TY - JOUR
AU1 - Hao, Ruixia
AU2 - Meng, Chengcheng
AU3 - Li, Jianbing
AB - Abstract Two main operating parameters (influent C/N ratio and electric current intensity) were examined for their impacts on the denitrifying bacterial community structure in an integrated system of three-dimensional biofilm-electrode reactor and sulfur autotrophic denitrification (3DBER-SAD). It was found that genus β-proteobacteria played a leading role under different operating conditions. The influent C/N ratio illustrated a great impact on denitrifying bacteria diversity. When the C/N ratio decreased from 1.07 to 0.36, the Shannon–Wiener index and Simpson index increased from 2.44 to 2.71 and from 0.89 to 0.92, respectively, while the proportion of heterotrophic denitrifying bacteria Thauera decreased from 61.4 to 21.1%, and the sulfur autotrophic denitrifying bacteria (e.g., genus Sulfuricella and Thiobacillus denitrificans) increased from 3.5 to 19.3%. In terms of the impact of electric current intensity, the Shannon–Wiener index and Simpson index decreased from 2.71 to 2.63 and from 0.92 to 0.90, respectively, as the current intensity increased from 60 to 400 mA. Introduction The coupled heterotrophic and autotrophic denitrification process in wastewater treatment has received increasing attention in recent years due to its improved nitrogen removal efficiency [1, 14]. Among various coupled processes, the three-dimensional biofilm-electrode reactor (3D-BER) integrated with sulfur autotrophic denitrification (SAD) represents a novel technology. This new process, abbreviated as 3DBER-SAD, is especially useful for the treatment of wastewater with low organic carbon content, such as groundwater or municipal wastewater treatment plant (WWTP) effluent [5]. In this process, the autotrophic denitrifiers utilize H2 and elemental sulfur as the electron donors, thus leading to reduced consumption of organic carbon. The H+ generated by sulfur autotrophic denitrification can also increase the generation of H2, and neutralize OH− resulted from heterotrophic and hydrogenotrophic denitrification processes. The improved pH buffer capability would then ensure an appropriate pH condition (i.e., around neutral pH values) for better performance of various denitrifiers [7]. Our recent study revealed that 3DBER-SAD process achieved a high and much stable denitrification performance under various operating conditions [5]. This technology is still at its early development stage, and is worth of further investigation from different perspectives, among which the analysis of the involved denitrifying bacterial community is of fundamental importance. The understanding of microbial components and structure has guiding significance for improving biological wastewater treatment process [13]. In general, the denitrifying bacteria can reduce nitrate (NO3−) into nitrogen gas (N2) under oxygen-limited conditions through four steps of enzymatic reaction: NO3− → NO2− → NO (g) → N2O (g) → N2 (g) [31]. Each step is catalyzed by a separate terminal reductase, including nitrate reductase (nar), nitrite reductase (nir), nitric oxide reductase (nor), and nitrous oxide reductase (nos). Among them, the enzyme catalysis of nir is of great importance on the entire reaction process because it determines the first denitrification step of producing a gaseous NO [3]. As a result, the nir functional genes have been commonly used to detect the presence of denitrifying bacteria [2]. Located in the periplasmic space, nir occurs in two major types with similar physiological function, including cytochrome cd1-type nitrite reductase (nirS) and copper (Cu)-type nitrite reductase (nirK), with nirS type gene accounting for the majority of denitrifiers studied so far [19, 20]. For example, Zhi and Ji [32] examined the denitrification functional genes in a tidal flow constructed wetland (CW), and found that the nirS gene was much more abundant than nirK during the entire CW operation period, indicating that nirS gene played a dominant role in nitrogen removal. The primers for nitrite reductase genes (nirS and nirK) have been previously described. These two gene types could be distinguished by PCR, but they have never been observed in the same strain [19]. For example, Yang et al. [28] conducted PCR amplification to identify potential enzyme genes involved in heterotrophic nitrogen removal process by strain Acinetobacter junii YB for nitrogenous wastewater treatment, and found that only the nirS gene was detected in the strain isolate. The denitrifying microbial community can be affected by various environmental factors [11]. Yang et al. [27] applied