The optimal ecological factors and the denitrification populationof a denitrifying process for sulfate reducing bacteriainhibition

SRB have great negative impacts on the oil production in Daqing Oil field. A continuous-flow anaerobic baffled reactors (ABR) are applied to investigate the feasibility and optimal ecological factors for the inhibition of SRB by denitrifying bacteria (DNB). The results showed that the SO42- to NO3- concentration ratio (SO42-/NO3-) are the most important ecological factor. The input of NO3- and lower COD can enhance the inhibition of S2-production effectively. The effective time of sulfate reduction is 6 h. Complete inhibition of SRB is obtained when the influent COD concentration is 600 mg/L, the SO42-/NO3- is 1/1 (600 mg/L for each), N is added simultaneously in the 2# and the 5# ABR chambers. By extracting the total DNA of wastewater from the effective chamber, 16SrDNA clones of a bacterium had been constructed. It is showed that the Proteobacteria accounted for eighty- four percent of the total clones. The dominant species was the Neisseria. Sixteen percent of the total clones were the Bacilli of Frimicutes. It indicated that DNB was effective and feasible for SRB inhibition.


Introduction
Sulfate reducing bacteria (SRB) is the sum of bacterias with sulfate reducing capability [1,2]. There are about 9 categories and 200 species of SRB [3,4]. The excessive reproduction of SRB in the oilfield would lead to lots of problems [5]. The abundance of SRB had led to the increase of sulfide and suspended solid concentration, collapse caused by electric field, and other serious production accidents in Daqing Oilfield, Lots of chemicals was added to inhibit the growth of SRB [6], which caused serious impacts and damages on the oilfield production and the ambient environment. Recently, researches on the application of microbial control methods for SRB inhibition are increasing [7]. These methods are preferred as they are safe, environmental friendly and cost-effective. Ecological control of SRB is achieved based on the theories of microbial ecology. Lots of researches have discussed denitrifying process mechanism of SRB inhibition. Five major mechanisms are listed. The first was competitive inhibition of substrate. Ricardo et al. [8] had pointed out that the DNB out competed the SRB when acetic acid was utilized as the substrate. The energy released by the nitrate reduction is higher than that of the sulfate reduction. Also, SRB required lower oxidation-reduction potential (ORP) (-100mV) than DNB (+100mV), the reduction of nitrate is prior to sulfate reduction. The second one is the inhibition by the intermediate metabolites of denitrifying reaction. Taylor et al. [9] indicated that the inhibition effects of NO 2 [12]. Eckford et al. [13] reported that the synergistic effectiveness of nitrate, nitrite and molybdate on SRB inhibition depended on the operational parameters of the process. The fourth mechanism is the anaerobic sulfur cycle formulated in the system. A possible anaerobic sulfur cycle model presented in the oilfield is proposed by Reinsel et al. [14]. The driving force is transferred or diffused from the surface layer to deep layer of the nitrate. Jenneman et al. [15] demonstrated that the yellow matters appeared in the nitrate added environment were polymeric sulfides, which suggested the present of sulfide cycle. The fifth mechanism is the control of ORP . Gevertz et al. [16] showed that the generation of intermediate metabolites (NO and N 2 O) by denitrifying reaction enhance the ORP and thus long-term inhibition sulfate reduction was obtained. Calcium nitrate is utilized for the treatment of the putrid and smelly drain sediment [17]. The results shows that as the ORP enhanced to positive value, the odor is controlled and the 99.5 % sulfide concentration is removed. The field investigation on the feasibility of nitrate dosage for the control of hydrogen sulfide production is conducted by He et al. in the Coleville Oilfield [18]. The growth of sulphur oxidising bacteria (SOB) and DNB are enhanced. The numbers of SRB keep still or reduced. Favorable efficiencies are also achieved through the dosage of nitrate in the Skjold, the Halfdan, the Gulfaks, the Veslefrikk and the Foinaven Oilfield [19]. However, researches on the ecological factors and the bacterial community in the denitrifying process for SRB inhibition are rare. Besides, most of the previous studies focus on the inhibition of the SRB number rather than their activities.
Nowever, the inhibition of SRB through the inhibition of their activities is explored in an anaerobic baffled reactor (ABR).Indigenous microorganisms would grows,, especially the DNB,could be stimulated with the dosage of N. So the sulfate reduction activities of the SRB are gradually declined. Finally, the SRB will stop the sulfate reduction reactions and thus the growth of SRB would be inhibited [20]. Thus, the inhibition strategy of SRB is converted from the simple kill of SRB to their activity inhibition. Besides, the optimal ecological factors and the microbial community structure in the ABR are also investigated.

