The Influences of PHA and Gram-Staining - ACS Publications

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Short-Chain Fatty Acid Production from Different Biological Phosphorus Removal Sludges: The Influences of PHA and GramStaining Bacteria Dongbo Wang, Yinguang Chen,* Xiong Zheng, Xiang Li, 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: Recently, the reuse of waste activated sludge to produce short-chain fatty acids (SCFA) has attracted much attention. However, the influences of sludge characteristics, especially polyhydroxyalkanoates (PHA) and Gram-staining bacteria, on SCFA production have seldom been investigated. It was found in this study that during sludge anaerobic fermentation not only the fermentation time but also the SCFA production were different between two sludges, which had different PHA contents and Gram-negative bacteria to Gram-positive bacteria (GNB/ GPB) ratios and were generated respectively from the anaerobic/oxic (AO) and aerobic/extended-idle (AEI) biological phosphorus removal processes. The optimal fermentation time for the AEI and AO sludges was respectively 4 and 8 d, and the corresponding SCFA production was 304.6 and 231.0 mg COD/g VSS (volatile suspended solids) in the batch test and 143.4 and 103.9 mg COD/g VSS in the semicontinuous experiment. The mechanism investigation showed that the AEI sludge had greater PHA content and GNB/GPB ratio, and the increased PHA content accelerated cell lysis and soluble substrate hydrolysis while the increased GNB/GPB ratio benefited cell lysis. Denaturing gradient gel electrophoresis profiles revealed that the microbial community in the AEI sludge fermentation reactor was dominated by Clostridium sp., which was reported to be SCFA-producing microbes. Further enzyme analyses indicated that the activities of key hydrolytic and acids-forming enzymes in the AEI sludge fermentation reactor were higher than those in the AO one. Thus, less fermentation time was required, but higher SCFA was produced in the AEI sludge fermentation system.

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more than two folds than the sole alkaline treatment (pH 10).11 The contents of sludge protein and carbohydrate, which are the two predominant organic compounds in sludge, significantly influenced SCFA generation,14 and SCFA production could be remarkably enhanced by the optimization of protein to carbohydrate ratio in sludge fermentation system.15 Apart from the operational conditions and the ratio of protein to carbohydrate, it is observed in our current study that the SCFA production is also affected by sludges with varied PHA contents and Gram-negative bacteria to Gram-positive bacteria (GNB/GPB) ratios which were generated from different biological phosphorus removal (BPR) processes. The classical BPR technology (i.e., the anaerobic/oxic (AO) process) is extensively investigated and widely applied.16 Since PHA is consumed for oxic phosphorus uptake, there is very low PHA contents left in sludge after phosphorus is taken up. Recently, another BPR technique (i.e., the aerobic/extendedidle (AEI) biological wastewater treatment process) has been

INTRODUCTION As a byproduct of biological wastewater treatment, excess sludge is inevitably produced in large quantities. Excess sludge is usually treated by the anaerobic digestion process to produce methane.1,2 Recently, the production of short-chain fatty acids (SCFA) from sludge has attracted growing concerns owing to the facts that the produced SCFA is a preferred carbon source for biological nutrient removal microbies or a raw material for microbial production of biodegradable plastics (such as polyhydroxyalkanoates (PHA)).3−6 Anaerobic SCFA production from sludge depends on two factors: the operating conditions of fermentation reactors and the characteristics of sludge. To date, however, most of the studies regarding SCFA generation from sludge have focused on the former. Several operating parameters, such as pH, temperature, and surfactant, have been reported to affect SCFA production.7−10 For example, compared with the control the SCFA production was improved by more than three times when sludge was fermented at pH 10.7 Besides, treating sludge by ultrasonic,11 ozone,12 and enzyme addition13 were also reported to obviously improve the SCFA production. It was found that combining the ultrasonic pretreatment and alkaline adjustment (pH 10) could increase the SCFA generation by © 2013 American Chemical Society