RFLP analysis and sequencing of nirS gene clone libraries to reveal the structure of denitrifying community in a shallow eutrophic lake, and found that environmental factors could regulate the composition and distribution of the functional bacterial groups. The new 3DBER-SAD process can achieve a higher and more stable denitrification performance as compared to conventional denitrification methods, especially when it is used for treating wastewater with low carbon/nitrogen (C/N) ratio [5]. Such process involves various biological denitrification mechanisms resulted from a very complex biofilm microbial community which can be affected by various factors (e.g., influent C/N ratio, electric current intensity, pH, reactor configuration) [4]. However, the information of the involved denitrifiers and their interaction with the operating condition is unclear, while few studies have been reported in the literatures to examine such interaction even for conventional BER. In fact, the identification of the relevant microbial structure and diversity as well as the understanding of their variation with operating parameters will be critical for further improving the reactor performance [7]. The objective of this study was then to investigate the impacts of two main operation parameters (i.e., C/N ratio, electric current intensity) on the denitrifying bacterial community structure (especially the proportion of bacterial types) in 3DBER-SAD process. In this process, nickel foam cathode and graphite anode were used, while the activated carbon (AC) mixed with granular elemental sulfur was filled to form a third electrode. The denitrifying bacterial community under different operating conditions was analyzed using nirS-based clone library method. The results will provide valuable information and guidance for designing more cost-effective wastewater denitrification systems. Materials and methods Experimental apparatus and procedure Figure 1 presents the experimental apparatus which is a denitrifying reactor using upflow biological filtration. With a diameter of 25 cm and a height of 140 cm, the plexiglass cylindrical reactor had an effective volume of 22 L. Its double-layer nickel foam cathode (height of 96 cm) was set surrounding the inner wall, and its graphite-rod anode (diameter of 5 cm and height of 130 cm) was set in the reactor center. The remaining space in the reactor was filled with granular activated carbon (AC) (size of 5–8 mm) when it was used as a 3D-BER, and with a mixture of granular AC and elemental sulfur (S0) granules (size of 3–5 mm) at a mixing ratio of 8:1 when it was used as a 3DBER-SAD. A DC regulated power supply (0–60 V, 0–2A, model HSPY-60-2, Beijing Hansheng Puyuan Technology Co., Ltd., China) was used for water electrolysis. Fig. 1 Open in new tabDownload slide Schematic diagram of 3DBER-SAD or 3D-BER (1DC regulated power supply; 2 anode; 3 filler (activated carbon particles for 3D-BER, mixture of activated carbon particles with elemental sulfur granules for 3D-BER-SAD); 4 cathode; 5 biofilm sampling port; 6 water distribution plate; 7 water outlet port; 8 peristaltic pump; 9 influent tank) The seed sludge taken from an anaerobic–anoxic–oxic (A/A/O) process based WWTP in Beijing was used for biofilm development in the reactor through gradually changing the electric current from 0 to 140 mA. After biofilm development, the reactor was operated using simulated WWTP effluent as the influent which had total nitrogen (TN) concentration of 35 mg L−1, a PO43−-P concentration of 3.5 mg L−1, and a pH of 7.0–7.5. CH3COONa was added to provide carbon source for biological denitrification according to the desired C/N ratio (i.e., ratio of TOC to TN). The detailed biofilm immobilization and development procedures as well as the preparation of WWTP effluent were described in our previous studies [4, 5]. In this study, the impacts of two main operating parameters (i.e., the influent C/N ratio and electric current intensity) on the performance of 3DBER-SAD were investigated when the hydraulic retention time (HRT) was kept at 12 h [5]. This was conducted at various operating conditions, including high C/N ratio and low current intensity (C/N = 1.07:1, I = 60 mA), low C/N ratio and low current intensity (C/N = 0.36:1, I = 60 mA), as well as low C/N ratio and high current intensity (C/N = 0.36:1, I = 400 mA). The variation of bacterial community under these operating conditions was then analyzed to reveal the corresponding microbiological impacts. Microbiological analysis About 50 mL of AC and sulfur granules filler sample was taken from the biofilm sampling port