3.1 Phosphorus removal and PHA synthesis in an anaerobic-oxygen limited process
An anaerobic-oxygen limited process started the SBR reactor. The variation of phosphate concentration and PHA content was shown in Fig. 1 after three days, eight days, and eleven operation days.
It can be seen from Fig. 1 that with the increase of working days, the phosphorus release amount in the anaerobic stage showed a continuous upward trend. After 11 days of operation under an anaerobic-oxygen limited process, the concentration of phosphate in water increased from 8.5 mg/L to 77.45 mg/L in the first half-hour of anaerobic treatment, and the total amount of TP. released at the end of the anaerobic process reached the maximum (85.4 mg/L). The abundant PHA synthetic bacteria cause this in the activated sludge of municipal wastewater treatment plant, among which PAOs is one of the common synthetic bacteria. Based on the principle of enhanced biological phosphorus removal from wastewater, under anaerobic condition[22], the polyphosphate accumulating bacteria can rapidly decompose the phosphorus accumulating in the cells, release a large amount of phosphate, and the concentration of phosphorus in the reactor rises quickly; in the oxygen limiting stage, the polyphosphate accumulating bacteria take molecular oxygen as electron acceptor and absorb excessive phosphorus from water to form polyphosphate. As shown in Figure 1, phosphorus in the system is almost completely absorbed at the end of oxygen limiting. It can be observed in Fig.1 that the variation of PHA content in activated sludge is highly correlated with the phosphorus release and absorption curve. With the increase of working days, the system's phosphorus removal effect is better, and PHA synthesis also increases. This is due to PAOs under anaerobic conditions. Many organic nutrients in wastewater are absorbed using the energy in glycogen and the hydrolysis of polyphosphate in cells. The external carbon source volatile organic acids (VFA) are converted into intracellular energy storage substances, thus accumulating PHA. In the oxygen limiting stage, PAOs absorb excessive phosphate from wastewater as energy storage and use free oxygen as an electron acceptor to oxidize the internal carbon source stored under anaerobic conditions. Therefore, PHA was slowly consumed. After 11 days, PHA content synthesized by activated sludge reached 20.19% of dry sludge weight. This shows that activated sludge has a high activity after anaerobic oxygen limiting operation. When the influent containing carbon source and nutrient solution enters the system, it can quickly absorb external carbon source and decompose phosphorus accumulation in cells, effectively remove phosphorus and complete a certain amount of PHA synthesis.
In conclusion, after 11 days of stable operation, the system can recover and synthesize a certain PHA amount while effectively removing phosphorus.
3.2 Influence of pH on the accumulation and synthesis of PHA
After the above 11 days of anaerobic-oxygeN-limiting operation, PHA content was measured at the end of the anaerobic period to account for 20.19% of the sludge's dry weight. Based on this, the change law of COD and PHA synthesis under the conditions of weak alkaline pH (7.5-8.5), pH=7, pH=6, pH=5 is further examined, and the results are shown in Fig. 2.
As can be seen from Fig.2, when entering the oxygen-limiting stage, the substrate is consumed rapidly. At this point, the COD degradation curve shows a clear downward trend, the microorganism is in a highly active state, and the substrate's utilization efficiency is also higher. After the oxygen- limit of 1h, the PHA content reached the highest value, accounting for 28.22% of the sludge's dry weight. Both the synthesis of PHA and the degradation of COD are affected when pH is checked. As shown in Fig.2, the residual part of COD in the oxygen-limiting end-stage system is not degraded, which directly affects PHA synthesis. It can be seen that the content of PHA has also been reduced, and at the time of anaerobic 1.5 h reached the highest value, accounting for 28.17% of the dry weight of sludge, under the condition of pH-6, the utilization rate of the system substrate is significantly inhibited, PHA synthesis has become irregular. Under the condition of pH-5, COD has been almost degraded, the amount of PHA is decreasing, and the system has gradually lost the synthesis ability of PHA.
The synthesis of PHA at different pH values is analyzed dynamically, and the results are shown in Table 4.
