Identification and Metabolic Mechanism of Non-fermentative Short-cut Denitrifying Phosphorus-removing Bacteria

To investigate the characteristics and metabolic mechanism of short-cut denitrifying phospho- rus-removing bacteria （SDPB） that are capable of enhanced biological phosphorus removal （EBPR） using nitrite as an electron acceptor, an aerobic/anoxic sequencing batch reactor was operated under three phases. An SDPB-strain YC was screened after the sludge enrichment and was identified by morphological, physiological, biochemical properties and 16S rDNA gene sequence analysis. Denitrifying phosphorus-removing experiments were conducted to study anaerobic and anoxic metabolic mechanisms by analyzing the changes of chemical oxygen demand （COD）, phosphate, nitrite, poly-fl-hydroxybutyrate （PHB）, and glycogen. The results show that strain YC is a non-fermentative SDPB similar to Paracoccus denitrificans. As a kind of non-fermentative bacteria, the energy of strain YC was mainly generated from phosphorus release （96.2%） under anaerobic conditions with 0.32 mg P per mg synthesized PHB. Under anoxic conditions, strain YC accumulated 0.45 mg P per mg degraded PHB, which produced most of energy for phosphate accumulation （91.3%） and a little for glycogen synthesis （8.7%）. This metabolic mechanism of strain YC is different from that of traditional phosphorus-accumulating organisms （PAOs）. It is also found that PHB, a kind of intracellular polymer, plays a very important role in denitrifying and accumulating phosphorus by supplying sufficient energy for phosphorous accumulation and carbon sources for denitrification. Therefore, monitoring AP/APHB and ANO2 -N/APHB is more necessary than monitoring AP/ACOD, ANO2 -N/ACOD, or AP / ANO2 -N.

Operation of three parallel AN/AO processes to enrich denitrifying phosphorus removing bacteria for low strength wastewater treatment

Three parallel anaerobic-anoxic/anaerobic-aerobic （AN/AO） processes were developed to enrich denitrifying phosphorus removal bacteria （DPB） for low strength wastewater treatment. The main body of the parallel AN/AO process consists of an AN （anaerobic-anoxic） process and an AO （anaerobic-aerobic） process. In the AO process, the common phosphorus accumulating organisms （PAOs） was dominate, while in the AN process, DPB was dominate, The volume of anaerobic zone（Vana）：anoxie zone（Vano） ： aerobic zone （Vaer） for the parallel AN/AO process is 1：1：1 in contrast with a Vana：Vaer and Vano：Vaer of 1：2 and 1：4 for a traditional biological nutrient removal process （BNR）. Process 3 excels in the 3 processes on the basis of COD, TN and TP removal. For 4 month operation, the effluent COD concentration of process 3 did not exceed 60 mg/L; the effluent TN concentration of process 3 was lower than 15 mg/L; and the effluent TP concentration of process 3 was lower than 1 mg/L.

Characterization of phosphorus removal bacteria in （AO）^2 SBR system by using different electron acceptors

Characteristics of phosphorus removal bacteria were investigated by using three different types of electron acceptors, as well as the positive role of nitrite in phosphorus removal process. An （AO）^2 SBR （anaerobic-aerobic-anoxic-aerobic sequencing batch reactor） was thereby employed to enrich denitrifying phosphorus removal bacteria for simultaneously removing phosphorus and nitrogen via auoxic phosphorus uptake, Ammonium oxidation was controlled at the first phase of the nitrification process. Nitrite-inhibition batch tests illustrated that nitrite was not an inhibitor to phosphorus uptake process, but served as an alternative electron acceptor to nitrate and oxygen if the concentration was under the inhibition level of 40mg NO2 - N·L^- 1. It implied that in addition to the two well-accepted groups of phosphorus removal bacterium （ one can only utilize oxygen as electron acceptor, P1, while the other can use both oxygen and nitrate as electron acceptor, P2 ）, a new group of phosphorus removal bacterium P3, which could use oxygen, nitrate and nitrite as electron acceptor to take up phosphorus were identified in the test system. To understand （AO）^2 SBR sludge better, the relative population of the different bacteria in this system, plus another A/O SBR sludge （ seed sludge） were respectively estimated by the phosphorus uptake batch tests with either oxygen or nitrate or nitrite as electron acceptor. The results demonstrated that phosphorus removal capability of （AO）^2 SBR sludge had a little degradation after A/O sludge was cultivated in the （AO）^2 mode over a long period of time. However, deuitrifying phosphorus removal bacteria （ P2 and P3 ） was significantly enriched showed by the relative population of the three types of bacteria, which implied that energy for aeration and COD consumption could be reduced in theory.