Received: Revised: Accepted: Published: 2688

November 15, 2012 January 31, 2013 February 11, 2013 February 11, 2013 dx.doi.org/10.1021/es304673s | Environ. Sci. Technol. 2013, 47, 2688−2695

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Table 1. Characteristics of the AO-Sludge and AEI-Sludgea parameter

unit

AO-sludge

AEI-sludge

total suspended solids volatile suspended solids total COD total protein total carbohydrate PHAb lipid and oil sludge TP content total bacteria GNB/GPB

mg/L mg/L mg/L mg/g VSS mg/g VSS mg/g VSS mg/g VSS mg P/g TSS copies/g VSS

14236 ± 396 11674 ± 215 14960 ± 230 556 ± 16 256 ± 12 25 ± 2 5.7 ± 0.5 55 ± 3 (3.24 ± 0.27) × 1010 2.19 ± 0.44

13292 ± 231 10634 ± 175 13720 ± 210 571 ± 20 152 ± 8 116 ± 5 5.6 ± 0.4 58 ± 5 (3.12 ± 0.31) × 1010 3.81 ± 0.73

a

Results are the averages and their standard deviations of triplicate measurements. bThe percentages of PHB, PHV, and PH2MV are 48.3%, 40.9%, and 10.8% of the total PHA in the AO-sludge and 52.8%, 41.8%, and 5.0% of the total PHA in the AEI-sludge, respectively.

sedimentation tank of a municipal wastewater treatment plant in Shanghai, China, and was simultaneously inoculated into two identical sequencing batch reactors (SBR) with a working volume of 50 L each. One was operated with the AEI regime, and the other was performed with the conventional AO regime. Both SBRs were operated with three cycles per day. According to the literature, AEI-SBR cycle consisted of a 210 min aerobic period, a 55 min settling period, 5 min decanting, and 210 min idle periods,18 while AO-SBR cycle consisted of a 120 min anaerobic period and a 180 min aerobic period, followed by 55 min settling, 5 min decanting, and 120 min idle periods.24 After settling period 25 L supernatant was discharged from both SBRs and was replaced with 25 L synthetic medium (detailed composition was listed below) during the first 5 min of aerobic period (AEI-SBR) and anaerobic period (AO-SBR), respectively. During the aerobic period, air was supplied into both SBRs at a flow rate of 50 L/min, and during the anaerobic period AO-SBR was mechanically stirred. The hydraulic retention time in the two SBRs was 16 h, while the sludge retention time (SRT) was maintained at approximately 14 d. Both SBRs were operated for over 240 d. In the AO-SBR, excess sludge was approximately wasted at the end of the aerobic period. After operation for 60 d the AO-SBR reached relatively stable, and then the wasted sludge was used for the following sludge fermentation tests. In the AEI-SBR, sludge was either discharged at the end of aerobiosis during the initial 80 d (step I) or wasted after 1 h of aeration during the remainder of the test (step II). After the effluent phosphorus concentration in step I and step II did not significantly change with operational time, the reactor performance was compared, and the evaluation of sludge discharging time on biological nutrient removal was made. The results in Table S1 (Supporting Information) showed that the removals of nitrogen and phosphorus in the AEI-SBR were not significantly affected by the time of sludge wasted (p > 0.05). Also it can be seen from Table S1 that the nutrient removal efficiency in the AEI-SBR was comparable with that in the AO-SBR. Thus, in the following sludge fermentation tests the AEI-sludge, unless otherwise described, was withdrawn from the AEI-SBR after aeration for 1 h. The synthetic medium was prepared daily and contained 265 mg of CH3COONa/L, 80 mg of CH3CH2COONa/L, and 10 mg of PO4−P/L, yielding an influent chemical oxygen demand (COD): PO4−P ratio of 30 mg COD/mg PO4−P. The concentrations of other nutrients in the synthetic medium were presented as below (per liter): 0.1 g of NH4Cl, 0.01 g of MgSO4·7H2O, 0.005 g of CaCl2, and 0.5 mL of a trace metals