of 3DBER-SAD (Fig. 1) under each operating condition. Following similar treatment procedure as described in Hao et al. [4], the sample was put in a 200-mL beaker to be stirred using a glass rod to strip off the biofilm from AC and sulfur granules. The obtained water-biofilm mixture was then subject to centrifugation (12,000 rpm for 5 min) to separate biofilm from the mixture. After centrifugation, the biofilm sample was stored in a 50-mL centrifuge tube at −20 °C. The sample was then used for the characterization of denitrifying bacterial community using DNA extraction, PCR amplification, clone library construction, colony PCR, nirS sequencing and phylogenetic analysis as described below [4]. The total community DNA was extracted from about 0.3 mg of biofilm sample using Ezup Soil Genomic DNA Extraction Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The primer pair, nirS1F [5′-CCTA(C/T)TGGCCGCC(A/G)CA(A/G)T-3′] and nirS6R [5′-A(C/G)(A/G)CGTTGAACTT(A/G)CCGGT-3′] which correspond to the 763–780 and 1638–1653 positions of the nirS gene of Pseudomonas stutzeri Zobell (X53676), was used for the PCR amplification of nirS genes [30]. The PCR mixture contained 1.5 μL of extracted DNA, 20 pmol of each primer (1 μL for each), 0.25 mM of deoxynucleoside triphosphate (dNTP) (4 μL), 5 μL of 10× PCR buffer (TaKaRa Inc., Dalian, China), and 5 U/μL concentration of Taq DNA Polymerase (0.2 μL) (TaKaRa Inc.). The mixture was added with sterile deionized water to obtain a final volume of 50 μL. The PCR amplifications were then conducted in 0.2-mL reaction tubes on a Thermal Cycler (Jinke, Hangzhou, China) [4]. A “touchdown” PCR was performed using an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C (30 s), primer annealing (40 s), and DNA chain extension (40 s) at 72 °C. The annealing temperature was decreased from 56 to 51 °C at a rate of 0.5 °C/cycle during the first ten cycles, and was kept at 51 °C during the remaining 25 cycles. The PCR was completed by a final DNA chain extension at 72 °C for 7 min. All PCR products were then analyzed on 1.2% (w/v) agarose gels. After agarose gel electrophoresis of the amplified nirS genes, PCR products were excised in the agarose gel between size 800 bp and 1000 bp [22]. The gel containing PCR products was cut with a sterile knife and was then put into 2-mL centrifuge tube for purification using the “SanPrep Gel and PCR Clean-Up System” (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The purified PCR product was cloned with the pMD 18-T Vector (Takala Inc., Japan), and the competent Escherichia coli JM109 cells (Takala Inc., Japan) were transformed according to manufacturer’s recommendations [22]. Aqueous LB broth was added to tubes which contained cells for cultivation in a shaking water bath at 37 °C for about 60 min at 200 r/min. The cell culture fluid was then distributed on the LB-Agar plate (containing AMP, X-Gel and TPTG), and was subject to inverted cultivation for 16 h at 37 °C. The blue and white screening of colony was then conducted to select recombinant transformants. Not all of the white clones have the target gene, and some of them may be false positive clones. As a result, the direct colony PCR was conducted to ensure positive clones using vector-targeted primers M13-47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and RV-M (5′-GAGCGGATAACA-ATTTCACACAGG-3′). The reaction system contained: 20 μM of each primer (0.5 μL for each), 0.25 mM of deoxynucleoside triphosphate (2 μL), 2.5 μL of 10× PCR buffer, and 5 U/μL of Taq DNA Polymerase (0.2 μL) (Takala Inc.). The PCR mixture was added with sterile deionized water to obtain a final volume of 25 μL. The PCR amplifications were performed in 0.2-mL reaction tubes on a Thermal Cycler (Jinke, Hangzhou, China) using similar programs described above, except that the annealing temperature was starting at 60 °C and then decreasing during the first ten cycles by 0.5 °C/cycle until it reached a touchdown at 55 °C, while the remaining 25 cycles were performed at an annealing temperature of 54 °C [4]. Agarose gel electrophoresis was then used to examine the PCR products for screening positive clone cells which correspond to the amplified fragments between size 800 and 1000 bp. In terms of nirS gene sequencing and phylogenetic analysis, three nirS gene libraries were constructed in total. They were designated in this study as library A, B and C, which corresponds to three different operating conditions of 3DBER-SAD: (A) high C/N and low current intensity, (B) low C/N and low current intensity, and (C) high C/N and low current intensity. A total of 57, 57 and 50 positive