAS pretreated with FA at different concentrations. The maximum SCFA yield obtained at 6 d in pH 9 reactor (169.4 mg COD/g VSS) was 2-fold of that achieved in the blank (84.6 mg COD/g VSS). When 3 d of FA pretreatment was applied, SCFA production was much greater than that from either the pH 9 or the blank reactor. When FA concentration ranged from 53.5 to 176.5 mg/L, the maximum production of SCFA achieved at 8 d (3 d pretreatment + 5 d fermentation) enhanced from 196.8 to 267.2 mg COD/g VSS.

When FA level was 237.8 mg/L, the maximum SCFA (226.9

mg COD/g VSS) generation showed a slight decrease, suggesting that the optimal FA pretreatment level was 176.5 mg/L.

From figure 5b, it can be clearly see that except for the blank, acetic acid was the predominant

SCFA while n-valeric was the lowest acid in each reactor. Further investigation exhibited that the average percentage of individual SCFA in these FA reactors was in the order of acetic > propionic > n-butyric > isobutyric > iso-valeric > n-valeric. COD mass balance analysis on the pH 9 and FA reactors at their optimal fermentation time was employed to further support the data presented in Figure 5.

The percentages of SCFA and hydrogen in all the

fermenters with FA addition were higher than those in the pH 9 fermenter, while the percentages of VSS and methane in the former fermenters were lower than those in the latter (Table S2). The results were consistent with the observation shown in Figure 5. It should be emphasized that the maximum SCFA yield from all the

15



Page 16 of 32

FA reactors investigated in this work was higher than that from pH 9 reactor. Additionally, pH in the FA reactors was controlled for only 3 days whereas constant pH control was required for the pH 9 reactor. All the facts presented above confirmed that using pertinent concentrations of FA to pretreat WAS for SCFA enhancement from anaerobic fermentation of WAS was feasible, and this FA-based strategy was easier controlled than alkaline fermentation, the widely accepted method for SCFA production. Implication for WWTPs

Most of the previous publications exhibited that FA would deteriorate the performance of anaerobic digestion[30-33]. Recently, however, Wei et al. reported that methane generation from anaerobic digestion of WAS pretreated with 420~680 mg NH3-N/L FA for 1 d was promoted by 22% [35].

This work

demonstrates that FA pretreatment can effectively promote SCFA yield, which expands the application potential of FA.

Furthermore, the current work also uncovers the details of how FA improves SCFA

generation. It was found that FA accelerated the breakdown of EPS and cell envelop of sludge cells, killed more live microbial cells, which thereby facilitated the organics released from WAS.

It was also

demonstrated that the presence of FA in the fermentation step resulted in severe inhibition to the SCFA consumption process (Table 1 and 2). Therefore, the findings obtained in paper also advanced the scientific understanding of the effect of FA on sludge anaerobic fermentation significantly. It is a very big challenge to achieve desirable nutrient removal and energy/resource recovery simultaneously in WWTPs, especially in such WWTPs treating wastewaters with low C/N ratios. The generation of SCFA from WAS may be a promising solution to this challenge, as the SCFAs produced can be utilized as not only preferred carbon sources for nutrient removal enhancement but also raw substrates for biodegradable plastic productions [48-50]. In previous study, several approaches such as ultrasonic, ozone, and alkaline were tested and proposed for enhancing SCFA production. Among them, alkaline fermentation

16


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is widely accepted to the most effective method, with the highest SCFA production (~250 mg COD/g VSS) achieved at constant pH 10 [13]. However, this method requires high input of alkalis and constant control of pH, which diminish the value of this method to some extents.