reported to achieve a satisfied phosphorus removal but with different inducing mechanisms.17,18 In the AEI process a strict anaerobic stage is not required, whereas an extended-idle period is operated (210−450 min) between the decanting and the next aerobic phases. During the initial 45−60 min of aerobic period a substantial quantity of PHA is synthesized, but a very low phosphorus is released.18 Fluorescent dye 4′,6diamidino-2-phenylindole (DAPI) staining further reveals that the sludge sample withdrawn at 1 h aeration contains a large number of intracellular polyphosphate granules (42.3 ± 5.5%, Figure S1, Supporting Information). The metabolic characteristic studies indicate that the AEI system can achieve a satisfied BPR performance together with the generation of PHA-rich excess sludge. PHA, an intracellular metabolic intermediate, can be rapidly and completely degraded under anaerobic conditions.19,20 Though the anaerobic degradation of PHA as an exogenous plastic material has been studied previously,19−21 the effect of varied PHA contents in sludges (i.e., PHA as an intracellular polymer) on anaerobic SCFA production has never been documented. In addition, our current study shows that the ratio of GNB/GPB in excess sludge generated from the AEI process is also higher than that generated from the AO process. It is known that Gram positive bacteria differ from Gram negative bacteria in the structure of their cell walls, and the thickness and length distribution as well as the cross-linking degree of peptidoglycan layer in Gram-positive cells is more extensive than those in Gram-negative cells.22,23 However, the potential impact of different GNB/GPB ratios in sludges on anaerobic SCFA production has not been reported. The purpose of this paper was to investigate the influences of two sludges with different PHA contents and GNB/GPB ratios, generated respectively from the AO and AEI wastewater BPR processes (hereinafter to be referred as the AO-sludge and AEIsludge, respectively), on SCFA production in both batch and semicontinuous tests at pH 10, which was demonstrated to be an efficient strategy for SCFA accumulation during sludge anaerobic fermentation.7 Also, the reasons for sludge PHA content and GNB/GPB ratio influencing the efficiency and rate of SCFA production were explored via the analysis of PHA degradation, bacterial cell disruption, diversities of microbial communities, and activities of key enzymes.

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MATERIALS AND METHODS The Sources of the AO-Sludge and AEI-Sludge. Two BPR (AEI and AO) bioreactors were operated to culture the sludges. Seed sludge was obtained from the secondary 2689