clones were randomly selected from library A, B, and C, respectively. These clones were then sequenced by a 3730 type DNA sequencing system (ABI, USA) using a BigDye Terminator v1.1 sequencing kit (ABI, USA) [4]. Multiple sequences were aligned using DNAMAN software package, and the operational taxonomic unit (OTU) was divided. A representative sequence in each OTU was then selected to compare with those in the sequence database from National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), and the high similarity sequence was downloaded for reference. Sequences determined in this study and those retrieved from the database were aligned using Clustal W software. The phylogenetic tree was then constructed by a neighbor-joining algorithm using the MegAlign software version 5.03 (DNAstar, USA) to obtain broader groupings for revealing the composition of bacterial community. As a result, the variation of the proportions of different bacterial classes in the biofilm community with operating condition can be quantified. However, the mass variation of bacterial population was not examined in this study although the 3DBER-SAD denitrification efficiency may serve as an indirect indicator of such variation. Scanning electron microscopy (SEM) analysis The surface morphology of microorganisms in biofilm was investigated by scanning electron microscopy (SEM). Activated carbon (AC) and sulfur (S0) granules with biofilm were taken from the sampling port of 3DBER-SAD (Fig. 1) at C/N ratio of 0.36 and current intensity of 400 mA. Both of the AC and S0 granules with and without biofilm were analyzed by SEM (HITACHIS-4300, Hitachi). The detailed sample treatment procedures for SEM analysis were described in our previous study [4]. Estimation of bacterial diversity Coverage (C) was used to measure the captured bacterial diversity. It is the proportion of nirS-based clone library microbial species (OTUs) in the sample as calculated below: C=1-n1N×100% $$ C = \left( {1 - \frac{{n_{1} }}{N}} \right) \times 100\;\% $$1 where N is the total sequenced number of clones in the library, and n 1 is the number of different OTU types from a clone library that were encountered only once. If coverage C is higher or reaches 100%, the capacity of clone library was considered sufficient [12]. The Shannon–Weaver index (H) and Simpson index (D) of diversity were used to determine the weighted diversity in each library, as calculated by the following equations: H=-∑niN×InniN $$ H = - \sum {\left[ {\frac{{n_{i} }}{N} \times {\text{In}}\left( {\frac{{n_{i} }}{N}} \right)} \right]} $$2 D=1-∑niN2 $$ D = 1 - \mathop \sum \nolimits \left( {\frac{{n_{i} }}{N}} \right)^{2} $$3 where n i is the number of species i in the sample. Results and discussion Denitrification performance of 3DBER-SAD As seen in Fig. 2, the 3DBER-SAD illustrated a consistently higher and more stable nitrogen removal performance than 3D-BER under various C/N ratio and electric current intensity conditions. The average TN removal rate reached 89, 86, and 85% for 3DBER-SAD under the three operating conditions (C/N = 1.07:1 and I = 60 mA; C/N = 0.36:1 and I = 60 mA; C/N = 0.36:1 and I = 400 mA), as compared to 73, 46, and 63% for 3D-BER, respectively. It can be found that the 3DBER-SAD performance was less affected by C/N ratio and electric current intensity, and it is thus particularly suitable for low C/N ratio wastewater treatment even under low electric current condition. Its TN removal rate was about 40% higher than that of 3D-BER at C/N = 0.36 and I = 60 mA. This is of particular importance for developing more cost-effective denitrification process for wastewater reclamation [5]. The variations of other parameters including pH and SO42− accumulation under different C/N ratio and electric current intensity conditions can be found in Hao et al. [5] which indicated that 3DBER-SAD process had a strong pH stabilization capability. The high and stable performance as well as the stronger pH buffer capability of 3DBER-SAD occurred due to the synergistic effect of heterotrophic, electrochemical hydrogenotrophic and sulfur autotrophic denitrification [5, 7]. The examination of its microbial ecology is then of necessity to reveal such synergistic effect. Fig. 2 Open in new tabDownload slide Variation of TN removal rate for 3D-BER-SAD and 3D-BER (other operating conditions: influent TN = 35 mg L−1, N:P = 10:1, HRT = 12 h, pH = 7.0–7.5, T = 15–20 ℃)) Surface morphology of biofilm in 3DBER-SAD Figure 3 presents the surface morphology of AC and S0 particle without and with biofilm. It can be found that the 3DBER-SAD filler particles have uneven