3.1 Enrichment of PHAs dominant strains and microbial community succession in activated sludge
3.1.1 Conventional index analysis
Due to the low content of carbon sources in urban sewage in southern China, the activated sludge gradually adapted to a high concentration of carbon sources after recovery and stabilization. Figure 3(a) shows the phosphorus content in the water at the end of the anaerobic-oxygen limiting stages during the enrichment period. PAOs in activated sludge releases intracellular phosphorus during the anaerobic phase and absorbs excess phosphorus in solution during the aerobic phase. The phosphorus content was increasing at the end of the anaerobic phase and was kept below 1 mg/L in effluent at the oxygen-limiting section during the period of sludge recovery. The phosphorus removal rate reached 99.8%, which suggests PAOs were enriched and their phosphorus removal ability was excellent.
With the rector running and the carbon source concentration increasing, SVI decreased from 155 to 72 and MLSS risen from 3800 to 4700. And the color of sludge becoming yellow. As shown in Fig. 3(b), the growth rate of activated sludge shows an increasing trend in the enrichment period, indicating that activated sludge had good adaptability to high carbon sources. It is conducive to the subsequent accumulation of PHAs and the recovery of PAOs.
The growth of activated sludge indicates that the carbon source was absorbed by the PHAs-accumulating bacteria and then assimilated for their growth and proliferation. There are two main ways for carbon sources to be absorbed:①being stored as internal carbon source PHAs;②being assimilated by cells for their growth and proliferation. For the process of PHAs accumulation, more carbon sources will be used for PHAs synthesis rather than cell proliferation [19].
During the enrichment of PHAs-accumulating bacteria, the increasing COD concentration improves the tolerance of high-load carbon of sludge, and then efficient PHAs-accumulating bacteria can be accumulated. As shown in Fig. 4, acetate was rapidly absorbed by microorganisms in the anaerobic section of this cycle. The COD concentration quickly decreased in the first 0.5h and was almost removed by the end of the anaerobic phase, while the content of PHAs and TP gradually increased. The oxygen-limiting section continues until the end of the cycle, accompanied by the decrease of TP and the consumption of PHAs. Though the conditions of influent substrate concentrations were different, the proportion of PHB is all about 75%. The PHAs synthesized by acetate as a carbon source are mainly PHB, and similar research results have also appeared in the report of Hong Chen[20]. PHV and PHB have the same trend. PHAs content gradually increased in the anaerobic stage, reaching a maximum of 23.9% at the end of the anaerobic phase, and was progressively consumed in the oxygen-limited section, and the final content was 7.28%.
According to the Mino model[21, 22]and Smolders model for acetate absorption[23], during the anaerobic section, the degradation of intracellular polyphosphate and a small amount of glycogen are hydrolyzed by PAOs to produce ATP and reducing power when acetate is the substrate. The energy is used for active transport to absorb external carbon sources such as VFAs. Then the acetate is converted to the precursor of PHB, acetyl CoA, and a small amount of the precursor of PHV, propionyl CoA. A large amount of phosphate that cannot be used for intracellular synthesis is accumulated and then diffuses to the water in the form of active transport, causing the TP to increase in the solution. In the aerobic section, O2 is used by PAOs to oxidize PHAs as the electron acceptor. The energy produced by the degradation of PHAs and the reducing power provided by reducing coenzyme II (NADH2) are used to actively transport phosphates from the water into the cells and forms polyphosphates. In this way, the effect of phosphorus removal is achieved.
3.1.2 Microbial community analysis
The microbial community in the sludge sample before and after enrichment were analyzed high-throughput 16S rRNA gene sequencing. Phyla Proteobacteria was enriched and increased from 32.04–69.66% of total OTUs, was the most abundant phylum. And Betaproteobacteria(31.34%), Gammaproteobacteria(19.91%), and Alphaproteobacteria(11.98%) are the main class. The bacterial cells showed in Fig. 5 are all affiliated with Betaproteobacteria and they are all belong to PHA-accumulating bacteria[24]. Phyla Bacteroidetes decreased from 32.40–22.03% after enrichment, and other phyla comprised 8.31% of total OTUs. The succession of the community and the change of mixed bacterial function is synchronous. It can be seen that the microbial community structure has a more obvious succession before and after the enrichment section.