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Batch Experiment of the Effect of Sludge GNB/GPB Ratio on Cell Lysis. From Table 1 it can be seen that besides GNB/GPB ratio, sludge PHA content was also different between the AO-sludge and AEI-sludge. It was reported that cells with higher PHA content became more fragile,26 which might influence cell lysis. To eliminate the PHA influence, in this batch test the excess sludge from the AEI-SBR was withdrawn at the end of aerobiosis (this sludge was defined as AEI-sludge-I). After concentrating at 4 °C for 12 h, it was detected that the AEI-sludge-I contained 10821 ± 197 mg/L VSS, 13990 ± 240 mg/L total COD, 581 ± 10 mg/g VSS total protein, 225 ± 9 mg/g VSS total carbohydrate, 26 ± 3 mg/g VSS PHA, (3.19 ± 0.35) × 1010 copies/g VSS total bacteria, and 3.77 ± 0.58 GNB/GPB ratio. By comparing the above data with those shown in Table 1, it can be found obviously that the AEI-sludge-I and AO-sludge contained different GNB/GPB ratios but approximately the same PHA content. The fermentation conditions were also the same as those depicted in the “SCFA Production from the AO-sludge and AEI-sludge” section. Analytical Methods. The analyses of COD, NH4+-N, NO3−-N, NO2−-N, PO4−-P, VSS, and total suspended solid (TSS) were conducted in accordance with standard methods.27 The measurements of sludge total phosphorus (TP), glycogen, PHA, SCFA, protein, carbohydrate, and lipid were the same as described in our previous publications.4,7 The activities of key hydrolytic enzymes (α-glucosidase and protease) were measured according to the method proposed by Goel et al.,28 and the key acid-forming enzymes (phosphotransacetylase (PTA), phosphotransbutyrylase (PTB), acetate kinase (AK), butyrate kinase (BK), oxaloacetate transcarboxylase (OAATC), and CoA transferase) were determined according to the methods reported in our previous publication.15 Molecular weight (Mw) distribution of the fermentation liquid was determined by gel-filtration chromatography analyzer (Shimadzu Co., Japan) according to the literature.29 Microbial extracellular polymeric substances (EPS) including loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS), humic acids, and DNA of activated sludge were measured according to the method described previously.30 Identification of the gram positive and negative bacteria in sludges was performed by classical gram staining. DAPI staining was used to determine the abundance of polyphosphate granules, and fluorescence in situ hybridization (FISH) was employed to quantify polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) in the AO-sludge and AEI-sludge. The microbial communities of two semicontinuous fermentation systems were assayed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technique, and the quantification of total bacteria in sludges was analyzed by the quantitative realtime PCR. All these methods were detailed in the Supporting Information. The dissociation and standard curves of the primers for total bacteria assay are provided in Figure S2 (Supporting Information). The oligonucleotide probes specific for PAO, GAO, and total bacteria are listed in Table S3 (Supporting Information). The sequences reported in this study have been deposited in the GenBank database under accession numbers JX484774 to JX484786. The closest matching sequences were searched using the BLAST program. Statistical Analysis. All measurements were performed in triplicate. An analysis of variance was used to evaluate the

solution. The trace metals solution was described in our previous publication.25 Characteristics of the AO-Sludge and AEI-Sludge. The excess sludge used in the following anaerobic fermentation tests was respectively withdrawn from the AO-SBR (at the end of aerobiosis) and AEI-SBR (at 1 h aeration) operated above and was concentrated at 4 °C for 12 h before use. The main characteristics of these concentrated sludges are detailed in Table 1. The top three organic compounds are proteins, carbohydrates, and PHA in the AO-sludge, while those are proteins, PHA, and carbohydrates in the AEI-sludge. Clearly, the AEI-sludge had a greater PHA content than the AO-sludge. Additionally, though the abundance of total bacteria was approximate in the two sludges, the AEI-sludge had a higher GNB/GPB ratio. SCFA Production from the AO-Sludge and AEISludge. It was reported previously that alkaline conditions were beneficial to SCFA generation from excess sludge, and the optimal pH was 10.7 Thus all the batch and semicontinuous tests in this study were conducted at this pH. The batch experiments were performed in two identical reactors with working volumes of 0.8 L at constant temperature (21 ± 1 °C) according to the method described previously.7 The long-term semicontinuous tests were conducted in two identical reactors with a working volume of 0.8 L each. The two reactors received 0.8 L of the AEI-sludge and AO-sludge, respectively, and the fermentation conditions were the same as described above. According to the results of batch tests the SRT was respectively maintained at 4 d and 8d in the AEI-sludge and AO-sludge fermentation reactors. Batch Test of the Effect of Sludge PHA Content on Cell Disruption, Solubilized Substrate Hydrolysis, and SCFA Production. The sludges used in this batch fermentation experiment should contain different sludge PHA contents but similar other characteristics such as sludge phosphorus content and GNB/GPB ratio to minimize the potential impacts of these parameters on SCFA generation. To obtain these sludges, the following tests were carried out. A total of 4.8 L sludge was taken from an aerobic activated sludge system in our laboratory which was used for PHA synthesis without enhanced biological phosphorus removal. The mixture was centrifuged at 4000 rpm for 5 min to remove the supernatant, and the sludge was resuspended in tap water with a final volume of 4.8 L before being divided equally into four reactors with a working volume of 1.2 L each. Influent COD concentrations (acetate to propionate rate was the same as the above synthetic medium) in these reactors were 150, 200, 250, and 300 mg/L, respectively. All other operational conditions were the same as those depicted above in the AEI process. After 1 h aeration, all sludge mixtures were centrifuged (4000 rpm for 5 min), then each sludge was resuspended in tap water with a volume of 0.4 L before being transferred into four identical fermentation reactors (the PHA content in these sludges was respectively 18 ± 1, 49 ± 3, 74 ± 4, and 105 ± 6 mg/g VSS (volatile suspended solids), and the main characteristics of these sludges were detailed in Table S2, Supporting Information). Clearly, a significant difference among these sludges was that they contained different PHA contents. The fermentation conditions were the same as those described in the “SCFA Production from the AO-sludge and AEI-sludge” section. It took about 180, 156, 144, and 120 h for these sludges to reach the maximal SCFA production, respectively. 2690

