Microcystis aeruginosa flour as carbon and nitrogen source for aerobic denitrification and algicidal effect of Raoultella sp. R11

Ecological Engineering 105 (2017) 162–169

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Microcystis aeruginosa flour as carbon and nitrogen source for aerobic denitrification and algicidal effect of Raoultella sp. R11 Jun feng Su a,b,∗ , Ting ting Lian a , Ting lin Huang a,b , Dong hui Liang a , Min Ma a , Jin suo Lu a,b a b

School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China Key Laboratory of Northwest Water Resources, Environment and Ecology, MOE, Xi’an 710055, China

a r t i c l e

i n f o

Article history: Received 1 December 2016 Received in revised form 12 March 2017 Accepted 2 April 2017 Keywords: Algicidal bacteria Aerobic denitrification Microcystis aeruginosa flour Response surface methodology (RSM) Fourier transform near infrared

a b s t r a c t Harmful algal blooms (HABs) could be deemed hazardous materials in aquatic environments. In this study, we investigated aerobic denitrification and algicidal effects of the Raoultella sp. R11. Based on PCR amplification, the denitrification genes napA was detected. A response surface methodology (RSM) analysis demonstrated that the maximum nitrate removal ratio was 96.06%, and optimal conditions occurred for inoculum of 13.02% (v/v), initial pH of 6.65, C/N ratio of 7.59 and temperature of 27.08 ◦ C based on a ridge analysis. When the Microcystis aeruginosa flour as carbon and nitrogen source for denitrification in aerobic conditions, the NO3 − -N and TOC removal ratios were 72.36% and 85.56%, respectively. Additionly, the denitrification rate was 0.0123 mg L−1 h−1 . Furthermore, the infrared spectrogram and SEM images showed the M. aeruginosa cell structure was destroyed, indicating that R11 could use algal death for denitrification. Therefore, Raoultella sp. R11 played a crucial role for simultaneous denitrification and algicidal effects in wastewater treatment. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Harmful algal blooms (HABs) have become a serious problem all over the world in recent years. Harmful algae often produce toxins to the environment, and the toxic products jeopardize marine organisms and destroy marine ecosystem balance (Glibert and Bouwman, 2012; Reichwaldt et al., 2013). Therefore, control of harmful algal blooms is crucial for maintenance of safe water supplies and also ecological health. In order to resolve the HABs and relieve the damage, several management strategies including physical, chemical, and biological have been tried. Although these methods exhibit algicidal effects to some extent in specific areas, their limitations were obvious and some adverse ecological consequences were shown (Jeong et al., 2000). Therefore, research on feasible and environmentally acceptable approaches to mitigate and control blooms has important theoretical and practical significance. However, the use of bacteria may be an effective method to control HABs. Several bacteria are known to have strong algicidal

∗ Corresponding author at: School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China. E-mail address: [email protected] (J.f. Su). http://dx.doi.org/10.1016/j.ecoleng.2017.04.014 0925-8574/© 2017 Elsevier B.V. All rights reserved.

activity (Dockyu et al., 2008; Kim et al., 2008a,b). Algicidal bacteria can inhibit algal growth or lyse cells, directly by physical contact or indirectly by excreting active compounds such as extracellular enzymes and algicidal compounds (Wang et al., 2010). As is known to all, nitrate is one of the most common contaminants in natural environment. However, biological denitrification is the most important method to remove nitrate. Biological denitrification occurs naturally when certain bacteria use nitrate as the terminal electron acceptor in their respiratory processes in the absence of oxygen (Zumft, 1997). Furthermore, most denitrifying bacteria are heterotrophic bacteria, which utilize organic carbon source as the electron donor. Therefore, biological denitrification was a good method for removing nitrate. (Moon et al., 2008; Kim et al., 2002). In this work, strain R11 denitrifying enzymes genes were investigated to explore the mechanism of aerobic denitrification. Meanwhile, Box-Behnken design and response surface methodology were used to design the experiments, and determine the optimum conditions for aerobic denitrification. The statistical design was based on four factors (inoculum, initial pH, C/N ratio and temperature). Furthermore, this paper presents an interesting study on the role of Microcystis aeruginosa flour as carbon and nitrogen source for denitrification in aerobic consditions. Finally, the FT-NIR was employed to reveal the mechanism of algae-lysing.

