Isolation of a bacterial strain, Acinetobacter sp. from centrate wastewater and study of its cooperation with algae in nutrients removal

Accepted Manuscript Isolation of a bacterial strain, Acinetobacter sp. from centrate wastewater and study of its cooperation with algae in nutrients removal Hui Liu, Qian Lu, Qin Wang, Wen Liu, Qian Wei, Hongyan Ren, Caibing Ming, MinMin, Paul Chen, Roger Ruan PII: DOI: Reference:

S0960-8524(17)30392-9 http://dx.doi.org/10.1016/j.biortech.2017.03.111 BITE 17815

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

31 January 2017 14 March 2017 17 March 2017

Please cite this article as: Liu, H., Lu, Q., Wang, Q., Liu, W., Wei, Q., Ren, H., Ming, C., MinMin, Chen, P., Ruan, R., Isolation of a bacterial strain, Acinetobacter sp. from centrate wastewater and study of its cooperation with algae in nutrients removal, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.111

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Isolation of a bacterial strain, Acinetobacter sp. from centrate wastewater and study of its cooperation with algae in nutrients removal Hui Liua,b,1, Qian Luc,1, Qin Wanga, Wen Liua, Qian Weia, Hongyan Renc, Caibing Minga, Min Minc, Paul Chenc, Roger Ruanc* a

Department of Environment Science and Engineering, Zhongkai University of Agriculture and

Engineering, Guangzhou 510225, China b

Guangdong Provincial Engineering and Technology Research Center for Agricultural Land

Pollution Prevention and Control, Guangzhou 510225, China c

Center for Biorefining, and Department of Bioproducts and Biosystems Engineering,

University of Minnesota, St. Paul, MN, USA 1

The first two authors contributed equally to this work

Corresponding author at: Yangtze Scholar Distinguished Guest Professor, Nanchang University, and Professor, Center for Biorefining, and Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108, USA. Tel.: +1 6126251710; fax: +1 6126243005. E-mail address: [email protected] (Roger Ruan)

Abstract Algae were able to grow healthy on bacteria-containing centrate wastewater in a pilot-scale bioreactor. The batch experiment indicated that the co-cultivation of algae and wastewater-borne bacteria improved the removal efficiencies of chemical oxygen demand and total phosphorus in centrate wastewater to 93.01% and 98.78%, respectively. A strain of beneficial aerobic bacteria, Acinetobacter sp., was isolated and its biochemical characteristics were explored. Synergistic cooperation was observed in the growth of algae and Acinetobacter sp. Removal efficiencies of

some nutrients were improved significantly by the co-cultivation of algae and Acinetobacter sp. After treatment, residual nutrients in centrate wastewater reached the permissible discharge limit. The cooperation between algae and Acinetobacter sp. was in part attributed to the exchange of carbon dioxide and oxygen between the algae and bacteria. This synergetic relationship between algae and Acinetobacter sp. provided a promising way to treat the wastewater by improving the nutrients removal and biomass production.

Keywords: Algae; Bacteria; Nutrients removal; Centrate wastewater; Acinetobacter sp.

1. Introduction Centrate wastewater, which is generated from the centrifugation of activated sludge in municipal wastewater treatment, contains sufficient nutrients for algae growth (Li et al., 2011). A study using a pilot-scale algae cultivation system showed that cultivate Chlorella sp. was able to remove 70% chemical oxygen demand (COD), 61% total nitrogen (TN), and 61% total phosphorus (TP) from centrate wastewater (Min et al., 2011). Because of the high concentrations of TN and TP in centrate wastewater (~131.5 mg/L TN and ~201.5 mg/L TP), microalgae cultivated in centrate wastewater have high biomass yield and can be converted into biofuel (Pittman et al., 2011). Given the great economic and environmental benefits, microalgae technology is regarded as a promising pathway to treat centrate wastewater. Wastewater-borne bacteria, which may compete for nutrients with other microorganisms in wastewater or release compounds toxic to algae, pose a serious threat to the growth of microalgae (Fergola et al., 2007). Various methods, such as the use of antibiotics, hightemperature treatment, and exposure to light with high intensity, have been investigated to control the bacteria in microalgae cultivation (Jemli et al., 2002; Lu et al., 2015). However, some studies revealed that microalgae and some bacteria may have synergistic cooperation. It was reported that the co-immobilization of Chlorella sorokiniana and Azospirillum brasilense could remove the nutrients in municipal wastewater more efficiently than individual microalgae

community (De-Bashan et al., 2004). Croft et al. (2005) improved algal biomass productivity by co-cultivating Chlorella vulgaris with Flavobacterium and Hyphomonas. Due to the possible coexistence and cooperation between algae and bacteria, more and more researchers are trying to utilize the algal-bacterial community to treat wastewater, rather than sterilizing the wastewaterborne bacteria. Because of the complexity of bacterial community in wastewater, recent studies mainly focus on the interaction between algae and individual bacterial strain. De-Bashan et al. (2008) found that Azospirillum sp. promoted the growth of algae and the co-cultivation improved removal of nitrogen. Research of Croft et al. (2005) identified a bacterial strain, Halomonas sp., which could cooperate with algae, from the medium. Compared with the studies on complicated bacterial community, studies on individual bacterial strain could identify exact cooperation model and metabolic mechanisms of algal-bacterial system (Buchan et al., 2014). In addition, isolated favorable bacterial strain could be used in algae cultivation for biomass improvement (DeBashan et al., 2008). Study on the interaction between algae and individual bacterial strain has become a critical pathway to realize the commercial use of algal-bacterial system for wastewater treatment. Cooperation models between algae and bacteria have been exploited in previous studies. Firstly, algae could utilize the energy provided by bacteria (Lu et al., 2013). Some bacteria could release extracellular enzymes, including amylase, lipase, and protease, which could convert highmolecular-weight organics into low-molecular-weight organics (Buchan et al., 2014; Pohlon et al., 2010). Due to the higher bio-digestibility of low-molecular-weight organics, algae grown with this kind of bacteria could have higher biomass yield. Secondly, some bacteria could release vitamins which are growth-promoting factors for algae (Croft et al., 2005). Thirdly, additional synergy can occur when algae utilize the in-situ carbon dioxide produced by bacteria as the carbon source while bacteria consume the in-situ oxygen released by algae (Boschker et al., 2005). Although some studies found that wastewater-borne bacteria in centrate wastewater may promote the growth of algae, the cooperation model between algae and bacteria in centrate wastewater has not been explored yet (Ma et al., 2014). The challenging questions about centrate wastewater treatment faced by researchers include: (1) Why could the algae and bacteria develop

