Effects of the inclusion of a mixed Psychrotrophic bacteria strain for sewage treatment in constructed wetland in winter seasons

Constructed wetlands (CWs) have been used globally in wastewater treatment for years. CWs represent an efficient ecological system which is both energy-saving and low in investment for construction and operational cost. In addition, CWs also have the advantage of being easy to operate and maintain. However, the operation of CWs at northern latitudes (both mid and high) is sometimes quite demanding, due to the inhibitory effect of low temperatures that often occur in winter. To evaluate the wastewater treatment performance of a culture of mixed Psychrotrophic bacteria strains in an integrated vertical-flow CW, the removal rates of ammonia nitrogen (NH3–N), chemical oxygen demand (COD), nitrite nitrogen (NO2−−N), nitrate nitrogen (NO3−−N) and total phosphorus (TP) were quantified at different bacterial dosages to determine the best bacterial dosage and establish kinetic degradation models of the mixed strains. The bacterial culture was made up of Psychrobacter TM-1, Sphingobacterium TM-2 and Pseudomonas TM-3, mixed together at a volume/volume ratio of 1 : 1 : 1 (at bacterial suspension concentrations of 4.4 × 109 ml−1). Results showed that the organic pollutants (nitrogen and phosphorus) in the sewage could be efficiently removed by the culture of mixed Psychrotrophic bacteria. The optimal dosage of this mixed bacteria strain was 2.5%, and the treatment efficiency of COD, NH3–N, NO2−−N, NO3−−N, total nitrogen and TP were stable at 91.8%, 91.1%, 88.0%, 93.8%, 94.8% and 95.2%, respectively, which were 1.5, 2.0, 2.1, 1.5, 2.2 and 1.3 times those of the control group. In addition, a pseudo-first-order degradation model was a good fit for the degradation pattern observed for each of these pollutants.

JC, 0000-0002-1277-7891 Constructed wetlands (CWs) have been used globally in wastewater treatment for years. CWs represent an efficient ecological system which is both energy-saving and low in investment for construction and operational cost. In addition, CWs also have the advantage of being easy to operate and maintain. However, the operation of CWs at northern latitudes (both mid and high) is sometimes quite demanding, due to the inhibitory effect of low temperatures that often occur in winter. To evaluate the wastewater treatment performance of a culture of mixed Psychrotrophic bacteria strains in an integrated vertical-flow CW, the removal rates of ammonia nitrogen (NH 3 -N), chemical oxygen demand (COD), nitrite nitrogen (NO − 2 −N), nitrate nitrogen (NO − 3 −N) and total phosphorus (TP) were quantified at different bacterial dosages to determine the best bacterial dosage and establish kinetic degradation models of the mixed strains. The bacterial culture was made up of Psychrobacter TM-1, Sphingobacterium TM-2 and Pseudomonas TM-3, mixed together at a volume/volume ratio of 1 : 1 : 1 (at bacterial suspension concentrations of 4.4 × 10 9 ml −1 ). Results showed that the organic pollutants (nitrogen and phosphorus) in the sewage could be efficiently removed by the culture of mixed Psychrotrophic bacteria. The optimal dosage of this mixed bacteria strain was 2.5%, and the treatment efficiency of COD, NH 3

Material and methods
2.1. Isolation, screening and identification of the mixed Psychrotrophic bacteria strain 2.1.1. Enrichment culture, separation, screening and purification of the Psychrotrophic bacteria Sediment samples, collected from Nansihu Lake in Shandong province, were incubated at a temperature of 2°C for 7 days in order to domesticate the microbes. Five grams of sediment was placed in each conical flask and shaken in an orbital shaker incubator (150 r.p.m.) at a temperature of (6 ± 1)°C. To allow the microbes to proliferate rapidly, we placed a few vitreous balls and 95 ml of sterile culture medium in each flask.
One millilitre of the nutrient solution was added to each test tube with 9 ml of sterilized water (1 : 10 dilution of the culture medium). In this way, dilution to 10 −2 , 10 −3 , 10 −4 , 10 −5 and 10 −6 times less than the original concentration of the culture medium could be easily obtained. Subsequently, 1 ml of the solution was taken from each flask and diluted 10 −4 , 10 −5 , 10 −6 times less than the original concentration. These solutions were then inoculated in three kinds of isolation media (with three parallels for each kind of medium), using agar plates. After that, the plates were placed into the incubator at a temperature of 6(±1)°C and growth was recorded constantly. The nine best-growing strains were selected and repeatedly purified to acquire a single colony. Then the colonies were inoculated in a slant culture medium and reserved in the refrigerator at 4°C.