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2. Materials and methods

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2.5. Aerobic denitrification performance using Microcystis aeruginosa flour as carbon and nitrogen source

2.1. Algal and bacterial culture Microcystis aeruginosa was provided from the Freshwater Algae Culture Collection of Institute of Hydrobiology (FACHB), Chinese Academy of Sciences (Wuhan, China). And, the Microcystis aeruginosa maintained as a unialgal axenic culture at 28 ◦ C,12:12 light/dark cycle with a white light intensity of 3300 Lux. The Microcystis aeruginosa cells were incubated in BG11 medium (Rippka et al., 1979). Before used as an inoculant, it was cultured for 7 d to reach the log phase. Strain R11(GenBank accession number KT005386) (Su et al., 2016) was isolated from a eutrophic Qu Jiang lake, Xi’an of China. In order to analyze algicidal and denitrification characteristic, and strain R11 was grown in sterilized LB (Luria-Bertani) medium and HM (heterotrophic medium), respectively. The LB medium was comprised of following reagents per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl. The final pH of LB was adjusted to 7.2. The HM (pH 7.0) included the following reagents per liter: CH3 COONa, 0.1 g; NaNO3 , 0.02 g; K2 HPO4 , 0.02 g; MgCl2 , 0.01 g; CaCl2 , 0.01 g and 2 mL of trace elements solution. The ingredients of TE (trace element) solution were as follows per liter: MgSO4 ·7H2 O, 0.5 g; EDTA, 1.0 g; ZnSO4 , 0.2 g; MnCl2 ·4H2 O, 0.1 g, FeSO4 ·7H2 O, 0.5 g; CuSO4 ·5H2 O, 0.5 g; CoCl2 ·6H2 O, 0.2 g. Here, strain R11 at exponential phase was used unless specially mentioned.

2.2. The preparation of Microcystis aeruginosa flour Microcystis aeruginosa flour was prepared by the following methods: the solution of Microcystis aeruginosa was centrifuged at 8000 rpm for 10 min. Then, the sediment was dried at 120 ◦ C for 3 h, the dry Microcystis aeruginosa should be ground into flour by mortar.

In order to investigate aerobic denitrification performance of strain R11, the Microcystis aeruginosa flour was added to the HM in replace of CH3 COONa and NaNO3 . And, the new medium was called MAFM (Microcystis aeruginosa flour medium), which was comprised of the following reagents per liter: Microcystis aeruginosa flour, 0.1 g; K2 HPO4 , 0.02 g; MgCl2 , 0.01 g; CaCl2 , 0.01 g and 2 mL of trace elements solution. 10% inoculum by volume was added to the 250 mL conical flask with 200 mL sterile MAFM. The conical flask was cultivated with constant shaking at 120 rpm and 30 ◦ C for 5 d. The samples were obtained from the conical flasks at 8-h intervals to determine the pH, the concentrations of NO3 − -N. Each experiment was performed in triplicate.

2.6. Fourier transform near-infrared spectroscopy Strain R11 was incubated in sterile LB medium at 30 ◦ C when it reached logarithmic growth phase. Then, 10%, 15%, 20%, 25% and 30% (v/v) inoculum of strain R11 were added to into Microcystis aeruginosa culture, respectively. Meanwhile, 10%, 15%, 20%, 25% and 30% (v/v) inoculum in sterile LB medium were added into Microcystis aeruginosa culture as control. The all algal-bacterial mixture of treatment and control were cultivated at 28 ◦ C,12:12 light/dark cycle with a white light intensity of 3300 Lux. After 8 days, the all algal-bacterial mixture of treatment and control were centrifuged at 8000 rpm for 10 min, and the sediment were washed with distilled water 3 times. The washing sediment should be centrifuged again and then to collect the algae cells and then the algae cells were made into algae flour by cryogenic freeze-drying machine. Finally, the all samples were measured by FT-NIR. The background scan was always conducted with a golden slit before acquiring the spectrum from each kernel.