the synergistic relationship in centrate wastewater? (2) What is the role of bacteria in the cocultivation system of bacteria and algae? (3) What are the characteristics of beneficial bacteria in the co-cultivation of algae and wastewater-borne bacteria? And (4) How can the conditions be optimized to promote the cooperation between algae and wastewater-borne bacteria for centrate wastewater treatment? By solving these challenging questions, researchers will have much deeper understanding of the biological treatment of centrate wastewater. This study, aiming at solving some of the critical challenging questions in centrate wastewater treatment, consists of four parts: Firstly, algae were used to treat centrate wastewater without sterilization in a pilot-scale bioreactor. Secondly, batch experiments in lab were conducted to measure the nutrients removal and biomass yield. Thirdly, a bacterial strain in centrate wastewater after treatment was isolated and identified. Finally, the cooperation between isolated bacterial strain and algae was explored through the co-cultivation experiments and the roles of bacteria and algae in nutrients removal were identified.

2. Materials and methods 2.1. Experimental design The experiments were carried out in four steps. In the first step, algae were cultivated on centrate wastewater in a pilot-scale bioreactor. The operation parameters of the bioreactor and the growth of algae were monitored in this step. The second step involved study of the roles of bacterial community and algal community in nutrients removal in a lab scale batch experiment. The parameters, including pH value, Oxidation-Reduction Potential (ORP), and nutrients removal, were monitored in this experiment. In the third step, a bacterial strain in algal-bacterial community was isolated. The isolated bacterial strain and its biochemical characteristics were identified. In the fourth step, the isolated bacterial strain was co-cultured with the algae in centrate wastewater during which nutrients removal and biomass production were monitored.

2.2. Centrate wastewater

Centrate wastewater was obtained from a municipal wastewater treatment plant in St. Paul (Minnesota, USA). Basic characteristics of centrate wastewater are shown in Table 1. The pH value of the wastewater was around 6.26, which is favorable to the growth of most algal strains. The wastewater contained various nutrients, including nitrogen, phosphorus and other organics, which are necessary for the growth of algae. The positive value (111.00±8.66 mV) of ORP showed that the centrate wastewater was in aerobic conditions.

2.3. Parameters measurement ORP and pH of centrate wastewater were measured by using ORP tester and pH tester, respectively. Values of ORP were expressed as mV. Nutrients, including chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), and ammonia (NH3-N), of centrate wastewater were measured daily in batch experiment using analysis kits purchased from Hach (USA). The measurement was conducted with a Hach DR 5000 Spectrophotometer according to previously published methods (Lu et al., 2016). Concentrations of nutrients were expressed as mg/L. The number of bacteria, which was expressed as colony-forming unit (CFU), was measured according to the method described by Liu et al. (2016). The biomass yield of algae was calculated based on the measurement of total volatile suspend solids (TVSSs) (Lu et al., 2015). Average growth rate (g/L/d) of algae was calculated according to Eq. (1). R = (Ta-T0)/t

Eq. (1)

where R is the average growth rate of algae; Ta and T0 are the TVSSs of algae at Day a and Day 0, respectively; t is the time interval (days).

2.4. Pilot-scale bioreactor The pilot-scale bioreactor consisted of a three-layer photo-bioreactor (PBR) and two retention tanks. Two pumps placed in retention tanks were used to continuously circulate the centrate wastewater in PBR (Figure 1). Volumes of PBR and two retention tanks were 900L and 400L,

respectively. Hydraulic retention time (HRT) of the pilot-scale bioreactor was 4 days. The pilotscale bioreactor was placed in a greenhouse where the temperature and relative air humidity were 28±5 oC and 50±10%, respectively. The algal strain used for centrate wastewater treatment in the pilot-scale bioreactor was Chlorella sp.

2.5. Batch experiment Chlorella sp. was cultivated in artificial medium. The chemical composition of artificial medium is listed as follows: NaNO3 (1500 mg/L), K2HPO4 (0.04), MgSO4·7H2O (75 mg/L), CaCl2·2H2O (36 mg/L), citric acid (6 mg/L), ferric ammonium citrate (6 mg/L), EDTA (disodium salt) (1 mg/L), Na2CO3 (20 mg/L), trace metal mix A5 (1.0 mL/L) (Hollinshead et al., 2014). Algal cells were washed with distilled water twice before inoculation into wastewater in batch experiment. Batch experiment was conducted in lab environment. Algae were cultivated in 250 mL Erlenmeyer flasks with 100 mL centrate wastewater. The light intensity, temperature, and relative air humidity were controlled at 120±10 µmol photons m-2 s-1, 25±1 oC, and 45±3%, respectively. The Erlenmeyer flasks were rotated continuously at 200 rpm. Density of algae inoculated into wastewater was about 0.275±0.025 g/L. Parameters measured and calculated in batch experiment included biomass yield, CFU, ORP, pH, and nutrient removal efficiencies.

