Environmental estrogens (EEs) are estrogen analogues, and estrogen-like chemical substances that are distributed in the natural environment and can interfere or harm the endocrine function of the body. Common and major environmental estrogens include estrone (E1), estradiol (E2), estriol (E3), bisphenol A (BPA), and so on (Darbre, 2014). In recent years, with the development of China’s economy and industry, the natural environment of the environment estrogen shows a wide distribution, the concentration of the trend. There are reports that the total concentration of estrogen in river water bodies could reach more than 100 ng/L (Zhou et al., 2011)
Existing research on environmental estrogen has focused on the effects of environmental estrogen on the physiological function of humans and animals, and there is ample evidence that EEs can cause abnormalities in the endocrine system, nervous system, and immune system (Hartmann, Beyer, & Harm, 2014). For a long time, it has been thought that estrogen only has an effect on higher organisms with its receptors, so the research involving microorganisms focuses on the use of microorganisms to degrade environmental estrogen, and there are few studies on the impact of estrogen on microorganisms. But there have also been reports in recent years showing that estrogen can also have a significant effect on microbes. In this regard 17β-estradiol is studied more environmental estrogen. The effects of E2 on methane bacteria in anaerobic sediments were studied and a certain concentration of E2 was found to affect the rate of methane and carbon dioxide production in sediments (Ruan et al., 2013). It has also been reported that E2 can also inhibit the growth of methane-producing bacteria while being degraded (Ruan et al., 2014). There are also reports that E2 inhibits the denitrification of microbes, but increases the proportion of N2O in the product (Wu et al., 2014b). It is concluded that the effect of environmental estrogen on microorganisms is present, but the mechanism of the action and the effect of all aspects are not known. In addition, heterogeneous nitrification bacteria have many advantages and high nitrification speed, and have become a hot research topic for biological nitrification in recent years (Mathieu & Meng, 2019). In natural waters, ammonia nitrogen can be used or oxidized by the nitrification of heterotrophic nitrifying bacteria, thus moving closer to a relatively reasonable level. High concentration of ammonia nitrogen in water is one of the main causes of water eutrophication (Giannopoulos et al., 2017). If the nitrification or proliferation of heterogeneous nitrification bacteria is abnormal, it will have an adverse effect on the nitrogen cycle of natural water bodies. Based on the current situation of this study, this study obtained the heterotrophic nitrifying bacterial flora from natural water bodies, explored the effects of E2 on its growth condition and nitrification, and obtained preliminary results.
The North Moat, which is located in Beijing, China, is a part of the water system of the North Canal, which basically covers the entire Beijing City North Second Ring Road. The Water System of the North Canal is an important water system in Beijing, where 70% of the population lives. Although the water quality of the North Canal system has improved in recent years, most of the water in the urban area of Beijing is in Chinese poor V-type standard, ammonia nitrogen concentration of 10 mg/L (Guo et al., 2012). This experimental sample was taken on October 20, 2019, at the North Moat in Beijing, at Tanxishengjing Park (116.3692˚E, 39.9486˚N) near the Water Tank, and obtained 50 mL of the bottom water, with a measured sampling depth of about 59.5 cm. Put the taken water samples in centrifuge tubes, refrigerated them in ice packs and transported them to the refrigerator, temporarily stored them at 4˚C, and carried out training work on the same day.
1) Enrichment culture: the water sample was shaked well, at room temperature activated 24 h, then added the water sample 5 mL to the enrichment culture liquid, at 25˚C, low light, 160 rpm concussion culture for 24 h. Rich culture formula: 10 g per liter with trypsin, 5 g of yeast extract, 5 g NaCl.
2) Preliminary screening: took the rich culture fluid 5 mL, added 100 mL BM culture fluid, in 25˚C, 160 rpm concussion culture 24 h. OD600 ≥ 1.00 indicates that the culture concentration is enough, otherwise needs to continue culture. The BM culture liquid is equivalent to the amino nitrogen concentration of 100 mg/L of ammonium sulfate as the only nitrogen source of the culture. BM culture solution is: per liter containing (NH4)2SO4 0.472 g, sodium butyrate 5.62 g, K2HPO4 0.147 g, NaCl 0.125 g, MgSO4 0.125 g, FeSO4 2.5 mg, MnSO4 2.5 mg (Zhang et al., 2019).
3) After the culture ending, take 5 mL culture fluid to 100 mL BM culture fluid, repeat step 2) in the culture method 2 times, get heterotrophic nitrifying bacteria flora.