surface with large specific surface area which can facilitate the attachment of microorganisms. Under low C/N ratio (i.e., 0.36:1) condition, the biofilm microorganisms were mainly in short rod (1–2 μm) shape on the surface of S0 granule (Fig. 3b), and in both short rod (1–2 μm) and axiolitic shape (0.5–1 μm) on the surface of AC granule (Fig. 3d). Both short rod and axiolitic shapes are the different morphologies of microorganisms, and our previous study [4] indicated that during denitrification process the Thiobacillus-like and Thauera-like bacteria were mostly short rod-shaped, while the axiolitic shaped microorganisms may be Enterobacter-like bacteria. Fig. 3 Open in new tabDownload slide Surface morphology of sulfur particle (a without biofilm, b with biofilm) and activated carbon particle (c without biofilm, d with biofilm) (3DBER-SAD operating condition: C/N = 0.36:1, I = 400 mA, influent TN = 35 mg L−1, N:P = 10:1, HRT = 12 h, pH = 7.0−7.5, T = 15−20 ℃) Denitrifying bacteria community under high C/N ratio and low current intensity condition Under the high influent C/N ratio and low current intensity operating condition (C/N = 1.07:1, I = 60 mA), a total of 15 OTUs were obtained from clone library A. The coverage (C) of library A was 92.98%, indicating that the clone library accounted for a high proportion of all the microbial species types in the 3DBER-SAD system. Figure 4 presents the summarized phylogenetic groups of the representative clones in each of the 15 OTUs, and Fig. 5 presents the phylogenetic dendrogram of nirS genes in 3DBER-SAD. It was found that all the clones of library A belong to genus β-proteobacteria, indicating that β-proteobacteria played a leading role in 3DBER-SAD system. In fact, β-proteobacteria has diversified metabolism types, including chemoorganoheterotroph, photolithoautotroph, chemolithoautotroph and methylotrophs [21]. Most bacteria species in β-proteobacteria obtain nutrients by breaking down organic matter, while some of them make use of hydrogen, ammonia, methane, and volatile fatty acids. Fig. 4 Open in new tabDownload slide Proportion of each denitrifying bacteria genus in clone library A Fig. 5 Open in new tabDownload slide Neighbor-joining tree showing positions of phylotypes affiliated with Thauera, Thiobacillus, Acidovorax, Azoarcus and Dechloromonas of clone library A (Z represents a clone; the numbers at the nodes indicate percentage of occurrence in 1000 bootstrapped trees, and the two clone groups represent closely related sequences) Genus β-proteobacteria has been found dominant in many denitrification systems [26, 29]. Thauera is a Gram-negative bacterium belonging to β-proteobacteria. It was reported that most Thauera species were heterotrophic bacteria in rod shape [18]. Under anaerobic respiration conditions, they can make use of benzoate, phenyl acetate, acetate and ethanol as electron donors for denitrification [16]. Liu et al. [10] investigated eight Thauera strains and proved the efficient denitrification ability of Thauera in wastewater treatment system. In this study, 35 clones (6 OTUs) had high similarity with Thauera in clone library A, accounting for 61.4% of the total clones (Fig. 4). This indicates that Thauera denitrifying bacteria is dominant under the condition of low current intensity and high C/N ratio. As shown in Fig. 4, the abundance of Thiobacillus denitrificans was 3.5% in clone library A. Thiobacillus denitrificans is a genus of Hydrogenophilaceae. [21]. Its metabolism type includes obligate autotrophy and facultative anaerobic. It can take advantage of elemental sulfur as its electron donor. Under anaerobic respiration conditions, Thiobacillus denitrificans can get energy through the oxidization of sulfur and make nitrate as the electron acceptor to achieve denitrification [9, 15, 24]. Although the proportion of Thiobacillus denitrificans was relatively small, it played an important role in stabilizing and improving denitrification efficiency of the 3DBER-SAD system. In addition, 11 clones (2 OTUs) of library A had certain similarity with Acidovorax, accounting for 19.3% of the total clones (Fig. 4). Acidovorax is sub-taxa of Comamonadaceae which is a kind of curved rod-shaped Gram-negative bacteria, with metabolism types of chemoorganoheterotroph or facultative chemolithoautotroph. Acidovorax has the ability of using both acetate and hydrogen for denitrification [21, 23]. Moreover, as shown in Fig. 4, there are five clones (two OTUs) having a similarity of 100% with Dechloromonas, and four clones (two OTUs) having certain similarity with Azoarcus. Dechloromonas can oxidize organic