2.6. Isolation and identification of a bacterial strain 2.6.1. Genetic identification After wastewater treatment, some bacteria became dominant in the complex microorganism community. To simplify the relation between algae and wastewater-borne bacteria, one bacterial strain was isolated and identified. At the end of batch experiment, centrate wastewater, which had been treated by algal-bacterial community for 5 days, was subjected to bacterial isolation. A bacterial strain was isolated according to the method described by Liu et al. (2016). Isolated bacterial strain was cultivated on solid beef extract culture-medium. Chemical composition of

the beef extract culture-medium was: beef extract (5.0 g/L), peptone (10.0 g/L), NaCl (15.0 g/L), and agar (20.0 g/L). The pH value of this medium was adjusted to 7.0 before use. DNA Extraction Kit (MP Biomedicals, USA) was used to extract the total DNA of isolated bacterial strain according to manufacturer’s instructions. Extracted total DNA was stored in dark at -20 oC before use. The primers used for polymerase chain reaction (PCR) amplification of 16S rRNA gene sequence are listed as follows: 27f (5’-AGRGTTTGATCMTGGCTCAG-3’) and 1492r (5’-GYTACCTTGTTACGACTT-3’). The PCR program used in this work has been reported in previous research (Liu et al., 2016). The Basic Local Alignment Search Tool (BLAST) was applied to compare 16S rRNA gene sequences with the reported gene sequences in GenBank database. A phylogenetic tree designed using MEGA software version 4.0 was used to illustrate the evolutionary trends.

2.6.2. Analysis of biochemical characteristics The biochemical characteristics observed in this study include cell shape and size, Gram reaction, glucose fermentation, oxidase synthesis, contact enzyme synthesis, nitrate reduction, nitrite reduction, oxygen demand, DL-lactate utilization, and citrate utilization. The shape and size of isolated bacterial cell was identified by using electron microscopy (JEOL, Germany) at magnifications of 3000 and 6000. Other tests were conducted according to previously published methods (Liu et al., 2016). An isolated bacterial strain was identified by comparing its biochemical characteristics with those reported bacterial strains (Buchanan & Gibbons, 1974).

2.7. Co-cultivation of algae and isolated bacterial strain Centrate wastewater was subjected to sterilization at 121 oC for 30 min before use. The bacterial strain identified and isolated through the experiments and algae were inoculated into sterilized centrate wastewater. Initial CFU of inoculated bacteria and TVSS of inoculated algae were 1.20×106±0.5×106 and 0.275±0.025 g/L, respectively. The co-cultivation conditions, including light intensity, temperature, and relative air humidity, were as same as the conditions in batch

experiment. Parameters, including biomass yield, CFU, ORP, pH, and removal efficiencies of nutrients, in the co-cultivation process were measured and calculated.

2.8. Statistical analysis The experiments at pilot-scale were performed only once while all tests and experiments in lab were conducted in triplicate. The results were expressed as means ± standard deviations.

3. Results 3.1. Wastewater treatment at a pilot-plant scale Concentrations of different nutrients (TN, TP, NH3-N, and COD) in the experimental periods are shown in Table 2. Nutrients in centrate wastewater had different initial concentrations in every experimental period since centrate wastewater was obtained from the wastewater treatment plant at different points of time. After one week treatment, average concentrations of TN, TP, NH3-N and COD were reduced to 65.7 mg/L, 15.0 mg/L, 41.1 mg/L, and 430 mg/L, respectively. Nutrients removal efficiencies changed dramatically with an upward trend (Figure 2(c)). Compared with the removal efficiencies in the first week, removal efficiencies of COD, TP, TN, and NH3-N in the final week were improved by 29.98%, 10.83%, 20.00%, and 12.92%, respectively. In the last three weeks, nutrients removal efficiencies remained stable. The main reason may be that algae and wastewater-borne bacteria established synergistic cooperation gradually during the experiment, which improved the nutrients removal efficiencies (de-Bashan & Bashan, 2010). At the end of the experiment, the cooperation system consisting of algae and bacteria reached stable status. In this way, the nutrients removal efficiencies remained stable. Algal biomass yield, bacterial growth, nutrients removal efficiencies, and changes in ORP are shown in Figure 2. Both bacteria and algae grew in the pilot-scale bioreactor during each experimental period. Figure 2(a) showed that the algae had growth (0.28~0.39 g/L) in this bioreactor. In the pilot experiment, CFU of wastewater-borne bacteria was improved by 15.1~26.3 times (Figure 2(b)). At the end of one-week cultivation, the CFU of bacteria reached

1.67~2.10×107. This result suggested that the algal community and the bacterial community in centrate wastewater could survive and grow in the same environment. Before algae inoculation, centrate wastewater was stored at condensation tank, which was under anaerobic conditions. When the centrate wastewater was pumped into bioreactor, it was exposed to an aerobic environment, in which the aerobic bacteria grew rapidly. Therefore, in the centrate wastewater treatment, under aerobic conditions, aerobic bacteria are dominant in the bacterial community. The decrease in ORP (Figure 2(d)), which ranged from 28 mV to 119 mV, suggested that the centrate wastewater treatment in the pilot-scale bioreactor is an oxygen consuming process although the wastewater remained under aerobic conditions. The reduction in ORP indicated that in the pilot-scale bioreactor, oxygen consumed by microorganisms is more than the oxygen produced by algae. Since photosynthesis of algal cells is an oxygen producing process, it is the activity of bacteria that contributed to the reduction in ORP in centrate wastewater. Therefore, in the pilot experiment, algal activity contributed to the oxygen production while bacterial activity is associated with the oxygen consumption. Batch experiment was conducted to explore the metabolisms of algal-bacterial community in centrate wastewater.