4) Save: the resulting bacteria liquid and glycerin 1:1 volume mixed, at −20˚C low temperature preservation.
Configured the BM culture fluid, took 5 mL of glycerin-preserved bacteria to add in 100 mL of BM culture solution, and immediately put the glycerin-preserved bacteria back into storage. The above BM culture fluid was treated at 25˚C to be treated with low light culture of 24 h, and the experimental bacteria liquid was obtained.
Divided tapered bottles into 5 groups, and added 100 mL BM culture fluid into each bottle. The purchased 17 β-estradiol (purity 99%, RUIBIO) was removed from the storage environment of 4˚C, a series of concentrations of solutions were immediately configured, 1 mL solution was dripped into the BM culture fluid of each group, and the BM culture fluid of the E2 concentration, which was finally used in the experiment. Added 1mL bacteria liquid to each bottle of culture, and set aside in a thermostatic incubator at 25˚C. In 1 d, 2 d, 3 d, 5 d, 7 d, respectively, the ammonia nitrogen concentration, nitrous nitrogen concentration, OD600 value of the culture fluid were determined. Reference to the State Environmental Protection Administration “Water and Waste Monitoring Analysis Method” (4th ed.), (Editorial Committee of Monitoring and Analysis Methods for Water and Wastewater, 2002) using the method of Nessler’s Reagents spectrophotometer to determine -N,N-(1-nixiamine)-ethyl diamine photometric method to determine -N, the method of UV spectrophotometry to determine OD600.
Average the two measured data for each set and graph and analyze it through Origin 2017 software.
3. Result and Analysis
3.1. Effects of E2’s Concentration on the Use of Ammonia Nitrogen by Nitrification Bacteria
Ammonia nitrogen is one of the common indicators of nutrients in water, which is directly related to water eutrophication. Tenfold gradient was set, then added E2 of 0, 0.1, 1, 10, 100 ng/L to the culture fluid and get the results below.
is the only initial nitrogen source in the solution, and its consumption can partly reflect the growth of bacteria. After 2d, the control group without E2 consumed 44.3% of ammonia nitrogen, whereas the E2 concentration of 1 ng/L group consumed 95.6% of ammonia nitrogen, and the E2-10 ng/L group also consumed 79.5%. E2-100 ng/L group was slightly higher than the control group, at 53.7%. In contrast, the E2-0.1 ng/L group consumed only 25.9%. Other data and overall trends are shown in Figure 1.
It can be found that, compared with the control group of E2-0, the -N using was faster with three groups of E2-1 ng/L and 10 ng/L. It shows that the utilization capacity of nitrification bacteria to ammonia nitrogen increased when E2 is 1 and 10 ng/L, and the utilization capacity of nitrification bacteria to ammonia nitrogen is reduced when E2 is 0.1 ng/L and 10 ng/L.
The impact of ammonia nitrogen utilization is also estimated. Since a group of ammonia nitrogen consumed by 95% at 48 h, only the first 48 h data were selected to get the figure below.
As can be seen from Figure 2, adding 0.1 ng/L concentration of E2 can reduce the daily average ammonia nitrogen utilization rate of 48 h by 41.6%. Therefore, the inhibition effect of ammonia nitrogen utilization of heterotrophic nitrifying bacteria at E2 concentration of 0.1 ng/L was significant. Correspondingly, the addition of 1, 10 ng/L E2 will be the use of ammonia nitrogen by heterogeneous nitrification bacteria to produce a certain role in promoting.
In the above, when the E2 concentration is 0 - 1 ng/L, the small concentration change will also have a significant effect on heterotrophic nitrifying bacteria.
As can be seen from Figure 3, when the E2 concentration is 10 - 100 ng/L, the change of E2 concentration will not make a significant change in the trend of ammonia nitrogen utilization of heterotrophic nitrifying bacteria, and the utilization of ammonia nitrogen varies slightly between the control groups. So compare with E2 of 0 - 1 ng/L, in the 10 - 100 ng/L concentration segments, the effect of E2 concentration change on heterotrophic nitrifying bacteria was less.
3.2. Effects of Different E2 Concentrations on Nitrification Products
In BM culture fluids, no is initially contained, so all comes from the nitrification of bacteria. Figure 4 shows that after the start of culturing 24 h, the concentration of in two groups of E2 with concentrations of 0 and 1 ng/L was lower, whereas the other groups were at 4 - 8 mg/L. Subsequently, the overall variation of the nitrogen concentration in each group was not significant, and eventually stabilized at 7 - 10 mg/L.