The screening of the mixed Psychrotrophic bacteria
After the enrichment cultivation, the nine single colonies were inoculated in the simulated wastewater while at a pH of 7.0. Inoculation quantity was controlled to within 10%. The solutions were statically cultured following 6 h of aeration, and the concentrations of COD, TP and NH 3 -N were measured every 12 h. According to the removal efficiency of the pollutants, the most efficient strains (1, 2, 3, 4 and 5) were selected.
These five bacterial strains, which had high-efficient degrading capability, were inoculated in lysogeny broth (LB) culture medium at different concentrations. The growth status of the mixed flora was recorded regularly. Meanwhile, the mixed flora was inoculated in the simulated wastewater at a pH of 7.0. The inoculation quantity was controlled within 10%. The solutions were then statically cultured after 6 h of aeration at 6(±1)°C, and the concentrations of COD, TP and NH 3 -N were measured every 12 h. According to the removal efficiency of the pollutants, the combination of the 1, 4 and 5 strains had the most notable effect on sewage disposal efficiency.

Identification of the mixed Psychrotrophic bacteria species
To identify the species of bacteria in the mixed Psychrotrophic bacteria, the 1, 4 and 5 strains were dyed, placed under the microscope and the morphological characteristics of these bacteria were recorded in table 1. A preliminary identification was carried out using physiological and biochemical characteristics of the bacterial strains.
Using a scanning electron microscope the 1, 4 and 5 strains were purified and inoculated on an LB solid culture medium. After incubation at 30°C for 24 h, the 1, 4 and 5 strains were selected, isolated and then rinsed several times with sterilized water. Firstly, the thalluses were fixed with 3% glutaraldehyde, then rinsed, and fixed with osmic acid. Finally, the bacteria were dehydrated with ethanol.

Integrated vertical-flow constructed wetland systems and operation
Six small-scale plots were constructed in March 2012. Each plot (2 m 2 ) was equally divided into two chambers: a down-flow chamber and an up-flow chamber, indicating the direction of the passing water. A collecting ditch, 100 mm deep, was installed at the bottom of the chambers. Each chamber comprised three different particle-size-distribution layers: 150 mm depth of gravel (40-50 mm diameter) on the bottom, 250 mm slag (5-10 mm diameter) in the middle and 300 mm (in the down-flow chamber) or 250 mm (in the up-flow chamber) brown soil (0-4 mm diameter) on the top. All the water pipes were   When the system worked, the sewage was first dosed evenly on the surface of the down-flow chamber, then the sewage flowed downward vertically, finally reaching the up-flow chamber through a connecting pipe. After reaching the up-flow chamber, it flowed upward vertically, and finally was discharged from the effluent pipes. In this experiment, the CW was designed to be a discontinuous-flow system and was operated in a non-saturated state. The detention time in the system was set to be 5 days.
The influent water was taken from the sewage treatment plant in Qufu. Parameters are listed in table 2. Preparation of bacterial suspension: the 1, 4, and 5 strains were incubated in the LB liquid culture medium until their absorbance reached 1.2. The solution was then centrifuged, then the supernatant was discarded and the bacteria thallus was diluted with a saline solution. This process was repeated    three times so that the nutrient of the medium could be fully removed. In the end, the absorbance of the solution was adjusted to 1.2 with the saline water. These bacterial solutions were then mixed together at a volume ratio of 1 : 1 : 1.
In early December 2014 (water temperature between 4 and 10°C), suspensions of mixed Psychrotrophic bacteria (4.4 × 10 9 ml −1 ) were injected into the IVCW system at a 0.5-5% volume/volume ratio to the sewage. We also included a control treatment that was not incubated with any Psychrotrophic bacteria. The concentrations of different parameters in effluent, including COD, NH 3 -N, NO − 3 −N, NO − 2 −N, TN and TP, were monitored constantly for three months.
All the parameters were analysed according to standard methods. Statistical analysis was performed using Origin8.6 and SPSS19 software.