3.2. Batch experiment Biomass yields, changes in ORP and pH, and nutrient removal efficiencies in batch experiment are shown in Figure 3. By eliminating the side effects of light intensity fluctuation, environmental temperature, and air conditions on experimental results, batch experiment could reflect the relationship between algae and wastewater-borne bacteria more accurately. TVSS of wastewater without algae, TVSS of wastewater with algae, and TVSS of wastewater with bacteria and algae are shown in Figure 3(a). Figure 3(d) showed that CFU of bacteria in wastewater without algae increased from 8.9×105 to 3.27×107 in five days. The concentration of bacteria in wastewater was improved by 36.7 times. However, TVSS of wastewater without algae remained stable, ranging from 0.92 g/L to 1.16 g/L. Therefore, in the algal-bacterial community, the growth of bacteria could not directly cause the improvement of TVSS. TVSS of wastewater with algae was improved by 0.51 g/L while TVSS of wastewater with algae and bacteria was improved by 1.02 g/L. This result indicated that algae had a higher biomass yield

when grown with wastewater-borne bacteria together. However, algae showed some inhibiting effects on the growth of bacteria since CFU of bacteria (2.31×107) in wastewater with algae was much lower than that of bacteria (3.27×107) in wastewater without algae (Figure 3(d)). The possible reason for this phenomenon is that algae compete with some bacteria for nutrients and slowed down the growth rate of bacteria. Figure 3(b) showed that the wastewater-borne bacteria consumed oxygen in centrate wastewater while the metabolic activity of algae released oxygen. Positive values of ORP in the experiment suggested that the wastewater was in aerobic conditions, which prohibited the growth of anaerobic bacteria. Therefore, aerobic bacteria, which could digest organics through oxygen consuming biochemical reactions, were the dominant strains in wastewater-borne bacteria (Chen et al., 2012). This result is in accordance with the result obtained from pilot-scale experiment. In the co-cultivation of algae and bacteria, ORP dropped on the first day but started to increase after the second day. Two metabolic mechanisms are proposed to explain this phenomenon. First, in the duration of lag phase, metabolisms, including photosynthesis, of newly inoculated algae were limited (Li et al., 2011). Accordingly, the oxygen releasing rate was low and the activity of wastewater-borne bacteria cause the reduction in ORP. After the lag phase, due to the fast growth of algae, oxygen releasing rate was improved and ORP started to increase on the second day of the batch experiment. Secondly, algae utilized organic carbon source in centrate wastewater by heterotrophic reactions at the beginning and started to utilize inorganic carbon source, carbon dioxide, by photosynthetic reactions after the digestible organics were exhausted. This explanation is supported by the changes in COD in batch experiment (Figure 3(f)). Therefore, algae consumed oxygen at the beginning of experiment and then started to release oxygen. Accordingly, ORP of wastewater dropped significantly in the first day and then increased gradually. Comparison of Figure 2(d) and Figure 3(b) showed that the average initial ORP (-22.85 mV) in pilot-scale experiment was much lower than the average initial ORP (110.00 mV) in batch experiment. The main reason for the difference is that the rotation of Erlenmeyer flasks promoted the solution of oxygen and maintained highly aerobic conditions in wastewater while the pilot-scale bioreactor did not have an aeration tank. Pilot-scale experiments indicate that aerobic bacteria are dominant in the bacterial community. To maintain the dominance of aerobic

bacteria, devices, such as aeration tank, which could improve the content of dissolved oxygen in wastewater should be added. The metabolic activities of both algae and wastewater-borne bacteria improved the pH of centrate wastewater (Figure 3(c)). The research of Vlek & Stumpe (1978) showed that ammonia volatilization and phosphorus sedimentation occurred when the pH of environment exceeded 9.5 (Vlek & Stumpe, 1978). In this study, pH of wastewater remained below 9.15, which is not enough for the phosphorus sedimentation and the ammonia volatilization. Since the environmental factors could not cause the removal of ammonia and phosphorus, it is the activity of bacteria or algae that contributed to the nutrients removal in wastewater. According to Figure 3(e), in the wastewater without algae, bacteria only removed 16.84% NH3-N, reducing the concentration of NH3-N from 31.30 mg/L to 26.03 mg/L. In the wastewater with algae and bacteria, removal efficiency of NH3-N reached 90.66% at the end of batch experiment. Similar phenomenon was observed in the removal of TN (Figure 3(g)). Wastewater-borne bacteria only removed 13.64% TN while algae removed 77.14% TN. Based on this result, it is concluded that in the process of removing TN in centrate wastewater, algae made greater contribution than bacteria. Compared with removal efficiency of TN (38.49~58.49%) in pilotscale experiment, removal efficiency of TN (79.49%) in batch experiment was higher. The main reason for the difference between removal efficiencies of TN in pilot-scale experiment and batch experiment is that the batch experiment in lab has much better growth conditions than pilot-scale experiment. For example, temperature in lab was strictly controlled while temperature in pilotscale experiment fluctuated daily. Good growth conditions in lab promoted the growth of algae. Biomass yield of algae in pilot-scale experiment was about 0.35 g/L while biomass yield in batch experiment reached 2.25 g/L. So in batch experiment, more nutrients, including nitrogen, were removed. Figure 3(f) showed that COD removal efficiencies in wastewater with bacteria and that in wastewater with algae were 72.56% and 74.57%, respectively. In the experimental period, 27.44% COD could not be removed by bacteria and 25.43% COD could not be removed by algae. This result suggested that for either bacterial community or algal community, the concentration of indigestible organics in centrate wastewater was high. The algal-bacterial community, which removed 93.01% COD, had much higher COD removal efficiency than either bacterial community or algal community. The percentage of COD, which could not be removed by algal-