Although nitrification bacteria may produce N2O, or directly assimilate nitrogen sources during nitrification, so the nitrogen concentration of nitrification cannot accurately measure nitrification, the overall trend of -N growth in Figure 4 is about the same. The data shows that the E2 concentration at 0 - 100 ng/L had little effect on the general trend of nitrous oxide production by heterogeneous nitrification bacteria.
Figure 1. Ammonia nitrogen concentration changes over time.
Figure 2. Degradation rate of ammonia nitrogen in each group for the first 48 h (mg·L−1·d−1).
Figure 3. Effect of 10 - 100 ng/L E2 on ammonia nitrogen utilization, got by same bacteria sample and experimental method.
Figure 4. Nitrogen concentration changes over time.
3.3. Effects of E2 Concentrations on OD600
OD600 is the absorbent value of liquid at a wavelength of 600 nm, it is positively correlated with the concentration of bacteria in the liquid, in this experiment, no non-life suspension or lactose was contained in the culture system, so OD600 can be used to reflect the total concentration of bacteria in the culture fluid.
As can be noted from Figure 5, compared to the control group without E2, after 72 h, E2 of 0.1 ng/L group’s OD600 is 29.8% lower, and E2 of 100 ng/L is basically the same. But E2 at 1 ng/L and 10 ng/L two groups are higher, with 96.1% and 69.7%. After 7d, E2 concentrations 0.1, 1, 10, 100 ng/L were 23.6%, 34.9%, 35.9% and 7.7% higher than the control group’s OD600.
In Figure 5, the OD600 values in two groups with E2 concentrations of 1 ng/L and 10 ng/L were always higher than in the control group, whereas the E2 of 0.1 ng/L group was lower for most of the previous period. In the first 72 h, the three groups with 1, 10 and 100 ng/L of E2 had higher OD600 values than the control group. It is worth noting that OD600 value of 0.1 ng/L E2 group increased faster than the control group on the 7th day, indicating that compared to E2 to the use of ammonia nitrogen inhibition of heterotrophic nitrifying bacteria, E2 to the proliferation of heterogeneous nitrification bacteria is limited and just in the first 3d of the shorter period, heterotrophic nitrifying bacteria seem to be somewhat “adapted” to E2.
In the previous ammonia nitrogen data, in all groups added E2, -N took advantage of two groups with E2 plus 1 ng/L and E2 to 10 ng/L, whereas E2 concentrations were relatively slow in groups with a concentration of 0.1 mg/L. plus is the only initial nitrogen source in the solution, and its consumption can partly reflect the growth of bacteria. This is also verified on the OD600 data.
When E2 is 10 - 100 ng/L, there is a more interesting phenomenon. In each group that E2 has been added, OD600 value generally increased and then decreased or slowed down. If it is considered that: 1) the resources and time consumed by each bacterial division once are the same, with 0.05 units of resources consumed and during 1 unit of time each bacterial divided into nt; 2) when resources are exhausted in the culture medium, the bacteria will no longer divide; 3) in this experiment, it can be roughly considered that the amount of nutrients (s) in each group is the same, set as 100 units, and the initial number of bacteria (n0) is the same, set as 1 unit, which can be obtained by using dev-c ++ programming simulation (see Table 1).
Figure 5. OD600 changes over time.
Table 1. Using programs to simulate bacterial split results.
g: number of last generations n: number of last bacteria.
Obviously, with limited nutrients, the faster bacteria divide, or the shorter the time it takes to divide each generation, the faster it will reach the peak and the peak of the total.
In Figure 6, almost every experience group’s OD600 values were greater than the control group, otherwise it was similar, indicating that E2 had a significant effect on the growth of heterotrophic nitrifying bacteria. If fitted to it. Figure 7 showed the same results as the OD600 compared with E2 concentrations over the time.
Select the peak of the fitted curve, map again with E2 concentration x-axis and fit linearly to get the following image. (The E2 of 75 ng/L group cannot get a peak, the measured maximum is 0.85, so estimated peak to be 1.00, which has been marked in Figure 8.)
Figure 6. OD600 changes over time when E2 is 10 - 100 ng/L (same experiment in Figure 3).
Figure 7. OD600 for different E2 concentrations changes over time (fitting).