Identification of mixed Psychrotrophic bacteria
Identification results of morphologic, physiological and biochemical characteristics of the bacterial strains at 6(±1)°C can be found in figure 2.
Under cold-temperature conditions (6(±1)°C), Strain 1 was composed of a Gram-positive translucent circular ivory colony, with a moist surface, and regular edge. Strain 4 was a Gram-negative opaque round milky colony with a moist surface, and irregular edge. Strain 5 was a Gram-positive translucent yellow colony with a moist surface and irregular edges. Results from SEM (scanning electron microscopy) are shown in figure 2. Strains 1, 4 and 5 were are all spherical with no flagella. According to the Shanghai Biological Engineering Co., Ltd, the specific 16S rDNA sequence of Strain 1 was 1498 bp (GenBank acceptance number KR083014), the sequence of Strain 4 was 1489 bp (GenBank acceptance number KR083015), and the sequence of Strain 5 is 1489 bp (GenBank acceptance number KR083016). A BLAST search was performed on the three strains, and the phylogenetic tree was analysed and constructed by using Clustal W and PHYLIP software. The phylogenetic tree is shown in figure 3.
According to the physiological and biochemical characteristics and the 16S rDNA of strains, strains 1, 4 and 5 could be preliminarily identified as cold Bacillus, sphingolipids of Bacillus and Pseudomonas. Strain 1 was named Psychrobacter TM-1, and strains 4 and 5 were named Sphingobacterium TM-2 and Pseudomonas TM-3, respectively.

Effect of simulated wastewater treatment
The degradation efficiencies of COD, NH 3 -N and TP from the simulated sewage at 6(±1)°C by these mixed Psychrotrophic bacteria cultures were 86.83%, 65.95% and 56.08%, respectively. Average removal efficiencies were 1.38, 1.17 and 1.11 times higher than the single strains of P. flava WD-3 (GenBank acceptance number JX114950) [20]. In addition, the removal performance was found to be very stable.

The effect of sewage treatment on an artificial wetland
When the hydraulic retention time (HRT) was 5 days and the temperature of water was 6-10°C, the water treatment efficiency in the CW with different inoculation amounts of low-temperature mixed flora was as shown in figures 3 and 4.  (e) ( f )  a, b, c, d, e and f ).
As shown in figure 3, between 0.5% and 5.0% bacteria the purgative efficiency of sewage in wetland system increased as the dosage of the mixed Psychrotrophic bacteria was increased. Indeed, when the amount of bacteria was increased from 0.5% to 2.5%, the sewage purification efficacy of the wetland system was significantly improved. When the amount of bacteria was increased from 2.5% to 5.0%, the    purification efficacy was improved; however, the improvement was not significant. When the combined bacterium dosage was 5.0% the removal of the pollutants was improved; however, the cost was too high to make it feasible. Considering the operating costs and the efficaciousness of sewage treatment, 2.5% should be chosen as the best dosage of the mixed Psychrotrophic bacteria for the CW. It can be seen in figure 4 that when the HRT was 5 days, the water temperature was 6-10°C and the dosage of combined bacterial liquid was 2.5%. As the hydraulic time was increased, the removal Our results showed that the removal efficiency of COD and NH 3 -N in wetland sewage was 6.4% and 6.5% higher than expected when the removal rate of the single P. flava WD-3 was 86.3% and 83.5% [20]. Other indicators of sewage treatment filtration did not show this level of improvement. Indeed, there were no significant differences among these indicators. Furthermore, the HRT was shortened by 5 days when the mixed Psychrotrophic bacteria was used in the CS (5 days versus the 10 days taken when using only P. flava WD-3 [20]). There this bacterial application both increased the efficacy of sewage treatment and reduced the cost of sewage purification.
Compared with one single low-temperature bacteria strain, the mixed bacteria strain better purified the sewage and shortened the HRT. In comparison, Bott & Love [21] treated aquaculture sewage with the mixed bacterial culture of Bacillus subtilis and photosynthetic bacteria, and found that the mixed bacterial culture had a noticeable effect on flocculation and could display synergistic properties. They found that the removal efficiencies for COD, TP and NH 3 -N were 62.35%, 66.78% and 52.60% after 48 h of treatment. Furthermore, sewage effluent transparency increased to 148.48% greater than the original, and dissolved oxygen (DO) increased 240%, compared to the original. Similarly, Moussavi & Heidarizad [22] added a mixture of bacteria to SBR directionally and found that the time of domestication and aeration was shortened, and the disposal ability and the effects of sequencing batch reactor (SBR) process were significantly improved, with a COD removal efficiency of greater than 92%. The wastewater purification abilities of B. subtilis, Saccharomyces, Lactobacillus and mixed strains were studied by Morikawa [23], and the results showed that the mixture of flora had a synergistic effect, improving the disposal capacity of the pollutants, with NH 3 -N and removal efficiencies reaching 93.2% and 97.8%, respectively. Therefore, a significant amount of prior research has shown that there are mutually beneficial relationships among the strains, and that mixing bacterial strains can have a synergistic effect in terms of contaminant removal efficacy. Moreover, these studies also indicated that there is potential to use these mixed bacterial strains to dispose of low-temperature sewage.