As the results shown in Fig. 5, the proportion of the PHA-accumulating bacteria in the initial sludge was deficient, only 0.4%. After bacteria enrichment, the abundance of dominant PHA-accumulating bacteria in sludge was significantly increased, which was reflected by increasing PHAs content. OTU4 (Hydrogenophaga genus) is a gram-negative hydrogen oxidizing bacteria with good PHAs synthesis ability. Koller[25] synthesized about 40% PHAs from isolated Hydrogenovora DSM 1749 using unhydrolyzed whey lactose as substrate, which verified the PHAs synthesis ability of the strain. In this experiment, the strain was acclimated and enriched (from 0.05–9.57%) under anaerobic and oxygen-limiting conditions. The result shows a certain effect of PHAs synthesis in macroscopic function and indicates that Hydrogenophaga is the dominant genus and plays a leading role in the process of PHA synthesis. Thauera[26] is a typical aerobic PHAs producing bacterium, which can synthesize PHAs by carbon source in the presence of oxygen. The abundance of OTU43 (Thauera genus) in the sludge was significantly improved compared with that in the initial sludge, indicating that there were aerobic PHAs-accumulating bacteria in the sludge after enrichment. They were the potential strains for the synthesis of PHAs in the oxygen-limiting feedstock whereafter.
Dechloromonas[27], Zoogloea[28], and Hydrogenophaga[29] were all reported as the dominant bacteria in enhanced biological phosphorus removal (EBPR). The changes in the abundance of these bacteria before and after the activity recovery were related to the effect of macroscopic phosphorus accumulation and release. OTU19、OTU53、OTU49、OTU86 all belong to Zoogloea. Zoogloea can secrete extracellular polymeric substances(EPS) that are composed of extracellular polysaccharides, proteins, nucleic acids, and other biological macromolecules. Then Zoogloea with compact structures and the strong ability to adsorb and oxidize organic matters is formed and it is of great significance to the settling ability of sludge.
3.2 Effect of feeding in the oxygen-limiting section on PHA production
3.2.1 Normal nutritional conditions
Figure 6 The changes of TP, COD, and PHAs in one-time and batch feeding when C/N = 20
As shown in Fig. 6, the changes of PHAs appear a different trend from before when the feeding modes were carried out in the oxygen-limiting section. In the experiment of one-time feeding in the oxygen-limiting section, the PHAs storage in the anaerobic section shows an upward trend. Still, the inflection point at the end of the anaerobic section is not the maximum value of this cycle. Acetate has been added again after 1h of oxygen-limiting operation, and the PHAs content increases rapidly again. After adding the carbon source for 0.5H (oxygen-limiting for 1.5H), it reached a new peak and then began to decrease. In the batch feeding operation, the first inflection point appeared after oxygen-limiting for 0.5h, and then PHAs were repeatedly synthesized and consumed with the batch addition of carbon source. The maximum content of PHAs in the two feeding modes is 26.52% and 34.98%, respectively, which were higher than the PHAs content without feeding mode.
The new trends indicate that feeding in the oxygen-limiting section can promote the increase of the content of PHAs, and this promotion performs at not only the end of the anaerobic section but also the end of a cycle. It has been documented[30] that under the anaerobic-aerobic mode, a kind of PAOs existing in activated sludge is in a "dormant state" in the anaerobic section, and does not degrade the substrate and synthesize PHAs. In contrast, in the aerobic stage, this kind of bacteria can quickly absorb carbon sources to synthesize PHAs and, at the same time, accumulate phosphorus to achieve phosphorus removal. However, according to the changes of TP and COD showed in the figure, it can be concluded that the bacteria that perform the PHAs synthesis in the oxygen-limiting section are not this kind of PAOs because the degradation of COD still occurred in the anaerobic section. And the phosphorus content in the microbial communities system was decreased compared with no feeding operation, which indicates that the overall activity of PAOs is reduced under the anaerobic-oxygen limiting feeding process. It is speculated that aerobic PHAs-accumulating bacteria such as Thauera plays the role of PHAs synthesis in the oxygen-limiting stage at this time. This bacteria is sensitive to the carbon source and can continue to synthesize PHAs within 30 minutes after feeding. It has been pointed out that PHB is the primary monomer component of PHAs synthesized by activated sludge using sodium acetate as the carbon source under aerobic conditions[30]. In this experiment, the main monomer of PHAs synthesized under the mode of feeding in the oxygen-limiting section was also PHB.