bacterial community, was only 6.99%. Similar result was observed in the removal of TP in wastewater. Figure 3(h) indicated that bacterial community and algal community removed 52.65% and 86.81% TP, respectively, while algal-bacterial community removed 98.78% TP. Therefore, in terms of COD removal and TP removal, algal-bacterial community is much better than either bacterial community or algal community alone. According to the batch experiment, in the treatment of centrate wastewater, algal-bacterial community has much better performance in the removal of nutrients, particularly COD and phsophorus. Two possible reasons contributed to this phenomenon. Firstly, the dominant bacterial community, aerobic bacteria, and algae provided carbon dioxide and oxygen to each other (Kumar et al., 2010; Su et al., 2011). Such a synergistic cooperation led to the higher biomass yield of algae which contributed to the higher nutrient removal efficiencies. Secondly, the cooperation between algae and wastewater-borne bacteria reduced the content of indigestible nutrients to a lower level. Since wastewater-borne bacteria removed some COD and phosphorus, which could not be digested by algae, the removal of COD and phosphorus in wastewater were much greater when both algae and bacteria were present (More et al., 2014). To explore the mechanism of synergistic cooperation between algae and beneficial wastewaterborne bacteria, a specific bacterial strain was isolated and identified and the biochemical characteristics of isolated bacterial strain were studied next.

3.3. Isolation and identification of bacterial strain 3.3.1. Genetic identification of isolated strain The homology between 16s rRNA gene consequences of isolated strain and GenBank strains (Table 3) indicated that the proximities between the isolated bacterial strain and Acinetobacter towneri, Acinetobacter genomic, and Acinetobacter haemolyticus reached 96.87%, 96.69%, and 95.13%, respectively. The phylogenetic tree of isolated strain and related bacterial strains is shown in Figure 5. The result of genetic identification suggests that the isolated bacterial strain has the highest similarity with Acinetobacter sp.

3.3.2. Morphological and biochemical characteristics As shown in Table 4, the isolated bacterial strain is a type of gram-negative bacilli without oxidase. Firstly, the test of glucose fermentation is negative. This result suggests that the isolated bacterial strain could not use glucose as the carbon source or energy source. The positive results in the tests of DL-lactate utilization and citrate utilization indicate that the isolated bacterial strain relies on the non-glucose carbon source. Secondly, due to the positive results in the tests of nitrate reduction and nitrite reduction, the isolated bacterial strain has the function of denitrification. Thirdly, the positive result in the test of oxygen demand suggests that the isolated bacterial strain needs oxygen in the metabolism. According to the Bergey’s Manual of Determinative Bacteriology, the isolated strain in this study is identical to Acinetobacter sp. Based on both genetic identification and morphological and biochemical characteristics, it is concluded that the bacterial strain isolated from algal-bacterial community is Acinetobacter sp. Acinetobacter sp., which is wide distribution in activated sludge, wastewater, and soil, is regarded as a group of non-fermentative aerobic microorganisms (Gao et al., 2014; Wang et al., 2013; Weon et al., 2002). Previous studies have developed various methods to enrich Acinetobacter sp. in wastewater for removal of phosphorus (Yang et al., 2015; Zhou et al., 2010). Acinetobacter sp. was also used to degrade some non-glucose carbon source, including oleic acid, phenol, acetate and ethanol, as well (Bouvet & Grimont, 1986; Li et al., 2001; Ying et al., 2007). However, the co-cultivation of algae and Acinetobacter sp. for nutrients removal in municipal wastewater has never been exploited in previous studies.

3.4. Co-cultivation of algae and Acinetobacter sp. in wastewater The batch experiment showed that the algal-bacterial community improved nutrient removal efficiencies in centrate wastewater. Since algal-bacterial community contains various bacterial strains with very different metabolic characteristics, it is impossible to explore the interaction between algae and the whole bacterial community. To simplify the model of algal-bacterial community, we explored the synergistic cooperation between algae and this isolated beneficial bacterial strain, Acinetobacter sp. To our knowledge, previous studies have never explored the cooperation between algae and aerobic bacteria in centrate wastewater treatment.

Performance of algae and Acinetobacter sp. in centrate wastewater treatment is shown in Figure 4. Figure 4(d) indicated that in the wastewater without algae, CFU of Acinetobacter sp. increased from 1.14×106 to 2.85×107. However, the growth of bacteria did not cause the improvement of TVSS. This result is in accordance with the result obtained from batch experiment. In wastewater inoculated with algae, CFU of Acinetobacter sp. reached peak value, 3.90×107, on the 2nd day but decreased slightly between the 3rd day and the 5th day. It is supposed that the competition between algae and Acinetobacter sp. for nutrients limited the growth of Acinetobacter sp. from the 3rd day to the 5th day. Biomass yield of algae grown in sterilized wastewater was 0.80 g/L while that of algae grown in unsterilized wastewater reached 1.02 g/L (Figure 4(a)). The existence of Acinetobacter sp. promoted the growth of algae, which contributed to the improvement of TVSS. Comparison between Figure 4(a) and Figure 3(a) revealed that algae grown with Acinetobacter sp. had much higher biomass yield than algae grown with the whole bacterial community. It is believed that in batch experiment, bacterial community contained some unfavorable bacteria which had negative effects on the growth of algae (Choi et al., 2010). It was reported that bacteria could limit the growth of algae by direct or indirect attack (Gan et al., 2014; Zhang et al., 2012). However, in the co-cultivation of algae and Acinetobacter sp., due to the sterilization process, those unfavorable bacteria were not present. Figure 4(b), which showed that ORP of wastewater ranged from 39 mV to 120 mV, indicated that the centrate wastewater was in aerobic conditions. Since the pH of wastewater was lower than 9.0, the slightly alkaline condition could not cause noticeable ammonia volatilization and phosphorus sedimentation (Figure 4(c)). In this experiment, it was the activity of microorganisms that removed the nutrients in wastewater. Figure 4(e) indicated that in the wastewater inoculated with Acinetobacter sp. and algae separately, the NH3-N removal efficiencies were 2.83% and 88.85%, respectively. This result suggested that under aerobic conditions, Acinetobacter sp. did not have the metabolic pathway to utilize NH3-N while algae mainly contributed to the removal of NH3-N. It was observed that concentration of nitrate remained at low level without significant fluctuation in the experiment. As shown in Figure 4(g), Acinetobacter sp. and algae could remove 13.54% and 73.10% of TN, respectively, while the co-cultivation system removed 79.12% of TN. This result suggests that synergistic co-cultivation in the removal of TN in centrate wastewater was limited. Removal of TP (Figure 4(h)) indicated that TP removal efficiencies in