Materials and methods
The vertical bafflers divided the ABR into seven serial chambers ( Figure 1). Each chamber had a separate upflow sludge bed. Most of the sludge was in granule or floc form. The ABR was in a rightangled trapezoid shape. The effective volume of each chamber is 4.8 L, the total effective volume of the reactor is 40 L. Heating device is utilized to keep the water temperature around 35±1 oC. The influent and the inhibition chemicals would been fed with the peristaltic pump. The inflow is 40 L/d and the hydraulic residence time is 12 h.
The entire operation of the ABR includes the start-up phase and stable operation phase. In the startup phase, the seed sludge pre-cultured for 30 d is inoculated evenly into each chamber of the ABR. The water quality during the start-up period is presented in The 2# and 5# Chamber Note: The amounts in tablewere based on 100L; NaNO 3 is added separately by pers added by peristaltic creep pump

Water for experiment
Water for experiment is synthesized by adding 180 gcane sugar and 90 g anhydrous Na 2 SO 4 , 5g fertilizer (supplying nitrogen and phosphate for microbial growth) and 130 g Na 2 HCO 3 (adjusting pH and neutralizing acid produced in the reactions) into 100 L tap water. The main inhibitory chemical is sodium nitrate, which is delivered according to the experiment demands. The average water quality of the influent of the ABR is COD of 1800mg/L, SO 4 2of 900 mg/L, pH 5.5-7.5 and alkalinity of 700mg/L.

Seed sludge
Seed sludge is collected from a secondary sedimentation tank in a sewage treatment plant. The collected sludge is precultured with water containing cane sugar and Na 2 SO 4 . The seeding amount of the sludge is 1.5 L in each chamber.

Analysis methods
SO 4 2is analyzed by absorption photometry; pH is detected with an acidity meter; alkalinity is measured with the potential titration method; S 2is analyzed with a sulfur ion selective electrode; Pb(NO 3 ) 2 is detected with a volumetric precipitation method; NO 3 is detected with a nitrate ion selective precipitation method; ORP is analyzed with a platinum electrode.

Construction of 16SrDNA clone library and sequence submissions to GenBank
The constructions of 16SrDNA clone library refer to the previous literature. The nucleotide sequences of 16S rDNA of the DNB have been deposited in the GenBank database under accession numbers from DQ450402 to DQ450424.

The performances of ABR during the rapid start-up phase
Stable performance of ABR is achieved rapidly with the abundant reproduction of SRB and the production of hydrogen sulfide. The removal efficiency of SO 4 2and S 2concentration are important indexes for the reactor operation. In the start-up phase, the concentrations of sulfate and S 2changed gradually. The removal efficiency of sulfate in the ABR reached 80 % after 10 d and ultimately reach 95%. The S 2concentrations exceed 110 mg/L . Thus, the reactor is successfully started. The other indexes of the influent and effluent are shown in Table 2.