In comparison, the strategy of FA-based

pretreatment, as developed in this work, not only saved alkali input but also simplified process control. For example, the maximum generation of SCFA (~267 mg COD/g VSS) was obtained in the fermenter fed with 176.5 mg/L FA treated sludge, which is comparable to that obtained from the constant pH 10 fermentation. However, the pH in the FA-based strategy was required to be controlled at 9 for only 3 d (i.e., the pretreatment time). It should be noted that reducing pH from 10 to 9 would save around 50% alkalis. Thus, the strategy proposed in this work advanced the application of alkaline fermentation significantly, which may have substantial benefits for the operation of WWTPs in the future. To reflect this viewpoint directly, we propose a promoted “nutrient removal-energy recovery” notion with the developed FA-based strategy for the operation of a WWTP (Figure S3). In the conventional operation paradigm, some external carbon sources such as acetate, propionate, and alcohol are usually added to maintain desirable nutrient removal when internal carbon sources in wastewater are insufficient. The useful substrates in WAS such as phosphorus and protein are often squandered, e.g., via burying dewatered sludge in landfills, in many developing countries such as China (Figure S3a). In the new operation paradigm with FA-based strategy integrated, WAS generated is first pretreated with FA for 3 d after concentrating in a thickener. The pretreated WAS is then transferred to the fermenter for anaerobic fermentation, during which SCFA is produced and the amount of WAS is reduced. After fermentation, the released nitrogen and phosphorus in the fermentation process are recovered from the SCFA-containing fermentation liquid by the formation of struvite precipitation, which not only reduces the nitrogen and phosphorus loading but also compensates a part of operational cost of the WWTP. A portion of the produced SCFA is introduced to the head of the WWTP

17



as supplementary carbon sources for enhancement of biological nutrient removal while the remaining SCFA is used to produce biodegradable plastic (Figure 3b). Therefore, the desirable BNR, more energy and resource recovery and high WAS reduction can be achieved, which strongly support the operation of WWTPs in a sustainable way. According to the findings reported in this study and previous literature, it can estimate that compared with the conventional operation paradigm, the FA-supported method may save ~$1.7 million per year in a wastewater treatment plants (Q = 105 m3/day), the details can be seen on Table S3. ACKNOWLEDGEMENTS

This work was supported by National Natural Science Foundation of China (51508178 and 51779089,). Dr Qilin Wang acknowledges the supports of Australian Research Council Discovery Early Career Researcher Award (DE160100667).

ADDITIONAL INFORMATION This document includes methods of supplementary, Tables S1 - S3, and Figures S1 - S3.

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Page 25 of 32

FIGURES

(a)

4000

Soluble COD (mg/L)

3500 3000 2500

53.5 mg FA/L 114.8 mg FA/L 176.5 mg FA/L 237.8 mg FA/L pH 9 Blank

2000 1500 1000 500 0

0

1

2

3

4

Time (d)

(b)

20 18 16

VSS reduction (%)

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


14 12 10 8 6 4 2 0

Blank

pH 9

53.5

114.8

176.5 237.8

FA concentration (mg/L)

Figure 1. The contents of soluble COD during pretreatment time (a) and VSS reduction on 3 d pretreatment

time (b). Error bars is standard error of multiple tests.

25



(a)

Extracted COD by heating method Soluble COD

1600

Soluble COD (mg/L)

1400 1200 1000 800 600 400 200 0

(b)

0h 1h 2h

0h 1h 2h

0h 1h 2h

Blank reactor

pH 9 reactor

FA reactor

140 120

LDH release (% of control)

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

100 80 60 40 20 0

0h

1h

2h

1h

2h

1h 2h

Control Blank reactor pH 9 reactor FA reactor

Figure 2. The contents of the soluble COD and the extracted COD by heating method (a) and the release of

LDH (b) during pretreatment time. Asterisks indicate statistical differences (p < 0.05) from the 0 h. Error bars is standard error of multiple tests.

26


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Figure 3. Visual images of the BacLight test using the sludge from the blank (a), pH 9 (b), and FA reactor (c) after 24 h pretreatment.

Live bacteria (fluorescent green): a1, b1, and c1; Dead bacteria (fluorescent red): a2,

b2, and c2; Overlay images: a3, b3, and c3.

27



Figure 4. The release of soluble proteins (a) and carbohydrates (b) in the initial 10 h pretreatment, and EEM profiles of liquid (c) at 24 h pretreatment. Error bars is standard error of multiple tests.

28


Page 28 of 32

Page 29 of 32

StepⅠ

StepⅡ

SCFA production (mg COD/g VSS)

(a)

Blank pH 9 114.8 mg FA/L 237.8 mg FA/L

300

53.5 mg FA/L 176.5 mg FA/L

240 180 120 60 0 0

2

4

6

8

10

12

14

16

Time (d)

(b) 120

n-valeric iso-butyric

iso-valeric propionic

n-butyric acetic

90

Percentage (%)

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


60

30

0 Blank

pH 9

53.5

114.8

176.5

237.8

FA concentration (mg/L)

Figure 5. Effect of FA pretreatment at different concentrations on SCFA production (a) and the fraction of individual SCFA under their optimal fermentation conditions (b). Error bars is standard error of multiple tests. Step 1: the sludge was pretreated by FA in the FA reactors during which pH was maintained at 9.0 ± 0.1; Step 2: pH was not controlled in the FA reactors.