Figure 8. peaks of OD600 and E2 concentration (left figure: all data; right figure: without E2 = 25 ng/L group’s data).
It can be seen that the maximum number of bacterial proliferation is approximately linearly positively correlated with E2 concentration. Among them, E2 of 25 ng/L group has a large deviation. If it is screened out for linear fitting again, a very ideal linear relationship can be obtained.
Similar phenomena have been observed in other reports, says the growth of vibrio in the water body has a good linear relationship with E2 concentration in 0 - 1 ng/L (Wei et al., 2016), which is similar to the conclusion obtained in this paper. It is speculated that E2 has a catalytic effect on the growth of many microbes in water, whereas different microbes are differently sensitive to E2, and vibrio may be more sensitive to E2 than the whole heterotrophic nitrifying bacterial population in the water body.
3.4. E2’s Influence on Nitrifying Bacteria, Base on the OD600 and Ammonia
Figure 9 showed, in both groups with higher utilization rates of 1 ng/L and 10 ng/L, there was a decrease of OD600 been observed, and group with 100 ng/L E2 had similar phenomenon. Considering these two groups had more than 90% of consumption at the turning points, it may be because of bacteria’s excessive proliferation at earlier stages, which made the concentrate of ammonia decrease sharply but the number of bacteria was big, resulting in that some of bacteria died for lack of ammonia in medium stage, and finally back to the equilibrium point.
The result of NH4-N and OD600 measures shows that E2 has the effect of “low concentration of E2 could inhibit bacteria, medium concentration of E2 could promote bacteria and high concentrate of E2 have lower promoting effect”. This has also been reported in other study. According to Lin’s research, one kind of composite E2 degradation bacteria grown better when the E2 was 5 mg/L and 10 mg/L, compared with that when E2 was 1 mg/L and 20 mg/L (Lin, 2016). So it can be assumed that E2 has the same effect on the proliferation and using of nitrifying bacteria. For the group of heterotrophic nitrifying bacteria in natural water, the turning points of E2’s effect might be between 0.1 - 1 ng/L.
Figure 9. OD600 (Abs) and NH4 consumption (%) of all groups.
This article has proved that the concentration of E2 in the range of 0 - 100 ng/L has a significant effect on the use of ammonia nitrogen by heterogeneous nitrification bacteria in natural water bodies over a period of one week. The detail influence as shown in that: compared with the control group without E2, heterotrophic nitrifying bacteria was inhibited when E2’s concentration was 0.1 ng/L, and its daily N- consumption speed had a 41.6% reduction. Heterotrophic nitrifying bacteria was promoted when E2’s concentration was 1 ng/L, 10 ng/L or 100 ng/L, and its daily N- consumption speed increased by 88.8%, 79.6% and 21.4%. But the influence of E2 on heterotrophic nitrifying bacteria wasn’t so significant.
In addition, over the one-week study period, the concentration of E2 in the range of 0 - 100 ng/L has a certain effect on the proliferation of heterogeneous nitrification bacteria in natural water bodies, which could be described as “low concentration of E2 inhibited it in a short time but promoted it later, medium or high concentration of E2 promoted it”. As shown in that: compared with the control group without E2, after 72 hours, the group with 0.1 ng/L of E2’s OD600 was 29.8% lower, but 1 ng/L, 10 ng/L and 100 ng/L; of these three groups, their OD600 was 96.1%, 69.7%, 29.5% higher than the control group. However, after 7 days, 0.1, 1, 10, 100 ng/L’s groups’ OD600 were all higher than the control group, which were 23.6%, 34.9%, 35.9%, 5.4% higher.
When E2’s concentration was in the range of 10 - 100 ng/L, its concentration influenced the N- consumption speed by nature water’s heterotrophic nitrifying bacteria, but the overall influence wasn’t huge. But in this range, heterotrophic nitrifying bacteria’s proliferation speed showed positive correlation with E2’s concentration, and the OD600 even showed linear relation to it (R2 = 0.67495).
E2 had been widely detected in Chinese natural water (Yao et al., 2018). E2, as one of the environmental estrogens, could not only influence human body and harm various kinds of animals, but lead to nitrifying bacteria growing abnormally, which results in the decline of nature water’s ammonia self metabolic ability, and the aggravating of its eutrophication. So the monitoring of E2 in the nature water should be intensified in the future.
This work was supported by the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2018ZX07111003).
*Changhao Xiao and Ziyi Dong contribute equally to this work.
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