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Figure 1. Comparison of the total SCFA production from the AO-sludge and AEI-sludge at different fermentation times (A) and the fraction of individual SCFA under the optimal conditions (B). Ace: acetic acid; Pro: propionic acid; i-But: iso-butyric acid; n-But: n-butyric acid; i-Val: isovaleric acid; n-Val: n-valeric acid. Results are the averages and their standard deviations of triplicate measurements.

significance of results, and p < 0.05 was considered to be statistically significant.

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Table 2. Comparison of Individual and Total SCFAs Production from the AO-Sludge and AEI-Sludge in the Semicontinuous Fermentation Tests after Steady-State Operationa

RESULTS AND DISCUSSION

Comparison of SCFA Production from the AO-Sludge and AEI-Sludge. As seen from Figure 1A in the AO-sludge reactor the maximal SCFA (231.0 mg COD/g VSS) appeared at time of 8 d. In the literature the maximal SCFA production and optimal fermentation time were respectively around 250 mg COD/g VSS and 8 d when sludge produced in the traditional biological nutrient removal process was fermented at pH 10,6,7 which were in agreement with the current study when the AO-sludge was fermented. During fermentation of the AEIsludge, as shown in Figure 1A, the SCFA was increased with time and reached the maximum at time of 4 d, which, however, was not significantly increased with further increasing the time from 4 to 10 d (p > 0.05). Obviously, the optimal fermentation time for producing SCFA from the AEI-sludge was 4 d, and the corresponding maximal SCFA was 304.6 mg COD/g VSS, which was significantly higher than that produced from the AOsludge (231.0 mg COD/g VSS). It should be emphasized that the SCFA produced from the AO-sludge at time of 4 d was only 155.7 mg COD/g VSS, which was approximately half of that produced from the AEI-sludge. Clearly, the AEI-sludge showed much greater SCFA production efficiency and much faster SCFA production rate than the AO-sludge. Further analysis showed that under the optimal conditions acetic acid was the most abundant SCFA in both AO- and AEI-sludge fermentation systems, and there was no significant difference in the percentage of individual SCFA (Figure 1B). Table 2 illustrates the individual and total SCFA production in two long-term semicontinuous sludge fermentation reactors after stable operation. The average SCFA production was 143.4 mg COD/g VSS in the AEI-sludge reactor, while it was only 103.9 mg COD/g VSS in the AO-sludge reactor. Apparently, even in the long term experiments the AEI-sludge still showed remarkably higher SCFA production than the AO-sludge. As can also be seen from Table 2 two reactors had almost the same SCFA composition. By comparing the data in Figure 1 and those in Table 2, it can be seen that the SCFA productions in the long term experiments were lower than those in the batch tests. The same observation was made in the literature. For example, Yuan et al. reported that about 250 mg COD/g VSS of SCFA was produced in batch tests, whereas Feng et al. found

acetic acid propionic acid iso-butyric acid n-butyric acid iso-valeric acid n-valeric acid total SCFA