Table 4 Kinetic parameters of PHA production under different pH.
nitrogen limiting
|
|
pH
|
%PHA
|
∆PHA (Cmmol/L)
|
∆S (Cmmol/L)
|
YPHA/S
|
rPHA
|
rS
|
5
|
19.31
|
9.47
|
NA
|
NA
|
4.73
|
NA
|
6
|
21.13
|
4.74
|
6.39
|
0.74
|
2.37
|
3.19
|
7
|
28.17
|
21.10
|
25.83
|
0.82
|
7.03
|
8.61
|
no control (7.5-8.5)
|
28.22
|
17.89
|
26.04
|
0.69
|
5.96
|
8.68
|
|
phosphorus limiting
|
|
pH
|
%PHA
|
∆PHA (Cmmol/L)
|
∆S (Cmmol/L)
|
YPHA/S
|
rPHA
|
rS
|
5
|
13.52
|
-2.68
|
-1.46
|
-1.84
|
-1.34
|
-0.73
|
6
|
18.39
|
-0.40
|
-8.54
|
-0.05
|
-0.20
|
-4.27
|
7
|
23.97
|
14.60
|
14.79
|
0.99
|
9.73
|
9.86
|
no control (7.5-8.5)
|
25.81
|
30.69
|
27.08
|
1.13
|
20.46
|
18.06
|
∆S: substrate consumption (Cmmol/L);∆PHA: PHA produced during the assay (Cmmol/L);%PHA:PHA content in biomass(%gPHA*g-1CDW);
rS:substrate consumption rate (Cmmol/h); rPHA:PHA production rate (Cmmol/h); YPHA/S:PHA to substrate stoichiometric yield (CmmolPHA/Cmmol S).
The main kinetic and stoichiometric parameters of PHA formation are shown in Tab 4. Under the condition of nitrogen limitation, when pH is not controlled, it is the most favorable for the total production of PHA. Considering the accumulation of PHA in this stage, it reached the highest value under several conditions, accounting for 28.22% of the cell dry weight. The substrate consumption raters of this stage also got the maximum value, which was 8.68 Cmmol S / L / h, which indicated that microorganisms were absorbing the carbon source to synthesize PHA. Besides, the PHA productivity rPHA reached 5.96 Cmmol PHA / L / h, and YPHA/S were also high, going 0.69 Cmmol PHA Cmmol/S. Under the condition of phosphorus limitation, when the pH was not controlled, the substrate consumption rate was the highest (rS = 18.06 Cmmol S / L / h), the production rate of PHA was the highest (rPHA= 20.46 Cmmol PHA / L / h), the maximum accumulation of PHA was 25.81% of the cell dry weight, and the storage capacity of PHA was 1.13 Cmmol PHA Cmmol/S, which indicated that carbon source was being used for the accumulation of PHA. The results also showed that the formation and substrate conversion of PHA was inhibited in the acidic pH range of less than 7. The overall production performance of PHA could be improved by controlling the pH in weak alkaline conditions. Consistent with the previous report of other authors, the substrate's polymer yield is higher than different pH values (0.33 Cmmol PHA Cmmol/S) at pH 8.8-9.2, indicating that carbon source is being used for PHA accumulation[14].
The above experimental results show that pH affects the metabolic process of MMC, so it has a significant influence on the synthesis. According to the experiment's kinetic analysis and component results, the accumulation capacity of PHA is the strongest under the weak alkaline condition without controlling the pH value. The synthesis amount tends to decrease with the decrease of pH value. This may be due to the following reasons:
(1) The effect of pH on enzyme activity is the main reason for decreasing PHA synthesis ability at low pH. As the water was developed with sodium acetate as a carbon source, it was weakly alkaline. The strain of PHA synthesis in activated sludge adapted to this condition had better adaptability to alkaline conditions. When the pH was adjusted to be acidic, the enzyme activity might be affected. The enzyme is the fundamental factor of a biochemical reaction. There are a lot of amino acid side chain groups in its molecule. The action of the enzyme is mainly through the dissociation of these side-chain groups. With different pH, the dissociation state of these side-chain groups will be other. The proportion of specific ionic groups with catalytic activity in the total amount of enzyme will be different, so the enzyme's catalytic ability will be different[23].