The performances of ABR during the stable operation phase
The inhibition of SRB in the ABR is achieved through the regulation and the control of external ecological factors and the addition of inhibiting chemicals. The effects of the SO 4 2-/NO 3 -, pH value, alkalinity and COD concentration on the denitrification inhibition to sulfate reducing are investigated. Based on these investigations, the feasibility and the optimal operational ecological factors of the denitrification inhibition to sulfate reducing are explored. 4 2-/ NO 3 concentration ratio Because of the respective electron receptors for the SRB and DNB's reduction process, the concentrations of SO 4 2-and NO 3 are crucial for the SRB and DNB's activity . The regulation and control of SO 4 2-/NO 3 are significant for the SRB inhibition through denitrification process. The concentrations' variations of S and N indicate that along with the flow of the water and the extension of the reaction time, the sulfide concentration increase stepwise at different SO 4 2-/NO 3 - (Figure 2 , Figure 3). The total sulfide concentrations are lower at lower SO 4 2-/NO 3 -(6:3-6:6) than that at higher SO 4 2-/NO 3 -(6:0-6:2). It indicates that the sulfate reduction can be inhibited by denitrification with the addition of sufficient nitrate. The reason for higher sulfide concentration at higher SO 4 2-/NO 3 is the rapid decline of the nitrate concentration along with the flow of water (Figure 3). The nitrate concentration in the 3# chamber of the ABR is extremely low and can not be detected in the

Regulation and control of pH value
When having one of the ecological factors, pH is very important on the bacterial growth. Variations in pH value would cause different degree of impacts on the SRB and DNB in the ABR. Besides, the alkalinity would neutralize the acid produced in the incipient acidification period and thus maintain the pH value. With the addition of NaHCO 3 and Na 2 CO 3 , the influent pH is 5.5-7.3 and the alkalinity was 400-900 mg/L ( Figure 4). The pH value of the effluent ranges from 6.2 to 6.7, and the alkalinity increases with the decrease of SO 4 2-/NO 3 -. When the SO 4 2-/NO 3 decrease from 6:1 to 6:5, the alkalinity of the effluent increase from 1100 mg/L to 1700 mg/L and remain stable when fixed conditions are provided. Extra alkalinity is produced with the addition of NaNO 3 and the subsequent denitrification. The ABR is resistant to certain pH and alkalinity variations. The production of extra alkalinity by the ABR is verified as the alkalinity of the effluent is always higher than that of the influent. This is in accordance with the theory that the sulfate reduction and the denitrification will generate alkalinity. The enhancement in pH value would increase the activities of SRB but reduce that of DNB. The enhancement range of effluent alkalinity is 200 mg/L higher to that of the influent. Also, the effluent nitrate concentrations also increase compared to the former phase, which are strongly supported that the activity of the SRB will be increased but that of the DNB would been reduced.

Concentration variations of effluent sulfate and sulfion
The concentrations of effluent sulfate and sulfion are important parameters for the evaluation of the SRB inhibition efficiency. The influent sulfate is kept stable around 600 mg/L. The trend for the concentration variations of effluent sulfate and sulfion is reverse, which is in accordance with the conservation of sulfur. When the SO 4 2-/NO 3 is 6:1-6:4, the S 2concentration reduced from 120 mg/L to 60 mg/L ( Figure 5). It suggested that the regulation and the control of SO 4 2-/NO 3 could influence the efficiency of sulfate reduction inhibition. The sulfion concentrations gradually increase with the flow of the water on different experimental stage. The possible explanation is that the activity of DNB dropped as NO 3 is almost depleted in the back of the ABR. Most of the NO 3 is consumed in the front of the ABR and thus the SRB returned its predominance at the following chamber of the ABR. When the SO 4 2-/NO 3 is 6/5, the sulfion concentrations rapidly increase to more than 100 mg/L at the end of the ABR. It shows that SRB is predominant in the back of the reactor. Growth of DNB is almost lagged with the shortage of NO 3 -.

Conclusions
(1) The SO 4 2-/NO 3 and COD are important ecological factors for the inhibition of sulfate reduction by dinitrification. The best performances of sulfate reduction are obtained with the SO 4 2to NO 3concentration ratio 1/1Lower COD concentrations are beneficial for sulfate reduction inhibition. Obvious inhibition of DNB on SRB are detected in the 3# chamber of the ABR. The effective action sections are 2# and 5# chamber. with the effective time of 6 h. Thus, the addition of indictors should be conducted in different sections to assure the inhibition efficiency. (2) In the continous-flow ABR, when the effluent COD concentration decrease to 600 mg/L and the nitrate is added at the 2# and 5# chamber simultaneously with the SO 4 2-/NO 3 of 1/1, the sulfate reduction is completely inhibited. Thus, the inhibition of SRB by denitrification is feasible.