29


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

Page 30 of 32

TABLES Table 1. The effects of FA on hydrolysis, acidogenesis, acetogenesis, homoacetogenesis, and methanogenesis processesa Operation

FA

Time

concentration

(d)

(mg/L)b

1

3

Group-I

Group-II

Group-III

Group-IV

Group-V

Dextran

Glucose

Propionic acid

Hydrogen

Acetic acid

Acetate

degradation

degradation

degradation

consumption

production

Degradation

(%)

(%)

(%)

(%)

(mg/L)

(%)

Blank

53.0 ± 1.5

55.8 ± 2.8

45.7 ± 4.5

24.0 ± 1.2

56.1 ± 4.1

18.7 ± 1.3

pH 9

48.2 ± 1.3

47.5 ± 1.7

43.9 ± 2.9

19.3 ± 3.1

50.6 ± 2.4

16.4 ± 2.2

61.5

45.2 ± 4.4

47.1 ± 1.5

43.4 ± 2.6

18.6 ± 1.2

43.3 ± 5.7

10.5 ± 1.2

101.2

39.2 ± 2.0

39.9 ± 2.1

41.2 ± 1.8

16.3 ± 2.6

39.2 ± 1.5

8.7 ± 2.6

138.8

33.6 ± 5.7

32.8 ± 2.4

40.5 ± 3.3

13.9 ± 1.4

31.9 ± 1.9

7.1 ± 1.1

178.9

32.5 ± 2.1

30.7 ± 1.3

40.4 ± 2.0

10.5 ± 1.9

28.1 ± 2.3

4.6 ± 1.5

Blank

89.5 ± 4.5

89.8 ± 4.4

48.8 ± 2.1

56.8 ± 4.4

151.9 ± 17.8

47.3 ± 3.9

pH 9

83.3 ± 3.0

82.1 ± 4.0

46.7 ±4.0

54.9 ± 3.8

140.8 ± 4.1

35.1 ± 3.3

61.5

81.3 ± 5.2

75.2 ± 2.5

45.6 ± 2.0

52.6 ± 3.2

130.0 ± 13.8

26.2 ± 2.8

101.2

80.3 ± 2.5

74.2 ± 3.9

45.1 ± 1.8

48.6 ± 2.7

110.2 ± 16.8

20.8 ± 3.1

138.8

77.1 ± 3.7

74.1 ± 3.2

44.4 ± 2.9

41.4 ± 4.3

102.5 ± 8.2

17.6 ± 2.5

178.9

76.1 ± 2.3

73.6 ± 2.2

43.2 ± 1.7

40.9 ± 1.4

95.3 ± 10.2

12.3 ± 3.1

a

Results are the averages and standard deviations of triplicate tests.

b

The FA levels utilized in this batch experiment, i.e., 61.5, 101.2 138.8, and 178.8 mg/L, were respectively the average FA levels determined from the fermenters filled with 53.5, 114.8, 176.5,

and 237.8 mg/L of FA treated sludge.

30


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Table 2. The inhibition kinetic parameters for FA inhibiting the degradations of dextran, glucose, propionate, hydrogen,

and acetate Xs,ia Substrate Dextran

101.2

138.8

178.9

(mg FA/L)

(mg FA/L)

(mg FA/L)

(mg FA/L)

0.5679

0.5152

0.4398

0.4267

Ks,ic 256.4

Glucose

0.7218

0.4916

0.4182

0.3872

0.3523

208.3

0.2839 0.2112

0.2345 0.1043

0.2135 0.0816

0.1754 0.0654

0.1522 0.0441

147.3 44.4

Xs,i is the degradation of the substrate when different FA concentration are added, and the unit is g/(L•d)

b c

0.7318

61.5

Hydrogen Acetate a

Xs,ob

Xs,o is the degradation of the substrate without the FA addition, and the unit is g/(L•d)

Ks,i is the related inhibition constant of FA, and the unit is mg/L

31



Graphic abstract

Synopsis: An innovative operation concept of a WWTP with FA-based pretreatment technology for enhancing both nutrient removal and resource recovery.

32


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Free Ammonia-Based Pretreatment Promotes Short-Chain Fatty Acid

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