acids (e.g., acetate) and reduce nitrate and nitrite, and it can also utilize sulfate, fumarate, chlorate or perchlorate as electron acceptor in addition to nitrate and nitrite. Azoarcus is a facultatively anaerobic, mesophilic and Gram-negative bacterium, and can grow with a variety of organic substrates such as short-chain fatty acids, alcohols, and amino acids with nitrate as an electron acceptor [6]. The results illustrated that the proportion of heterotrophic denitrifying bacteria (e.g., Thauera) was much greater than that of sulfur autotrophic denitrifying bacteria (e.g., Thiobacillus denitrificans) and other denitrifying bacteria when the 3DBER-SAD system was sufficient in organic carbon source. The heterotrophic denitrifying bacteria played the dominant role in the denitrifying process of 3DBER-SAD under high C/N ratio condition. This is in agreement with the previous studies. For example, Sun and Nemati [17] reported that the microbial reaction rate of converting nitrite into nitrogen was faster when the electron donor was acetate, and lower when the electron donor was elemental sulfur. Denitrifying bacteria community under low C/N ratio and low current intensity condition Under the low influent C/N ratio and low current intensity operating condition (C/N = 0.36:1, I = 60 mA), a total of 18 OTUs were obtained from clone library B. The coverage (C) of library B was 84.21%. Figure 6a, b present the distribution of β-proteobacteria, α-proteobacteria and γ-proteobacteria as well as the proportion of each denitrifying bacteria genus in clone library B, and Fig. 7 presents the corresponding phylogenetic dendrogram of nirS genes. Fig. 6 Open in new tabDownload slide Distribution of proteobacteria in a clone library B and c clone library C and as well as the proportion of each denitrifying bacteria genus in, b clone library B, and d clone library C Fig. 7 Open in new tabDownload slide Neighbor-joining tree showing positions of phylotypes affiliated with bacteria such as Thauera, Thiobacillus, Sulfuricella, Azoarcus and Dechloromonas of clone library B (L represents a clone; the numbers at the nodes indicate percentages of occurrence in 1000 bootstrapped trees) Similar to the high influent C/N ratio and low current intensity condition, it can be found that β-proteobacteria also played a leading role in 3DBER-SAD system under low influent C/N ratio and low current intensity condition. About 68.4% of the clones of library B belong to β-proteobacteria, while the proportion of α-proteobacteria (1.8%) and γ-proteobacteria (1.8%) was much smaller (Fig. 6a). As shown in Fig. 6b, genus Thauera, Dechloromonas, Sulfuricella and Thiobacillus denitrificans accounted for 21.1, 22.8, 12.3 and 7.0% of the total clones in library B, respectively. Similar to Thiobacillus denitrificans, Sulfuricella is another kind of sulfur autotrophic denitrification bacteria. Sulfuricella denitrificans strain skB26 (Fig. 7) is a rod-shaped, motile and Gram-negative bacterium. It is a psychrotolerant sulfur oxidizer recently reported to be isolated from a freshwater lake, and represents a new genus in the class of β-proteobacteria. Its identified genes include those required for sulfur oxidation, denitrification, and carbon fixation [8, 25]. Genus Alicycliphilus, Pseudomonas, Paracoccus were not existing in clone library A, but they were contained in library B, each accounting for 1.8% of the total clones (Fig. 6b). Alicycliphilus belongs to Comamonadaceae that is in the class of β-proteobacteria, while Paracoccus and Pseudomonas belong to α-proteobacteria and γ-proteobacteria, respectively. Some species of these bacteria are known as the hydrogenotrophic bacteria [23]. The Uncultured bacterium accounts for 28.1% in library B (Fig. 6b), and their physiological properties need further research. Denitrifying bacteria community under low C/N ratio and high current intensity condition Under the low influent C/N ratio and high current intensity operating condition (C/N = 0.36:1, I = 400 mA), a total of 18 OTUs were obtained from clone library C. The coverage (C) of library C was 88.0%. Figure 6c, d present the distribution of β-proteobacteria and α-proteobacteria as well as the proportion of each denitrifying bacteria genus in clone library C, and Fig. 8 presents the corresponding phylogenetic dendrogram of nirS genes. It can be found that β-proteobacteria still played a leading role under these two operating conditions, but its proportion in the community decreased at lower C/N ratio. Fig. 8 Open in new tabDownload slide Neighbor-joining tree showing positions of phylotypes affiliated with bacteria