Characteristics of anoxic phosphors removal in sequence batch reactor

The characteristics of anaerobic phosphorus release and anoxic phosphorus uptake were investigated in sequencing batch reactors using denitrifying phosphorus removing bacteria （DPB） sludge. The lab-scale experiments were accomplished under conditions of various nitrite concentrations （5.5, 9.5, and 15 mg/L） and mixed liquor suspended solids （MLSS） （1844, 3231, and 6730 mg/L）. The results obtained confirmed that nitrite, MLSS, and pH were key factors, which had a significant impact on anaerobic phosphorus release and anoxic phosphorus uptake in the biological phosphorous removal process. The nitrites were able to successfully act as electron acceptors for phosphorous uptake at a limited concentration between 5.5 and 9.5 mg/L. The denitrification and dephosphorous were inhibited when the nitrite concentration reached 15 mg/L. This observation indicated that the nitrite would not inhibit phosphorus uptake before it exceeded a threshold concentration. It was assumed that an increase of MLSS concentration from 1844 mg/L to 6730 mg/L led to the increase of denitrification and anoxic P-uptake rate. On the contrary, the average P-uptake/N denitrifying reduced from 2.10 to 1.57 mg PO4^3--P/mg NO3^--N. Therefore, it could be concluded that increasing MLSS of the DEPHANOX system might shorten the reaction time of phosphorus release and anoxic phosphorus uptake. However, excessive MLSS might reduce the specific denitrifying rate. Meanwhile, a rapid pH increase occurred at the beginning of the anoxic conditions as a result of denitrification and anoxic phosphate uptake. Anaerobic P release rate increased with an increase in pH. Moreover, when pH exceeded a relatively high value of 8.0, the dissolved P concentration decreased in the liquid phase, because of chemical precipitation. This observation suggested that pH should be strictly controlled below 8.0 to avoid chemical precipitation if the biological denitrifying phosphorus removal capability is to be studied accurately.

Anoxic Biological Phosphorus Uptake in A^2O Process

A lab-scale anaerobic-anoxic-oxic (A2O) process used to treat a synthetic brewage wastewater was investigated. The objectives of the study were to identify the existence of denitrifying phosphorus removing bacteria (DPB), evaluate the contribution of DPB to biological nutrient removal and enhance the denitrifying phosphorus removal in A2O bioreactors. Sludge analysis confirmed that the average anoxic P uptake accounted for approximately 70% the total amount of P uptake, and the ratio of anoxic P uptake rate to aerobic P uptake rate was 69%. In addition, nitrate concentration in the anoxic phase and different organic substrate introduced into the anaerobic phase had significant effect on the anoxic P uptake. Compared with conventional A2O processes, good removal efficiencies of COD, phosphorus, ammonia and total nitrogen (92.3%, 95.5%, 96% and 79.5%, respectively) could be achieved in the anoxic P uptake system, and aeration energy consumption was saved 25%. By controlling the nitrate recirculation flow in the anoxic zone, anoxic P uptake could be enhanced, which solved the competition for organic substrates among poly-P organisms and denitrifiers successfully under the COD limiting conditions. Therefore, in wastewater treatment plants the control system should be applied according to the practical situation to optimize the operation.

Denitrification and Dephosphatation by Anaerobic/Anoxic Sequencing Batch Reactor

Removal of denitrifying phosphorus was verified in a laboratory anaerobic/anoxic sequencing batch reactor (A/A SBR). The results obtained demonstrated that the anaerobic/anoxic strategy can enrich the growth of denitrifying phosphorus removing bacteria (DPB) and take up phosphate under anoxic condition by using nitrate as the electron acceptor. The phosphorus removal efficiency was higher than 90% and the effluent phosphate concentration was lower than 1 mg·L-1 after the A/A SBR was operated in a steady-state. When the chemical oxygen demand(COD) of influent was lower than 180mg· L-1, the more COD in the influent was, the higher efficiency of phosphorus removal could be attained under anoxic condition. However, simultaneous presence of carbon and nitrate would be detrimental to denitrifying phosphorus removal. Result of influence of sludge retention time (SRT) on denitrifying phosphorus removal suggested that the decrease of SRT caused a washout of DPB and consequently the enhanced biological phosphorus removal decreased with 8 days SRT. When the SRT was restored to 16 days, however, the efficiency of phosphorus removal was higher than 90%.