(2)The electronegativity of bacteria changed. The lower pH has reached the electric point of some bacteria, and the surface charge of bacteria has changed from negative to positive. Some extracellular secretions and active substances adsorbed on the surface of bacteria dissociate into the solution, leading to the unstable COD change. The bacteria can not absorb the carbon source matrix to synthesize PHA, so the synthesis ability is reduced. As shown in[24], when the acid pH values were 4.0, 5.5, and 6.5, PHA accumulation was the lowest, and no apparent PHA accumulation phenomenon was observed. This phenomenon can be explained as follows: under low pH value, acetate is still in the form of undistributed, and this undivided acetic acid will rapidly diffuse into bacterial cells, and then it dissociates and exerts an intracellular proton load, thus reducing the intracellular pH value. This decrease in internal pH may result in a reduction of PHA production.
3.3 Influence of nutrient ratio on PHA synthesis by activated sludge
The ratio of C / N and C / P is a significant factor affecting PHA content synthesized by activated sludge. Under the condition of limiting nutrient ratio, the content of PHA synthesized by microorganisms is higher. In this section, pH = 7.5 is controlled under anaerobic oxygen limiting process because previous results (Section 3.2) have shown that weak alkaline conditions without pH control under this process are most conducive to PHA production. The second group of tests was conducted by gradually reducing nitrogen and phosphorus concentration in the influent water. Variation of PHA content in a running cycle is shown in Fig. 3.
It can be seen from Fig.3 that in the process of limiting phosphorus source, the content of PHA showed a rising trend in the anaerobic stage but gradually decreased in the oxygen limiting stage. Since in the anaerobic phase, microorganisms use the energy released from polyphosphate esters to absorb organic substrates (VFAs) and store them in the internal carbon source (PHA). The oxygen limiting section consumes the inner storage material PHA when the external carbon source is insufficient, so the synthesis trend is shown in the figure. It can be seen from Fig.3 that when the C: P ratio increases from the initial 100 to 150, the PHA content presents a significant upward trend and reaches the highest value at the end of anaerobic treatment, at this time, the content accounts for 36.07% of the dry sludge weight; if the C: P ratio continues to increase, the content of PHA is gradually decreased. When the C: P ratio increased to 200, 250, and 500, the content of PHA did not fall immediately at the initial stage of oxygen limitation but continued to increase. The following analysis is made on this situation. Due to the continuous reduction of phosphorus source in the system, the absorption and utilization of carbon source by activated sludge are hindered, and part of carbon source will remain at the end of the anaerobic process. Microorganisms make extensive use of organic matter, which can accumulate intracellular storage material PHA and synthesize polyphosphate ester and glycogen and other energy storage materials at the same time. Therefore, a large amount of oxygen needs to be consumed. At the initial stage of oxygen limitation, the reactor's actual dissolved oxygen concentration is always deficient, which is actually in micro oxygen or low oxygen state. Some studies have demonstrated that the micro oxygen state is more beneficial to PHA accumulation[25]. Therefore, microorganisms use some of the capacity provided by dissolved oxygen to convert the residual organic matter into PHA, resulting in the phenomenon of PHA synthesis after the start of the oxygen limiting stage. The results showed that limiting phosphorus concentration significantly affected PHA accumulation by activated sludge mixed bacteria. However, excessive phosphorus limitation will lead to the deterioration of sludge settling performance, increased flocculent sludge viscosity, and decreased synthesis. With the increase of the limiting ratio, the extracellular substances with high water content were secreted by the microbial micelles, which caused the non-filamentous sludge bulking to hinder the further improvement of PHA synthesis[26].