Under the average nutrient ratio (C/N/P = 100/20/1), the COD degradation rate and phosphorus removal capacity of the two feeding modes were all reduced, but the influence on PAOs of batch feeding in the oxygen-limiting section was more pronounced. Due to the existence of external carbon sources almost in the whole oxygen-limiting section, the heterotrophic bacteria in the sludge can grow and proliferate by degrading external carbon sources in the presence of oxygen. At the same time, the process of PAOs to decompose PHAs and synthesize polyphosphate would be inhibited. It would not only lead to the increase of effluent phosphorus content and the loss of total phosphorus content in the microbial communities system (effluent phosphorus reached 7.46mg/L, which is similar to phosphorus in inflow). And it would result in less phosphorus release in the anaerobic section of the next cycle. But also it leads to the incomplete degradation of carbon source in the effluent. In the one-time feeding mode, though the process of O2 being used by PAOs to oxidize PHAs as the electron acceptor and the process of the energy being used by PAOs to absorbs the phosphorus is inhibited in the oxygen-limiting stage. And other heterotrophic bacteria absorb carbon sources to proliferate and compete with PAOs. PAOs still show a good effect on phosphorus removal and COD degradation.
Although PAOs have been affected to a certain extent in batch feeding mode, the comparison in Fig. 6 shows that the maximal content of PHAs obtained by batch feeding is significantly higher than that of onetime-feeding under normal nutrient conditions. Since PHAs-accumulating bacteria are domesticated and enriched in the screening environment of "Feast-Famine", the condition that carbon source always exists in the water under batch feeding mode makes the process of bacteria decomposing PHAs and continuing to grow and proliferate inhibit. The lower consumption and degradation of PHAs in the oxygen-limiting section leads to the accumulation of PHAs in the cell, which increases the initial content of PHA in the next cycle and makes PHAs continuously get higher accumulation.
3.2.2 Restricted nutritional conditions (nitrogen-limited)
As shown in Fig. 7, the effects of two feeding modes in the oxygen-limiting section on the synthesis of PHAs were compared under three different nitrogen-limiting conditions (C/N = 60, 100, 150).
In the operation of one-time feeding in the oxygen-limiting section, the trend of PHAs content was also increased in the anaerobic section. PHAs were consumed first in the oxygen-limiting section and then generated again after feeding. In the process of increasing the C/N ratio, all the peak values and final values of PHAs contents are rising.
Under nitrogen-limiting conditions, the synthesis of essential proteins related to growth and proliferation in microorganisms, such as the synthesis of DNA and RNA, will be blocked. That would negatively affect the tricarboxylic acid cycle(TCA cycle) of cells. Under optimal microbial growth conditions with average nutrient ratios[31], the CoASH produced in the TCA cycle inhibits the production of 3-ketoacyl-CoA thiolase. And 3-ketoacyl-CoA thiolase is essential for the synthesis of PHAs because it can catalyze the formation of the precursor acetyl-CoA of PHAs. The imbalance of microbial growth caused by nitrogen restriction would increase intracellular NADH+, which would bring CoASH to the non-inhibitory level and then remove the restriction of PHAs accumulation. At the same time, the increase of NADH+ can also help the synthesis of PHAs by inhibiting the activity of citrate synthase in microorganisms and promote the accumulation of PHAs in microorganisms[32].