such as Thauera, Thiobacillus, Sulfuricella and Dechloromonas of library C (H represents a clone; the numbers at the nodes indicate percentages of occurrence in 1000 bootstrapped trees) About 68.0% of the clones of library C belong to β-proteobacteria, while the proportion of α-proteobacteria (6.0%) was much smaller than that of β-proteobacteria (Fig. 6c). As shown in Fig. 6d, genus Comamonadaceae bacterium (belonging to Comamonadaceae), Alicycliphilus and Acidovorax accounts for 6, 4, and 8% of the total clones in library C, respectively. These hydrogenotrophic bacteria (i.e., a total of 18%) can use hydrogen for denitrification. Genus Thauera, Sulfuricella, Azoarcus, Alicycliphilus, Thiobacillus and Acidovorax accounted for 12, 22, 4, 4, 2 and 8% of the total clones in library C, respectively. These six genera were also present in library A or library B (Figs. 4, 6b). However, some species of bacteria in library C (e.g., Magnetospirillum, Dechlorosoma suillum and Dechlorospirillum) were not found in library A and library B, even though their proportion was relatively low. There have been few studies about the physiological characteristic of these three kinds of bacteria, and thus their denitrification function in 3D-BER-SAD is unclear. Impact of C/N ratio on denitrifying bacteria community structure The above results indicated that the operating condition of 3DBER-SAD could affect the diversity and composition of denitrifying bacterial community. The influent C/N ratio had a great influence on the bacteria community structure although the TN removal rate was less affected by C/N ratio as seen in Fig. 2. Table 1 presents the Shannon–Wiener and Simpson index of clone library A, B and C. When the C/N ratio decreased from 1.07 to 0.36, the Shannon–Wiener and Simpson index of the nirS-based clone library increased from 2.44 to 2.71 and from 0.89 to 0.92, respectively. This demonstrates that the diversity of denitrifying bacterial community in 3DBER-SAD was relatively higher at a lower C/N ratio, probably due to enriched particular heterotrophic denitrifying microorganisms at high C/N ratio. For example, all the clones of library A belong to β-proteobacteria, but only 68.4% of the clones in library B belong to β-proteobacteria (Fig. 6a). Library B also contained α- and γ-proteobacteria. Although β-proteobacteria was dominant in the 3DBER-SAD biofilm, its proportion decreased with C/N ratio. Shannon–Wiener and Simpson index of clone library A, B and C Index . Library A . Library B . Library C . Shannon–Wiener 2.44 2.71 2.63 Simpson 0.89 0.92 0.90 Index . Library A . Library B . Library C . Shannon–Wiener 2.44 2.71 2.63 Simpson 0.89 0.92 0.90 Open in new tab Shannon–Wiener and Simpson index of clone library A, B and C Index . Library A . Library B . Library C . Shannon–Wiener 2.44 2.71 2.63 Simpson 0.89 0.92 0.90 Index . Library A . Library B . Library C . Shannon–Wiener 2.44 2.71 2.63 Simpson 0.89 0.92 0.90 Open in new tab The C/N ratio also affected the proportion of denitrifying bacteria with different nutritional types in the 3DBER-SAD system. For example, Thauera denitrifying bacteria using organic carbon as electron donor had a significantly lower proportion at lower C/N ratio, and its proportion in library A and library B was 61.4 and 21.1%, respectively (Figs. 4, 6b). Thiobacillus denitrificans using sulfur as electron donor accounted for 3.5% in library A (Fig. 4), and the sum of Sulfuricella and Thiobacillus denitrificans using sulfur accounted for 19.3% in library B (Fig. 6b). In addition, the proportion of Acidovorax using both organic carbon and hydrogen was 19.3% in library A (Fig. 4), but no Acidovorax were found in library B (Fig. 6b). This probably means that Acidovorax bacteria were mostly using organics as the electron donor. However, other genus of hydrogenotrophic bacteria, such as Alicycliphilus, Pseudomonas and Paracoccus, were found in library B, and their total proportion was 5.3% (Fig. 6b). In library A, Dechloromonas accounted for 8.8%, but its proportion was 22.8% in library B (Figs. 4, 6b). Figure 9a presents the probable distribution of heterotrophic and sulfur autotrophic denitrifying bacteria in library A and library B. When the influent C/N ratio decreased from 1.07 to 0.36, the proportion of heterotrophic denitrifying bacteria had a significant decrease, while sulfur autotrophic denitrifying bacteria had a significant increase. Fig. 9 Open in new tabDownload slide Comparison of denitrifying bacteria distribution, a heterotrophic and sulfur autotrophic denitrifying bacteria in clone library A and library B, b hydrogenotrophic and