Similar to the phosphorus limit test, it can be seen from the figure (b) that the content of PHA synthesized by activated sludge keeps increasing when the C: N ratio increases from 20 to 150. When the C: N ratio increased from 20 to 150, PHA reached the maximum at 0.5 h of oxygen limitation, which accounted for 50.39% of the cell dry weight. It have shown that under the condition of low nutrient concentration, microorganisms will be stimulated to enter the metabolic pathway of PHA accumulation rather than their biomass growth[20]. Under the condition of limited nutrients, the biosynthesis process of cellular materials is also inhibited to a certain extent, and Acetyl CoA, the intermediate product of metabolism, is transformed from the material needed to flow to the TCA cycle to form synthetic bacteria into the PHA synthesis pathway. The experimental results are similar to those obtained by[23] ,which indicates that excessive limitation of nitrogen concentration will lead to the proliferation of filamentous bacteria, resulting in poor sludge settleability and sludge bulking, thus reducing the PHA content. The results showed that no matter which nutrient concentration was limited, the PHA synthesis capacity of activated sludge was more substantial than that of the initial acclimation stage, which helped improve the synthesis capacity of PHA.
Besides, combined with this paper's study, it was found that the maximum synthesis of PHA under nitrogen limitation (50.39%) was higher than that under phosphorus limitation (36.07%), indicating that nitrogen limitation was more conducive to the accumulation of PHA than phosphorus limitation. As reported in[14,19], higher PHA content was obtained under N restriction than p restriction. (51% and 59% respectively). However, excessive nitrogen and phosphorus limitation will result in sludge bulking. It is an important problem to control sludge bulking while PHA production is high.
3.4 Influence of N-limitation and P-limitation on monomer components
At present,a report founds PHA synthesis from activated sludge is mainly short-chain PHA, and the main PHA monomers are 3-hydroxybutyric acid (PHB) and 3-hydroxyvaleric acid (PHV)[27]. According to the monomer structure or content,the properties can vary from hard to soft to elastic. For example, PHB is a homopolymer with poor mechanical properties, easy pyrolysis, poor solvent resistance, and high crystallinity, which makes the material hard and brittle, which is not conducive to processing and production, and limits its commercial application; In contrast, the homopolymer PHV has good physical flexibility and can be widely used as a good processing material. To adjust and control PHA with different performance to meet additional production requirements, the monomer composition of PHA reached the maximum synthesis under five different nutrient ratios (C / N ratio of 20, 60, 100, 120, 150; C / P ratio of 100, 150, 200, 250, 500) was further analyzed, and the influence of different nutrient ratio on the monomer composition of PHA was evaluated. The results are shown in Fig.4.
Fig.4 compares and analyzes the proportion of PHB and PHV under the limitation of two nutrient sources. With the decrease of phosphorus concentration, the content of PHB and PHV showed no obvious change rule when the synthesis of PHA was the highest. Since the composition of PHA is mainly determined by the type of substrate, acetic acid is used as a carbon source in the experiment, so PHB is the main product of PHA synthesized by activated sludge. When the C / P ratio was 100, PHB accounted for 67.86%. When the C / P ratio was 150, 200, 250 and 500, the proportion of PHB / PHA was 66.01%, 66.49%, 66.70% and 66.19%, respectively. The ratio of PHV / PHA was 32.14%, 33.99%, 33.51%, 33.3% and 33.81%, respectively. Under the condition of nitrogen limitation, it can be seen from the figure that the decrease of nitrogen concentration has an observable promoting effect on the accumulation of PHV when the C / N ratio increases from 20 to 150, the proportion of PHV / PHA is continually increasing, from 33.79% to 49.14%. However, the proportion of PHB / PHA decreased gradually with the C / N ratio increase. When the C / N ratio was 20, 60, 100, 120 and 150, the proportion of PHB / PHA was 66.21%, 65.42%, 60.59%, 53.81% and 50.86%, respectively.
The results showed that PHB was the principal monomer for cell synthesis, no matter the nitrogen or phosphorus limitation. Increasing the C / N ratio is beneficial to increase the proportion of PHV in PHA, but with the increase of the C / P ratio, the change of PHB and PHV is not apparent. Therefore,the final product of PHA was realized, which also provided the possibility of controlling the composition of PHA product by regulating the substrate composition in the future.