wastewater inoculated with Acinetobacter sp. and algae separately reached 48.75% and 83.43%, respectively. However, the TP removal efficiency was improved to 96.26% by the algal-bacterial community. Similar phenomenon was observed in the removal of COD (Figure 4(f)). Algae alone could only remove 63.91% of COD in wastewater while co-cultivation of algae and Acinetobacter sp. improved the COD removal efficiency to 79.11%. Therefore, synergistic cooperation between algae and Acinetobacter sp. contributed to the improvement of nutrient removal in centrate wastewater. At the end of 5-days cultivation, concentrations of COD, TN, and TP were 178 mg/L, 8.99 mg/L, and 0.97 mg/L, respectively. According to the requirement of municipal wastewater discharge standards, maximum concentrations of COD, TN, and TP are 200 mg/L, 25 mg/L, and 1 mg/L, respectively. So after co-cultivation of algae and Acinetobacter sp., centrate wastewater reached the permissible discharge limit.

3.5. Synergistic cooperation between algae and Acinetobacter sp. Experiment reveals that the presence of Acinetobacter sp. promoted the growth of algae and nutrients removal. Based on the biochemical characteristics of Acinetobacter sp. and cocultivation system, synergistic cooperation between algae and Acinetobacter sp. is discussed as follows. Firstly, it was observed that Acinetobacter sp. contributed to the removal of phosphorus. Therefore, it is assumed that Acinetobacter sp. has the pathway to utilize phosphorus in centrate wastewater. This assumption is supported by previous publications, which applied Acinetobacter sp. in the removal of phosphorus (Akpor & Muchie, 2010; Wang et al., 2008). In addition, Acinetobacter sp. promoted the growth of algae, which contributed to the removal of phosphorus. Therefore, in this work, Acinetobacter sp. had two pathways to promote the removal of phosphorus in centrate wastewater. Secondly, in the first two days of the wastewater treatment process, both algae and aerobic bacteria had good growth. Hence, it is supposed that algae and Acinetobacter sp. may not compete seriously for nutrients at the initial stage of wastewater treatment. Accordingly, the cocultivation of algae and Acinetobacter sp. could create a better growth conditions without intensive competition. In addition, algal-bacterial community, which has much wider utilization

range of organic carbon source than the separate algal community and separate bacterial community (Mellado et al., 2013; Snellman et al., 2002), contributes to the higher removal efficiency of COD in centrate wastewater. Thirdly, at the later stage of the wastewater treatment process, with the exhaustion of organic carbon source, algae will start to utilize inorganic carbon source, transferring from heterotrophic metabolism to phototrophic metabolism (Deng et al., 2012; Wang et al., 2011). Due to the fast growth of algae at the initial stage of wastewater treatment process, algal community has the ability to produce more oxygen (Price et al., 2012), which is necessary for the metabolisms of Acinetobacter sp. Acinetobacter sp. utilized the oxygen produced by algae and algae utilized the carbon dioxide released by Acinetobacter sp. Under this condition, the co-cultivation of algae and Acinetobacter sp. improved the utilization efficiency of carbon source and reduced the dependence on oxygen supply from external environment. Fourthly, compared with the cultivation of Acinetobacter sp., co-cultivation of algae and Acinetobacter sp. has much higher efficiencies of TN and NH3-N removal. This result suggests that in the practice, in terms of nitrogen removal, co-cultivation of algae will have much better performance than the aerobic fermentation. The main reason is that contribution of Acinetobacter sp. in the removal of nitrogen source is limited while algae could remove the nitrogen efficiently (Vardon et al., 2011). With the improvement of algal biomass yield, the NH3-N and TN removal efficiencies were improved.

4. Discussion Accordingly, high concentrations of nutrients were left in centrate wastewater after algae cultivation. In this study, it was observed that removal efficiencies of nutrients in centrate wastewater treated by algae were low and concentrations of residual nutrients were high. This result is in accordance with the research of Min et al., (2011), which showed that removal efficiencies of COD, TP and TN in centrate wastewater during 11 days were 86.3%, 58.1% and 34.8%, respectively. Due to the low removal efficiencies of nutrients, particularly TN and TP, concentrations of residual nutrients in centrate wastewater could not reach the permissible discharge limits. In the practice, further treatment after algae cultivation should be conducted to

reduce the concentrations of nutrients (Liu et al., 2016). Cost of further treatment, however, could improve the total cost of wastewater treatment and limit the application algae technology in wastewater treatment (Min et al., 2011). This study solved the problem caused low removal efficiencies of nutrients by developing an integrated algal-bacterial system for the treatment of centrate wastewater. According to the discharge standards, residual nutrients in centrate wastewater treated by the integrated system of algae and Acinetobacter sp. reached the permissible discharge limits. The integrated system of algae and Acinetobacter sp. not only reduced the pollutants in wastewater, but also promoted resources recovery. Previous studies revealed that some nutrients were embedded into large solid particles, which could not be absorbed by algal cells (Lu et al., 2012; Wei et al., 2013). Accordingly, some resources in centrate wastewater could not be recovered by algae for biomass production. In the integrated algal-bacterial system, Acinetobacter sp. digested some organic carbon, which could not be algae, and produce carbon dioxide. Algae used the carbon dioxide released by bacteria for growth (Buchan et al., 2014). Such an integrated algal-bacterial system improved the recovery of carbon resource in centrate wastewater significantly. In addition, as a phosphorus-accumulating bacterium, Acinetobacter sp. in the algal-bacterial system provided another pathway to recovery of phosphorus. These are the main reasons why utilization efficiencies of resources, particularly COD and TP, in centrate wastewater were improved by the algal-bacterial system. The increase of resources recovery contributed to the improvement of biomass yield of algae. So the integrated algal-bacterial system, which reduced concentrations of nutrients in centrate wastewater to the permissible discharge limit and improved biomass yield of algae by improving the resources recovery, is a promising method to promote the commercial application of microorganisms in wastewater management.