2.3. PCR amplification of nitrate reductase genes In order to determine whether strain R11 contained nitrate reductase gene, the primers NAP1/NAP2 were used for napA amplification, and conducted as described (Huang et al., 2015).

2.4. Box-Behnken design for optimizing the environmental factors The RSM was applied for evaluation of the effects of aerobic denitrification by strain R11 and their optimization for various responses. In this work, the three levels, four factors (inoculum, C/N ratio, initial pH and temperature) were chosen. The levels of four independent variables at three levels (+1, −1, 0) were defined according to the Box-Behnken design, and 29 experiments were required for the procedure. A series of experiments were carried out with different inoculum (v/v; 5%, 10%, 15%); initial pH (5, 7, 9); C/N ratio (3, 6, 9) and temperature (25 ◦ C, 30 ◦ C, 35 ◦ C). DesignExpert (version 8.06) software was used for the statistical design of experiments and data analysis. The experimental data were fitted to second-order polynomial function to obtain the regression coefficients (b). The function for the four factors is expressed as follows: Y i = b0 +



bi x i +



bii xi 2 +



bij xi xj

Where Yi is the nitrate removal ratio of strain R11, Xi and Xj are variables, bo is the constant, bi is the linear coefficient, bij is the interaction coefficient, and bii is the quadratic coefficient.

2.7. Analytical methods and statistical analysis The liquid samples were filtered through 0.45 ␮m membrane for analysis. NO3 − -N, NO2 − -N and NH4 + -N were determined by ultraviolet spectrophotometer (DR5000, HACH, American) according to standard methods. The concentration of chlorophyll-a was measured according to the method (Yang et al., 2007). TOC was measured by a TOC analyzer (jena multi N/C 3000, Germany). The pH value was determined by a pH meter (MM10, HACH, American). The nitrate and chlorophyll-a removal ratio formula was (C0 − Cn )/C0 × 100%. C0 is initial concentration of nitrate and chlorophyll-a. Cn is final concentration of nitrate and chlorophylla. Data in this experiment was analysed by Microsoft excel and Origin9.0 software.

3. Results and discussion 3.1. PCR amplification of denitrification genes As shown in Fig. 1, 877 bp of the napA gene fragment was amplified from strain R11, suggesting that strain R11 exhibits nitrate reduction under aerobic conditions. NAP could play a role in both aerobic and anaerobic conditions, but is more important in aerobic conditions. NAP is essential for the conversion of nitrate under aerobic conditions and is often used as a functional marker to identify aerobic denitrifying bacteria (Huang et al., 2013).

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Fig. 1. The amplification of napA gene of Raoultella sp. R11. Mark: DL 2000 DNA Marker (TaKaRa, Japan).