sulfur autotrophic denitrifying bacteria in library B and library C Impact of current intensity on denitrifying bacteria community structure The electric current intensity also had a considerable influence on the denitrifying bacteria community in 3DBER-SAD system, although the TN removal rate was less affected by this factor as seen in Fig. 2. As shown in Table 1, the Shannon–Wiener and Simpson index of the nirS-based clone library decreased from 2.71 to 2.63 and from 0.92 to 0.90, respectively, as the current intensity increased from 60 to 400 mA, illustrating that the diversity of denitrifying bacterial community in 3DBER-SAD was relatively lower at higher current intensity. This is probably because the strong electric current (i.e., 400 mA) had a negative effect on the metabolism of some denitrifying bacteria species, and thus decreased their diversity. For example, β-proteobacteria accounted for 68.4 and 68.0% in library B and library C (Fig. 6a, c), respectively, showing that β-proteobacteria played a key role in 3DBER-SAD. Both of α- and γ-proteobacteria accounted for 1.8% in library B (Fig. 6a). In library C, α-proteobacteria occupied a proportion of 6%, but no γ-proteobacteria was present (Fig. 6c). Similar to the effect of influent C/N ratio, the electric current intensity affected the proportion of denitrifying bacteria with different nutritional types. As the current intensity increased, the proportion of Thauera and Thiobacillus denitrificans in library B and library C decreased from 21.1 to 12% and from 7 to 2%, respectively (Fig. 6b, c). However, there was little variation in the proportion of Sulfuricella with the increase of current intensity. In addition, Dechloromonas which accounted for 22.8% of the total clones in library B (Fig. 6b) was not present in library C, while other species (e.g., Comamonadaceae bacterium, Magnetospirillum, Dechlorosoma suillum and Dechlorospirillum) contained in library C (although with low proportion) were not found in library B (Fig. 6b). Figure 9b presents the probable distribution of hydrogenotrophic and sulfur autotrophic bacteria in library B and library C. It can be seen that the proportion of hydrogenotrophic bacteria was obviously increased along with the rise of current intensity due to increased production of H2. The changed denitrifying bacteria community structure with operating conditions in 3DBER-SAD could ensure its improved and stable denitrification performance. Conclusion The nirS clone library analysis method was used to examine the denitrifying bacterial community in 3DBER-SAD. Although the influent C/N ratio and electric current intensity had little impacts on the denitrification performance of this novel process, they greatly affected the denitrifying bacterial community structure in the biofilm. In general, both the C/N ratio and electric current showed a negative impact on the bacterial diversity, with high diversity associated with low C/N ratio and low current intensity. The operating parameters also affected the proportion of denitrifying bacteria with different nutritional types. When the C/N ratio decreased, the proportion of heterotrophic denitrifying bacteria also decreased but sulfur autotrophic denitrifying bacteria increased. When the current intensity increased, the proportion of hydrogenotrophic denitrifying bacteria increased. Such changed composition of denitrifying bacteria community with operating conditions in 3DBER-SAD could ensure its improved and stable denitrification performance. Acknowledgements This research was supported by the Natural Science Foundation of China (No. 51378028). JL acknowledges the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program that enabled him to participate in the research collaboration. The authors thank the anonymous reviewers for their comments and suggestions that helped in improving the manuscript. Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest All authors declare that they have no competing interests. References 1. Capua FD , Papirio S, Lens PNL, Esposito G Chemolithotrophic denitrification in biofilm reactors Chem Eng J 2015 280 643 657 10.1016/j.cej.2015.05.131 Google Scholar Crossref Search ADS WorldCat 2. 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TI - Impact of operating condition on the denitrifying bacterial community structure in a 3DBER-SAD reactor
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-016-1853-4
DA - 2017-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/impact-of-operating-condition-on-the-denitrifying-bacterial-community-8a06mmmIyc
SP - 9
EP - 21
VL - 44
IS - 1
DP - DeepDyve
ER -