5. Conclusions It was concluded that (1) A bacterial strain was isolated from centrate wastewater after treatment and identified as Acinetobacter sp.; (2) Co-cultivation of algae and Acinetobacter sp. significantly improved the removal of COD and TP in the centrate wastewater; (3) After

treatment, residual nutrients in centrate wastewater reached the permissible discharge limit; (4) In this case, the synergistic cooperation between algae and wastewater-borne bacteria was attributed in part to the exchange of carbon dioxide and oxygen between bacteria and algae; (5) Given the synergistic relationship, Acinetobacter sp. could be co-cultivated with algae for both wastewater treatment and biomass production.

Acknowledgements This work was in part supported by grants from Guangdong Province Natural Science Foundation of China (2015A030313596), International Science and Technology Cooperation Program of Guangzhou Project (2017) and Guangdong Province Agriculture Development (2016), the State of Minnesota through the Minnesota Environment and Natural Resources Trust Fund (ENRTF), and University of Minnesota Center for Biorefining.

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Figure Captions Figure 1. Pilot-scale bioreactor for centrate wastewater treatment Figure 2. Biomass yield of algae, growth of bacteria, nutrients removal efficiencies, and changes of ORP Figure 3. Biomass yield of algae, changes of ORP and pH, and removal efficiencies of nutrients in batch experiment Figure 4. Biomass yield of algae, changes of ORP and pH, and removal efficiencies of nutrients in the combined system of algae and Acinetobacter sp. Figure 5. Phylogenetic tree of isolated strain and related bacterial strains

Table 1. Basic characteristics of centrate wastewater Parameter TN NO3-N ORP CFU pH

Values 42.87±5.62 mg/L 1.01±0.11 111.00±8.66 mV 0.96×106±0.04×106 6.26±0.46

Parameter NH3-N COD TP TVSS

Values 31.72±0.38 mg/L 836.67±11.24 mg/L 36.91±3.77 mg/L 1.12±0.18 mg/L

Table 2. Concentrations of nutrients in wastewater (mg/L)

Initial Final Initial TP Final Initial NH3-N Final Initial COD Final TN

1st week 131.0 80.6 57.4 16.2 99.8 55.6 1704 753

2nd week 203.5 123.6 112.4 36.4 120.6 68.9 1832 795

3rd week 124.9 72.8 59.6 12.8 67.5 40.5 1027 300

4th week 89.1 50.4 39.8 5.8 61.1 35.4 1272 270

5th week 102.2 46.3 101.4 11.0 65.4 29.2 2113 351

6th week 109.0 50.2 120.8 11.1 68.7 33.7 2184 287

7th week 86.2 35.8 66.3 11.5 57.6 24.7 1801 256

Table 3. Homology between 16s rRNA gene sequences of isolated strain and GenBank strains rRNA length (bp)

Relevant bacterial strains

Proximity

1417

Acinetobacter towneri (AB 1110) Acinetobacter genomic (ATCC 17979) Acinetobacter haemolyticus (ATCC 17906) Acinetobacter calcoaceticus (DSM 30006) Acinetobacter baumannii (ATCC 19606) Acinetobacter lwoffii (ATCC 17925)

96.87% 96.69% 95.13% 95.31% 96.65% 95.23%

Acinetobacter baylyi (CCM 7195) Acinetobacter junii (LMG 998)

96.84% 96.01%

Table 4. Morphological and biochemical characteristics of isolated strain Characteristics Shape Size Glucose fermentation Contact enzyme Nitrite reduction DL-lactate utilization

Results Rod (1.0-1.5)µm × (1.5-2.5)µm + + +

Characteristics Gram reaction

Results -

Oxidase

-

Indole production Nitrate reduction Oxygen demand Citrate utilization

+ + +

(b)

(a) Figure 1

2.0

30

Initial CFU Final CFU

1.8 25 1.6 20

1.2

6

CFU (10 )

TVSS (g/L)

1.4

1.0

TVSS of wastewater Initial TVSS Final TVSS TVSS of algae

0.8 0.6

15

10

5 0.4 0.2

0 1

2

3

4

5

6

7

1

2

Experimental period (weeks)

3

4

5

6

7

Experimental period (weeks)

(a)

(b) 200

100

COD TP TN NH3-N

90

Initial ORP Final ORP 150

70

ORP (mV)

Removal efficiency (%)

80

60

100

50

50 40

30

0 1

2

3

4

5

6

7

1

Experimental period (weeks)

2

3

4

5

Experimental period (weeks)

(c)

(d) Figure 2

6

7

160

3.2

Bacteria Algae Algal-bacterial system

2.8

Bacteria Algae Algal-bacterial system

140

120 2.4

100

ORP (mV)

TVSS (g/L)

2.0

1.6

80

60

1.2

40

0.8

20

0

0.4 0

1

2

3

4

0

5

1

2

3

4

5

Experimental period (days)

Experimental period (days)