3.2. Box-Behnken design for optimization of environmental factors Twenty-nine sets of experiments were conducted by using the Box-Behnken method. By applying multiple regression analysis methods, the final model was as follows: Y = 92.55 + 9.94X1 − 1.75X2 + 6.87X3 − 15.56X4 − 6.84X1 X1 − 15.56X2 X2 − 17.06X3 X3 − 47.71X4 X4 + 7.50X1 X2 + 9.56X1 X3 − 9.00X1 X4 + 15.19X2 X3 − 5.81X2 X4 − 6.00X3 X4 where Y is the predicted response of nitrate removal ratio, X1 , X2 , X3 and X4 are the inoculum, initial pH, C/N ratio and temperature, respectively. The t-values and corresponding P-values, along with the regression coefficient, are given in Table 1. Temperature had the greatest effect on nitrate removal ratio and the P-value of 17.70 demonstrated the overall model was significant, as there was only a 0.01% chance that t-value this large could occur to noise. The nitrate removal ratio was modeled using response surface methodology with Design Expert, as shown in Fig. 2. The response 3D-surface of the nitrate removal ratio as a function of inoculum (v/v) and pH as independent variables is shown in Fig. 2(a). In general, the nitrate removal ratio increased as the inoculum (v/v) and pH increased and then subsequently declined. Fig. 2(a) shows that the nitrate removal ratio gradually increased as the inoculum (v/v) increased from 5% to 11.03%, and then gradually decreased at inoculum (v/v) above 11.03%. The nitrate removal ratio

Table 1 The least-squares fit and the parameter estimates. Term

Regression coefficient

t value

P value

constant X1 X2 X3 X4 X1 X1 X2 X2 X3 X3 X4 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4

92.55 9.94 −1.75 6.87 −6.84 −15.56 −17.06 −47.71 −15.56 7.50 9.56 −9.00 15.19 −5.81 −6.00

17.702 13.155 0.408 6.296 32.262 3.366 17.425 20.947 163.916 2.498 4.060 3.597 10.242 1.500 1.598

<0.0001*** 0.0027** 0.5333 0.0250* <0.0001*** 0.0879 0.0009*** 0.0004*** <0.0001*** 0.1360 0.0640 0.0790 0.0060** 0.2409 0.2268

Coefficient of correlation (R2 ) = 0.9465. Coefficient of determination (Adj R2 ) = 0.8931; Coefficient of variation = 16.80%. * Significant (0.01 < p value <0.05). ** Very significant (0.001 < p value <0.01). *** Vitally significant for (p value <0.001).

increased slightly when the pH was increased from 5.00 to 6.37, and a pH of greater than 6.37 did not result in further improvement in nitrate removal ratio. For strain R11, the highest nitrate removal ratio was 90.13% at an inoculum (v/v) 11.03% and initial pH 6.37. The experimental results suggest that variation in culture media pH within the 90% aerobic denitrification activity range of P.aeruginosa (pH 6.5–7.1) (Klenner et al., 2004). The major end production of

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Fig. 2. (a) Response 3D-surface showing the effect of inoculum (v/v; %) and pH on nitrate removal ratio by strain R11. (b) Response 3D-surface showing the effect of inoculum (v/v; %) and temperature on nitrate removal ratio by strain R11. (c) Response 3D-surface showing the effect of C/N ratio and temperature on nitrate removal ratio by strain R11.

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Fig. 3. Changes of Nitrate, Nitrite, TOC and pH by Raoultella sp. R11 in HM containing Microcystis aeruginosa flour as carbon and nitrogen source at aerobic condition.