The increase of the C/N ratio within a specific range would stimulate the accumulation of PHAs[33]. In addition, it was observed that the variation of the PHAs content in the anaerobic section showed a decrease in PHAs when C/N is 100 and 150. This phenomenon also appears in the study reported by Wen [33], indicating that it is related to the degree of nitrogen restriction. Under the condition of a high C/N ratio, the metabolism of microorganisms was greatly affected, and there were complex mixed bacteria in activated sludge. It is speculated that the synthesis of protein and nucleic acid is blocked in some microorganisms due to the lack of nitrogen[34], which affects the influence of cell growth and then decomposes their cellular materials under endogenous respiration. Therefore, the synthesis and decomposition PHAs fluctuated in different microorganisms during the whole operation cycle. And the addition of carbon sources made the non-PHAs accumulating bacteria also absorb carbon sources for life activities, which affected the PHAs-accumulating bacteria and increased the complexity of the change of PHAs content. In addition, the settling ability of activated sludge was poor at the high C/N ratio, and the SVI of sludge reached 124 and 165 when the C/N was 100 and 150, respectively.
The nitrogen-limited reactor was operated by gradually reducing the amount of nitrogen source. At the high C/N ratio, the sludge-water mixture was difficult to separate, and the color of the sludge in the reactor started to turn white. Part of the sludge was discharged with the supernatant after the end of the reaction, resulting in the reduction of the sludge volume in the system. It has been reported[35] that sludge bulking of activated sludge would appear when the condition of a high C/N ratio. Due to the lack of nitrogen source, the growth of bacteria in the mixed culture system is inhibited, and a large number of bacterial micelles are in the endogenous metabolic period. Filamentous bacteria with heavy concentrations and high DO affinity peculiarities compete for carbon source and oxygen electron acceptor under nutrient-limited conditions. And then, they proliferate to become dominant bacteria which would lead to sludge bulking. In conclusion, increasing the C/N ratio can effectively promote the accumulation of PHAs in microbial communities system. But the problem of the sludge being discharged in the process of removing the supernatant caused by the deterioration of sludge settling ability cannot be ignored. Therefore, the C/N ratio is not as high as possible.
In batch feeding mode, the change of PHAs becomes more complex with the increase of the C/N ratio. Under nutrient imbalance, the PHAs content did not show a continuous upward trend in the anaerobic section. PHAs were decomposed in the anaerobic section, and the maximum content of PHAs appeared at 0.5h after the end of the anaerobic section. In the subsequent oxygen-limiting section, there was also a higher PHAs content when the C/N ratio was higher. But the variation of the PHAs content was also more changeable, and there were multiple inflection points in the operation process.
The variation of the PHAs content has been fluctuating when C/N = 60 in batch feeding mode, indicating that some PHAs-accumulating bacteria in mixed microbial cultures began to carry out endogenous respiration. PHAs were synthesized and decomposed at the same time, which makes the change rule of PHAs intricate. And the settling ability of activated sludge also started to get worse and SVI was 102. The trend of PHAs content became gentle from C/N = 100 in the oxygen-limiting section. There was no significant increase in the content of PHAs because the degradation and synthesis of PHA kept a balance. Due to the presence of external carbon sources throughout the cycle in batch feeding mode, the PHAs-accumulating bacteria could not eliminate other non-PHAs synthetic bacteria under the condition of "Feast-Famine". It indicates that the PHAs-accumulating bacteria have lost their dominant position in the uptake of carbon sources and the utilization of oxygen in the environment. It has been reported[34] that the overall PHAs content of the system can reach a higher level in activated sludge with more abundant and dominant PHAs-accumulating bacteria because both the yield and biomass of PHAs are stimulated to different degrees under the condition of nutrient restriction. The accumulation of PHAs has reached saturation when the C/N ratio is between 100 to 150 because PHAs content was no significant increase. Similar to one-time feeding, there was sludge bulking in the sludge system. The poor settling ability (SVI = 205) leads to part of the sludge was discharged with the supernatant and the PHAs-accumulating bacteria lose their dominant position in the sludge system.
In the process of increasing the C/N ratio, the maximum content of PHAs in the two feeding methods has been improving continuously. However, the activity of PAOs in the one-time feeding mode is less affected than in batch feeding mode when the C/N ratio is high, and the PHAs-accumulating bacteria can still occupy the dominant position in the screening environment of "Feast-Famine" and obtain more PHAs. But the PHAs-accumulating bacteria lost the dominant position in the batch feeding mode. Therefore, the mode of one-time feeding in the anaerobic-oxygen limiting process has a higher PHAs synthesis potential, and the PHAs accumulation performance is better under the nitrogen-limited condition with C/N = 150.