Wastewater treatment dynamics equation
The study of kinetics could optimize the technology and the methods associated with the biochemical disposing process. Indeed, the patterns of the microbial degradation could be predicted by establishing kinetic degradation models and simulating degradation with modern technology. The models used in the construction of the CWs were typically first-order kinetic models. This basic design equation (first-order kinetic model) has been widely applied in Australia, Europe and the USA for the prediction of bacterial treatment results. In spite of certain limitations, first-order kinetic models are still regarded as the most suitable for describing the process of degradation for pollutants in CWs. This is because calculation parameters can be worked out easily and the calculation process is simple. Jou et al. investigated the feasibility of using a CW to restore a heavily polluted creek, and estimated the reductions in biochemical oxygen demand (BOD) and nitrogenous biochemical oxygen demand (NBOD) using the first-order kinetic model in a laboratory wetland system, which provided some basis for using this model when designing the constructed wetlands [24].
As for the first-order kinetic equation applied in CWs, the main consideration was the relationship between the disposal load and the processing efficiency. The derivation of the model used here was based on the degradation of the matrix following the first-order reaction kinetics. The first-order kinetic model for the degradation of contaminants in CW was [25,26] as follows:    The volume degradation rate k v represents the treatment efficiency of the contaminants. Mixed Psychrotrophic bacterial flora had a noticeably high disposal rate for pollutants at a HRT of 5 days. Furthermore, the degradation rates of COD, NH 3 -N, NO − 2 −N, NO − 3 −N, TN and TP significantly increased as the dosage of bacteria was increased. The correlation coefficients (R) of each k v reached 0.9816, 0.9597, 0.9473, 0.9934, 0.9267 and 0.9183. Through the analysis of treatment efficiency for the pollutants in the CW at different dosages, it is evident that the removal kinetics of contaminants in the CW was in accordance with the first-order kinetics model.

Conclusion
(1) The nine bacterial strains, isolated from artificial wetland sediments, were cold-resistant bacteria.
Following screening, a combination of the best strains (1, 4 and 5) had the highest removal efficiency for COD, NH 3 -N and TP in wastewater. Removal rates for this mixed Psychrotrophic bacteria strain were 86.83%, 65.95% and 56.08%, respectively. The strains 1, 4 and 5 were preliminarily identified as cold Bacillus, sphingolipids of Bacillus and Pseudomonas, and were named Psychrobacter TM-1, Sphingobacterium TM-2 and Pseudomonas TM-3. (2) The effectiveness of the IVCW system with the mixed Psychrotrophic bacteria strain in winter conditions was demonstrated through the increased removal of COD, NH 3 -N, NO − 2 −N, NO − 3 −N, TN and TP. At high concentrations of organic substrate, the dosage of the mixed Psychrotrophic bacteria strain and the removal rate of the contaminants were positively correlated and followed a first-order rate equation. We also evaluated the effects of different bacterial dosages and determined that the optimal dosage of this mixed Psychrotrophic bacteria strain was 2.5% for these CW systems when considering biological effect, time, cost and resource consumption.