AO-sludge

AEI-sludge

43.7 ± 2.1 18.5 ± 1.1 8.4 ± 0.4 15.2 ± 0.5 10.8 ± 0.8 7.3 ± 0.4 103.9 ± 5.2

62.5 ± 1.8 21.1 ± 0.4 10.0 ± 0.2 26.2 ± 1.4 16.3 ± 1.1 7.2 ± 0.1 143.4 ± 2.9

a

The data are the averages and their standard deviations of triplicate measurements, and the unit is mg COD/g VSS. The average percentages of acetic, propionic, iso-butyric, n-butyric, iso-valeric, and n-valeric acids in the AO-sludge reactor were respectively 42.1%, 17.8%, 8.1%, 14.6%, 10.4%, and 7.0%, which were 43.6%, 14.7%, 7.0%, 18.3%, 11.4%, and 5.0% in the AEI-sludge reactor.

that only 95 mg COD/g VSS of SCFA was generated in semicontinuous long-term experiments.7,31 All the above results clearly showed that use of the AEI-sludge, as compared with the AO-sludge, to produce SCFA not only significantly enhanced SCFA production but also greatly accelerated SCFA accumulation. In the following text the reasons for the AEI-sludge exhibiting higher SCFA production efficiency and greater SCFA production rate were explored. Mechanisms of the AEI-Sludge Showing Greater SCFA Production Efficiency and Rate than the AO-Sludge. The previous studies showed that the production of SCFA at pH 10 was directly relevant to the metabolism of sludge protein and carbohydrate.7 Apart from protein and carbohydrate, as seen in Table 1, PHA was also one of the main organic compounds in the two sludges, which was observed to be decreased with time during the fermentation of both the AEI-sludge and AO-sludge (Figure S3, Supporting Information). As PHA in the AEIsludge was much higher than that in the AO-sludge (116 versus 25 mg/g VSS), its influence on SCFA production was investigated first. PHA can be biologically converted to SCFA under anaerobic conditions.19,21 As is known that PHA is an intracellular product. Before it is fermented to produce SCFA, the microbial cell needs to be disrupted. The cell disruption can be estimated by the measurement of intracellular substrate release.32 Since 2691

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the sludges used in the batch test of the effect of sludge PHA content on SCFA generation had almost the same protein and carbohydrate contents in EPS (Table S2, Supporting Information), the variations of soluble protein (carbohydrate) to total protein (carbohydrate) ratio was applied in this study to indicate cell breakage. As seen in Table 3, with the increase Table 3. Ratio of Soluble Protein (Carbohydrate) to Total Protein (Carbohydrate) at 1 d of Fermentation Time in the Batch Fermentation Tests Using Sludge with Different PHA Contenta PHA content in original sludge (mg/g VSS) 18 ± 1 49 ± 3 74 ± 4 105 ± 6

soluble protein to total protein (%)

soluble carbohydrate to total carbohydrate (%)

± ± ± ±

4.3 ± 0.3 6.1 ± 0.4 9.3 ± 1.1 11.5 ± 1.1

13.9 15.6 19.8 22.6

0.8 1.1 1.5 1.9

The data are the averages and their standard deviations of triplicate measurements.

Figure 2. The relationship between sludge PHA content and percentages of soluble substrates with Mw < 1000 at 2 d of fermentation time. The data are the averages and their standard deviations of triplicate measurements.