denitrification was nitrogen at the optimum pH of 7 (Zhang et al., 2012). As shown in Fig. 2(b), strain R11 exhibited good denitrification performance between 28.60 ◦ C and 30.50 ◦ C and inoculum (v/v) from 12.05% to 15.00%, with a nitrate removal ratio of 90.05%. The three-dimensional response surface was plotted to directly display the effects of inoculum and incubation temperature on nitrate removal. As shown in Fig. 2(b), the nitrate removal ratio was largely affected by temperature, and appropriate temperature was essential for bacterial growth. Aerobic denitrification was seriously affected by low temperature (below 10 ◦ C) (Xie et al., 2003). Fig. 2(c) demonstrates the interactive effects of C/N ratio and temperature on nitrate removal process. The maximum nitrate removal ratio of 90.00% was achieved at a C/N ratio of 5.93–8.21 and temperature of 27.36 ◦ C to 31.25 ◦ C. The optimal C/N ratio was 8 with a maximum nitrate removal rate of 254.6 mg L−1 h−1 (Kim et al., 2008a, 2008b). At the same time, the optimum conditions for nitrate removal by strain R11were a C/N ratio of 6.92 and 29.87 ◦ C, with maximum nitrate removal ratio of 91.32%. As shown in Fig. 3, a high C/N ratio (above 7.86) did not result in improvement in nitrate removal ratio, and this can probably be explained that high levels of CH3 COONa. For numerical optimization, a minimum and a maximum level must be provided for each parameter. In this study, response surface methodology was used to optimize combinations of inoculum, initial pH, C/N ratio, and temperature. The optimum conditions were an inoculum of 13.02% (v/v), initial pH of 6.65, C/N ratio of 7.59 and temperature of 27.08 ◦ C to achieve maximum nitrate removal ratio of 96.06%. 3.3. Aerobic denitrification using Microcystis aeruginosa flour as carbon source and nitrogen source As shown in Fig. 3, strain R11 was able to utilize Microcystis aeruginosa flour instead of CH3 COONa and NaNO3 , as carbon and nitrogen sources for denitrification in aerobic conditions. During strain R11 in MAFM (Microcystis aeruginosa flour medium), the NO3 − -N concentration decreased throughout the denitrification process. Fig. 3 shows that the NO3 − -N concentration decreased obviously within 20 h, likely reflecting the higher organic carbon source at the initial stage. Thereafter, the NO3 − -N concentration decreased slowly from 20 to 72 h. Lastly, the NO3 − -N concentration

increased slightly from 72 to 96 h, and the NO3 − -N removal ratio was 72.36% with a denitrification rate of 0.0123 mg L−1 h−1 . The NO2 − -N concentration was maintained at approximately 0.9 mg/L during denitrification, suggesting that strain R11 prefers nitrate as a nitrogen source relative to nitrite. During the whole aerobic denitrification process, the TOC decreased significantly from 0 to 48 h and then decreased slowly from 48 to 96 h, the TOC removal ratio was 85.56%. These results demonstrated that strain R11 could use carbon released by algal for denitrification (Su et al., 2016). Previous studies (Manage et al., 2000; Mayali and Doucette, 2002; Su et al., 2007) have demonstrated that algicidal bacteria can use the compounds released from cyanobacterial (or algal) cell lysis. Moreover, pH can be used to demonstrate the characteristics of biological reactions. The pH increased from 6.66 to 7.22. Then, pH maintained a certain range. The increase in pH was probably due to the increasing alkalinity in the process of denitrification (Van et al., 2006). The psychrobacter sp. S1-1 had pronounced aerobic denitrification ability between pH 6.0 and 7.5 (Zheng et al., 2011). 3.4. Fourier transform near-infrared spectroscopy analysis Fig. 4 shows the infrared spectrogram of Microcystis aeruginosa cells treated with strain R11, beef extract peptone broth and normal Microcystis aeruginosa cells with different inoculum (10%, 15%, 20%, 25%, 30%; v/v). The main absorption peak position of infrared spectrogram of Microcystis aeruginosa cells in control and treatment groups were generally consistent. However, compared with infrared spectrogram of normal Microcystis aeruginosa cells, the relative intensity of absorption peak was lower for each treatment group. Bands on infrared spectrograms of the analyzed samples were weaker for Microcystis aeruginosa cells treated with strain R11 than for control group at 2100 cm−1 − 3900 cm−1 . The intensity of the O H stretching vibration area decreased and appeared as sharper peaks at 3300 cm−1 − 3600 cm−1 , probably due to the destruction of O H in the polysaccharide and protein components of Microcystis aeruginosa cells. Furthermore, the absorption peak at 1650 cm−1 in each treatment group was C O stretching vibration, and the absorption peak decreased. The absorption peak around 1540 cm−1 N H bending vibration and C N stretching vibration, however, was not obvious, suggesting that the carbon-hydrogen bond (C H) of protein structure was destroyed. For strain R11 inoculum sizes