Denitrifying phosphorous removal in anaerobic/anoxic SBR system with different startup operation mode

To achieve stable and efficient nitrogen and phosphorus removal and to investigate the characteristics of the A/A SBR enriched with denitrifying phosphorus removal bacteria(DPB),the whole course of startup was studied with two reactors operated in different mode.The reactor I was operated under anaerobic/settling/anoxic/settling mode,and the reactor II was operated under anaerobic/anoxic/settling mode.Differences between the two reactors in removal efficiency of COD,nitrogen and phosphorus were examined.The results indicated that efficient performance could be achieved in both reactors with different startup operation mode,while the phosphorus removal efficiency was improved sooner in reactor I than in reactor II,which suggested that reactor I would supply a more favorable condition for DPB proliferation.Meanwhile,it was observed that the amount of organic substrates consumption had a linear correlation to that of phosphorus release in anaerobic phase when DPB was accumulated in the A/A SBR denitrifying phosphorus removal system.

Effect of carbon source and nitrate concentration on denitrifying phosphorus removal by DPB sludge

Effect of added carbon source and nitrate concentration on the denitrifying phosphorus removal by DPB sludge was systematically studied using batch experiments, at the same time the variation of ORP was investigated. Results showed that the denitrifying and phosphorus uptake rate in anoxic phase increased with the high initial anaerobic carbon source addition. However once the initial COD concentration reached a certain level, which was in excess to the PHB saturation of poly-P bacteria, residual COD carried over to anoxic phase inhibited the subsequent denitrifying phosphorus uptake. Simultaneously, phosphate uptake continued until all nitrate was removed, following a slow endogenous release of phosphate. High nitrate concentration in anoxic phase increased the initial denitrifying phosphorus rate. Once the nitrate was exhausted, phosphate uptake changed to release. Moreover, the time of this turning point occurred later with the higher nitrate addition. On the other hand, through on-line monitoring the variation of the ORP with different initial COD concentration, it was found ORP could be used as a control parameter for phosphorus release, but it is impossible to utilize ORP for controlling the denitrificaion and anoxic phosphorus uptake operations.

Nitrate removal and extracellular polymeric substances of autohydrogenotrophic bacteria under various pH and hydrogen flow rates

In recent years there has been an increasing interest in the use of autohydrogenotrophic bacteria to treat nitrate from wastewater. However, our knowledge about the characteristics of extracellular polymeric substances（EPS） releasing by these activities is not yet very advanced. This study aimed to investigate the change in EPS compositions under various p H values and hydrogen flow rates, taking into consideration nitrogen removal. Results showed that p H 7.5 and a hydrogen flow rate of 90 m L/min were the optimal operating conditions, resulting in 100% nitrogen removal after 6 hr of operation. Soluble and bound polysaccharides decreased, while bound proteins increased with increasing p H. Polysaccharides increased with increasing hydrogen flow rate. No significant change of bound proteins was observed at various hydrogen flow rates.

Influence of carbon source and temperature on the denitrifying phosphorus removal process

To supply the valuable operating parameters for the popular usage of the new denitrifying phosphors removal process,it is essential to study the dominant biochemical reactions and the characteristics of denitrifying phosphorus removing bacteria(DPB).Thus,parallel batch experiments using DPB sludge were carried out to assess the effect of substrates(sewage,HAc,and endogenous carbon source)on denitrifying dephosphorus removal efficiency in this study.The results showed that the initial specific phosphorus release rate increased with the high concentration of the short-chain volatile fatty acids ratio in the influent,and sufficient phos-phorus was released by DPB.This improved the subsequent denitrification and phosphorus uptake efficiency.The specific endogenous denitrification mainly relies on the internal carbon source(PHB)stored by poly-P bacteria.Denitrifying phosphorus removing bacteria were very hungry when the internal PHB was consumed.Consequently,the specific endogenous denitrification rate was low and the phosphorus uptake did not happen.On the other hand,in the experiment,the denitrifying phosphorus removal performance under two temperature conditions(8-10°C and 25-26°C)was also investigated and analyzed.It was found that the lower temperature decreased the specific phosphorus release and uptake rate,but did not inhibit the denitrifying phosphorus removal completely.Therefore,the negative influence of the low temperature on the overall phosphorus removal was not significant.