of PHA both the ratios of soluble protein to total protein and soluble carbohydrate to total carbohydrate were increased. It was reported that the microbial cells became more fragile as there is more intracellular PHA.26 From the current study it is obvious that the increase of sludge PHA was beneficial to cell disruption, which thereby caused more soluble substrate productions for subsequent acidification in the increased sludge PHA fermentation systems. In this study PHB and PHV are the main composition of PHA (they accounted for around 90% of total PHA in both sludges, Table 1), and the metabolic pathways for anaerobically converting PHB and PHV to SCFA under anaerobic conditions are proposed in Figure S4 (Supporting Information). The polymer PHA is first degraded to monomers such as 3hydroxybutyrate and 3-hydroxyvalerate by depolymerases, and then the monomers are activated by CoA transfer. The resultant 3-hydroxybutyryl-CoA and 3-hydroxyvaleryl-CoA are further metabolized to acetyl-CoA, propionyl-CoA, butyrylCoA, and valeryl-CoA, which are finally bioconverted to acetate, propionate, butyrate, and valerate, respectively. It was noted that the percentages of PHB and PHV in the AO-sludge were almost the same as those in the AEI-sludge (Table 1), thus the influence of PHA composition on SCFA production was not considered in this study. The batch fermentation experiment using activated sludges with different PHA contents showed that most of sludge PHA polymer was decomposed during the initial 2 d of fermentation time no matter what the original PHA content in sludge was, and at this fermentation time SCFA generation was found to be unaffected by sludge PHA content (Table S4, Supporting Information). However, further study revealed that the percentages of small soluble substrates (Mw < 1000) at 2 d of fermentation time exhibited well positive correlation with sludge PHA content (R2 = 0.97, Figure 2). Due to the fact that soluble substrates in these fermentation reactors contained nearly the same amount of SCFA (Table S4, Supporting Information), it seems that the hydrolysis process of solubilized substrates was accelerated when sludge PHA content was increased. Moreover, since the sludge with higher PHA content had lower protein and carbohydrate (Table S2, Supporting Information), it can be deduced that the anaerobic hydrolysis rate of PHA was faster than that of other main cell constituents

such as protein and carbohydrate. It is known that before the solubilized organic matters with large Mw are utilized to produce SCFA, they require to be hydrolyzed. If the hydrolysis rate is accelerated, the fermentation time will be saved. Therefore, it can be easily understood that when the AEIsludge and AO-sludge was fermented, respectively, the former produced more SCFA but required less fermentation time as its PHA content was higher. As shown in Table S5 (Supporting Information), the increase of sludge PHA not only shortened SCFA accumulation time but also improved SCFA production, which directly supported the above deduction. Though the abundance of total bacteria in the AO-sludge and AEI-sludge was almost the same, it can be seen from Table 1 that the two sludges had different ratios of GNB/GPB, which was another obvious difference between them. Gram positive bacteria had different cell wall structure from Gram negative bacteria, thus the potential impact of sludge GNB/GPB ratio on SCFA generation was also investigated. A batch test using two sludges (i.e., the AO-sludge and AEI-sludge-I) with different GNB/GPB ratios (2.19 ± 0.44 versus 3.77 ± 0.58) but with similar PHA contents (25 ± 2 versus 26 ± 3 mg/g VSS) and total bacteria amounts ((3.24 ± 0.27) × 1010 versus (3.19 ± 0.35) × 1010 copies/g VSS, Figure S5A and S5B), was performed. The results in Figure 3A showed that the ratios of soluble protein to total protein and soluble carbohydrate to total carbohydrate in the AEI-sludge-I were much greater than those in the AO-sludge. As the AO-sludge and AEI-sludge-I had almost the same EPS quantity and constituent (Table S6, Supporting Information), it seems that cell lysis in the AEIsludge-I was easier than that in the AO-sludge. This deduction can be further supported by the measurement of VSS reduction (Figure 3B). It was found that the VSS reduction in the AEIsludge-I was also higher that in the AO-sludge. In the literature, it was observed that the disruption of model Gram positive bacteria (e.g., Bacillus subtilis) is harder than that of Gram negative bacteria (e.g., Escherichia coli) due to the stronger peptidoglycan barrier,22,23 which was in accord with the observation in our study. It is known that Gram positive cell walls are composed of a thick (20−80 nm) and multilayered peptidoglycan sheath, while Gram negative cell walls contain an outer membrane that surrounds a thin (1−7 nm) peptidoglycan

a

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Figure 3. Comparison of the soluble protein and carbohydrate release ratios (A) and VSS reduction (B) when the AEI-sludge-I and AO-sludge were fermented for 1 d. Error bars represent standard deviations of triplicate measurements.