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(v/v) of 20% to 30%, the amide bond (C O) intensity at 1500 cm−1 decreased significantly, which showed protein amide bond (C O) was damaged. In each treatment group, many small sharp peaks appeared at 2100 cm−1 − 2400 cm−1 , probably due to the Microcystis aeruginosa cells structure and absorption of the overflow of the cytoplasm. Fig. 5(a) shows that Microcystis aeruginosa cells were intact before treatment, and the cell wall and membrane

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were closely combined. However, after treatment, the Microcystis aeruginosa cells were thoroughly decomposed, leading to the release of intracellular substances, such as proteins and carbohydrates. The strain s7 had strong algicidal effect, based on infrared spectroscopy, it destroyed the protein of Microcystis aeruginosa (Luo et al., 2010).

Fig. 4. Infrared spectrogram of Microcystis aeruginosa treated by strain R11, beef extract peptone broth and normal Microcystis aeruginosa cell with different inoculum (10%, 15%, 20%, 25%, 30%; v/v).

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Fig. 4. (Continued)

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Fig. 5. SEM images of Microcystis aeruginosa. (a) before treatment (b) after treatment by Strain R11.

4. Conclusions (1) The Raoultella sp. R11 had pronounced aerobic denitrification and algicidal effects, and the nitrogen removal mechanism of strain R11 was determined by napA gene amplification. (2) Response surface methodology (RSM) analysis demonstrated that the maximum nitrate removal ratio was 96.06%, and optimal conditions occurred were determined to be inoculum of 13.02% (v/v), initial pH of 6.65, C/N ratio of 7.59 and temperature of 27.08 ◦ C by ridge analysis, where inoculum and temperature had biggest influence on nitrate removal. (3) When the Microcystis aeruginosa flour as carbon and nitrogen sources for denitrification in aerobic conditions, the NO3 − -N and TOC removal ratio were 72.36% and 85.56%, with a denitrification rate of 0.0123 mg·L−1 ·h−1 . These results demonstrated that strain R11 could use carbon released by algal death for denitrification. (4) The Microcystis aeruginosa cells were treated by strain R11 with different inoculum (10%, 15%, 20%, 25%, 30%; v/v). In each treatment group, many small sharp peaks appeared at 2100 cm−1 − 2400 cm−1 , probably due to the Microcystis aeruginosa cells structure were destroyed. Acknowledgements This research work was partly supported by the National Natural Science Foundation of China (NSFC) (No. 51678471), the National Key Research and Development Project (NO. 2016YFC0200706). References Dockyu, K., Kim, J.F., Yim, J.H., Kwon, S.K., Lee, C.H., Hong, K.L., 2008. Red to red- the marine bacterium Hahella chejuensis and its product prodigiosin for mitigation of harmful algal blooms. J. Microbiol. Biotechnol. 18 (10), 1621–1629. Glibert, P., Bouwman, L., 2012. Land-based nutrient pollution and the relationship toharmful algal blooms in coastal marine systems. Loicz Newslett. Inprint 2, 5–7. Huang, X., Li, W., Zhang, D., Qin, W., 2013. Ammonium removal by a novel oligotrophic Acinetobacter sp: Y16 capable of heterotrophic nitrification-aerobic denitrification at low temperature. Bioresour. Technol. 146 (10), 44–50. Huang, T.L., Guo, L., Zhang, H.H., Su, J.F., Wen, G., Zhang, K., 2015. Nitrogen-removal efficiency of a novel aerobic denitrifying bacterium, Pseudomonas stutzeri strain ZF31, isolated from a drinking-water reservoir. Bioresour. Technol. 196, 209–216. Jeong, J.H., Jin, H.J., Sohn, C.H., Suh, K.H., Hong, Y.K., 2000. Algicidal activity of the seaweed Corallina pilulifera against red tide microalgae. J. Appl. Phycol. 12 (1), 37–43.

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