(a) TVSS of algae

(b) ORP of wastewater

10.0

45

9.5

40

9.0

Bacteria Algal-bacterial system

35

8.5

30

pH

6

CFU (10 )

8.0 7.5

Bacteria Algae Algal-bacterial system

7.0

25 20 15

6.5

10

6.0

5

5.5

0

5.0 0

1

2

3

4

5

0

1

Experimental period (days)

2

3

4

5

Experimental period (days)

(d) CFU

(c) pH of wastewater 35

900

Bacteria Algae Algal-bacterial system

30

Bacteria Algae Algal-bacterial system

800 700

25

COD (mg/L)

NH3-N (mg/L)

600

20

15

500 400 300

10 200

5 100

0

0

0

1

2

3

Experimental period (days)

(e) Removal of NH3-N

4

5

0

1

2

3

Experimental period (days)

(f) Removal of COD

4

5

45

45

Bacteria Algae Algal-bacterial system

40

Bacteria Algae Algal-bacterial system

40 35

35

30

TP (mg/L)

TN (mg/L)

30

25

20

25 20 15

15

10

10

5 0

5 0

1

2

3

4

0

5

1

2

3

Experimental period (days)

Experimental period (days)

(h) Removal of TP

(g) Removal of TN

Figure 3

4

5

160

3.2

Bacteria Algae Algal-bacterial system

2.8

Bacteria Algae Algal-bacterial system

140

120 2.4 100

ORP (mV)

TVSS (g/L)

2.0

1.6

80

60

1.2 40 0.8

20

0.4

0 0

1

2

3

4

5

0

1

Experimental period (days)

(a) TVSS of algae

4

5

(b) ORP of wastewater Bacteria Algae Algal-bacterial system

9.5

Bacteria Algal-bacterial system

40

9.0

35

8.5

30

8.0

25

6

CFU (10 )

pH

3

45

10.0

7.5

20

7.0

15

6.5

10

6.0

5 0

5.5 0

1

2

3

4

5

0

1

Experimental period (days)

2

3

4

5

Experimental period (days)

(c) pH of wastewater

(d) CFU

40

900

35

Bacteria Algae Algal-bacterial system

800

30

700

20

15

600

COD (mg/L)

Bacteria Algae Algal-bacterial system

25

NH3-N (mg/L)

2

Experimental period (days)

500

400

10

300

5

200

100

0 0

1

2

3

4

Experimental period (days)

(e) Removal of NH3-N

5

0

1

2

3

Experimental period (days)

(f) Removal of COD

4

5

45

45

Bacteria Algae Algal-bacterial system

40

Bacteria Algae Algal-bacterial system

40 35

35

30

TP (mg/L)

TN (mg/L)

30

25

20

25 20 15

15

10

10

5 0

5 0

1

2

3

4

5

0

Experimental period (days)

1

2

3

Experimental period (days)

(g) Removal of TN

(h) Removal of TP Figure 4

4

5

Acinetobacterhaemolyticus ATCC 17906T（Z93437） Acinetobacterbeijerinckii NIPH 838T (AJ626712)

20

Acinetobacter genomic sp. 16 ATCC 17988 (Z93451) 8

56

Acinetobacter genomic sp. 15bj 79 (Z93452) Acinetobacter schindleri NIPH 1034T (AJ278311)

4 31 44

Acinetobacter johnsonii ATCC 17909T, RUH 2231T: (Z93440) Acinetobacter bouvetii 4B02T (AF509827) Acinetobactertjernbergiae 7N16T (AF509825)

55

Acinetobacter tandoii 4N13T (AF509830)

38

Acinetobacter gyllenbergii NIPH 2150T (AJ293694) Acinetobacter parvus NIPH 384T (AJ293691) 5

Acinetobacter genomic sp. 17 942 (Z93454)

21

7

14 33

Acinetobacter genomic sp. 14BJ 382 (Z93453) Acinetobacter genomic sp. 13BJ14TU ATCC 17905 (Z93447) Acinetobacter lwoffii ATCC 17925 (Z93441) Acinetobacter genomic species 6 ATCC 17979 (Z93439)

53

Acinetobacter ursingii NIPH 137T (AJ275038) Acinetobacter genomic sp. ‘close to 13TU’ 10090 (Z93449) 23

Acinetobacter calcoaceticus DSM 30006T (X81661)

7

88 89 39

Acinetobacter genomic sp. 3 ATCC 17922 (Z93436) Acinetobacter genomic sp. ‘between 1 and 3’ 10095 (Z93450) Acinetobacter genomic sp. 10 ATCC 17924 (Z93443)

97 48

Acinetobacter genomic sp. 11 DSM 590 (X81659) Acinetobacter gerneri 9A01T (AF509829) 100

100

40

4

Acinetobacter baumannii ATCC 19606T (Z93435) Acinetobacter genomic sp. 13TU ATCC 17903 (Z93446) Acinetobacter junii LMG 998T (AM410704) ‘Acinetobacter venetianus’ RAG-1 (AJ295007)

18

Acinetobacter baylyi CCM 7195T (AM410709)

100 48

60

Isolated strain (FJ 645593)

Acinetobacter towneri AB 1110T (AF509823) 100 Acinetobacter genomic sp. 15TU 151a (Z93448) Acinetobacterradioresistens 17694T (Z93445) Alkanindiges illinoisensis MVAB Hex1T (AF513979) Psychrobacter immobilis ATCC 43116T (U39399) Moraxella lacunata ATCC 17967T (AF005160)

0.01

Figure 5

Highlights Algae were able to grow healthy on bacteria-containing centrate wastewater A strain of beneficial aerobic bacteria, Acinetobacter sp., was isolated Co-cultivation of algae and bacteria improved the nutrient removal efficiencies Synergistic cooperation was observed in the growth of algae and Acinetobacter sp.