layer (Figure S5C and S5D).22 It has been reported that the strength and rigidity of cell wall results from a layer of peptidoglycan, which is a covalent macromolecular structure of stiff glycan chains that are cross-linked by flexible peptide bridges.33 Thus, the increase of GNB/GPB accelerates sludge cell lysis, which thereby provides more substrates for subsequent acidification. It can be concluded that another reason for greater SCFA production observed in the AEI-sludge fermentation reactor was due to the increased GNB/GPB ratio. SCFA production from sludge at pH 10 was mainly dominated by biological effects,7 and microbial community assay was proven to be a useful tool for understanding the performance of anaerobic SCFA generation systems.8 Therefore, the reason for the AEI-sludge showing greater SCFA production was also explored by determining the microbial community via PCR-DGGE analysis, and the results were shown in Figure 4 and Table S7 (Supporting Information). The AO-sludge reactor contained some typical SCFA-producing microbes such as Paludibacter sp. (band 1) and Clostridium sp. (band 3).34,35 Also from Figure 4, it was found that the bacterial community structures involved in the AO-sludge and AEI-

sludge reactors showed significantly different. Clostridium sp. (bands 3, 6, and 8) was the predominant bacterium in the AEIsludge reactor, which was reported to be able to produce acetate under obligately alkaliphilic.35 Band 4 was affiliated to Alkalif lexus imshenetskii which was reported to be capable of growing at high pH values with acetate, propionate, and succinate as the main fermentation products.36 This bacterium was found to be present in the AEI-sludge reactor but was outcompeted in the AO-sludge reactor. Bacillus sp. (band 7), which was reported to be the dominant bacteria in microbial fermentation systems and able to produce and secrete large quantities of extracellular enzymes,37 appeared in the AEIsludge reactor but not in the AO-sludge reactor. Accordingly, the observed SCFA production in the AEI-sludge fed reactor was higher than that in the AO-sludge fed reactor. The generation of SCFA during sludge anaerobic fermentation at pH 10 is mainly relevant to sludge hydrolysis and acidification because the process of methanation is inhibited.7 Measurement of enzymes activities is an alternative method to assess microbial activity.38 Thus, the activities of key hydrolytic and acid-forming enzymes were further assayed to reveal the microbial activities of key hydrolytic and SCFA-producing microbes. Protease and α-glucosidase have been reported to play main roles in hydrolysis of protein and carbohydrate, respectively.28 As shown in Table 2, acetic, propionic, and butyric acids are the three major SCFA in both fermentation reactors. Thus, only some key enzymes relevant to these three SCFA generations were measured. PTA and AK are the key enzymes responsible for the transformation of acetyl-CoA to acetic acid, while PTB and BK are the key enzymes that convert butyryl-CoA to butyric acid.15 Additionally, OAATC and CoA transferase are directly related to propionic acid production.15 As seen in Figure 5, the activities of protease, α-glucosidase, PTA, AK, PTB, BK, OAATC, and CoA transferase in the AEIsludge reactor were higher than those in the AO-sludge reactor during the long-term semicontinuous tests, which was consistent with the results of SCFA production in these two sludge fermentation reactors.

Figure 4. DGGE profile of bacterial communities in two semicontinuous sludge fermentation reactors after stable operation. 2693

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Figure 5. Comparison of the relative activities of key hydrolytic and acid-forming enzymes between the two semicontinuous fermentation reactors after stable operation. Error bars represent standard deviations of triplicate measurements.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

This file contains additional analytical methods, Tables S1−S7, and Figures S1−S5. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Phone: +86-21-65981263. Fax: +86-21-65986313. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Hi-Tech Research and Development Program (2011AA060903), NSFC (51178324 and 51278354), Shanghai Postdoctoral Scientific Program (12R21415700), and China Postdoctoral Science Foundation (2012M510888).

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