The Feasible Research of Lakes Eutrophication Using the Ecosystemized multi-Probiotics

Abstract
The emerging environmental issue of lake eutrophication was recognized as a water pollution problem in lakes and reservoirs in the mid-20th century, and the situation has become more serious currently worldwide. Human activities accelerate the rate of nutrients entering ecosystems, and the trend is now approaching ecologically critical levels. This project examines whether the water quality can be remediated by adding biologics agents – Ecosystemized multi-Probiotics – to ponds with eutrophication. Three trials, (3 out-door ponds with a capacity of 2000, 100, 1.2 m³, respectively) were run for 2 months with the same treatments (~6 ppm biologics agents added). Biological Oxygen Demand, Suspended Solids, Ammonia Nitrogen/NH₄⁺ and pH value were taken as indicators of water quality. The indicators showed a significant improvement of water quality in all three trials; The results showed that the pond gradually became clearer and more transparent. Thus, Ecosystemized multi-Probiotics is a potential solution to help mitigate algal blooms.
Introduction
The technique of inducing restoration of a micro-ecosystem using multi-Probiotics is a new aquatic ecology engineering method used to treat eutrophication. The Ecosystemized multi probiotics strategy combines various probiotics strains and culture mediums, which is similar to an ecosystem community of living organisms in conjunction with the nonliving components of their environments. Ecosystemized multi-Probiotics consist of various probiotics with appropriate proportions of aerobic, anaerobic and facultative anaerobe.
This work aims to explore the effectiveness of introducing the Ecosystemized multi-Probiotics into water with Eutrophication and examines its impact on Biological Oxygen Demand, Suspended Solids, Ammonia Nitrogen/NH₄⁺ and pH value of lake water. It was hypothesized that if Ecosystemized multi-Probiotics are added into water with Eutrophication, then pH value will decrease and the water would be clearer.
Background Information
Literature Review

1. Lake Eutrophication – A Causality of Human’s Pollution

Eutrophication is an emerging environmental issue currently impacting the water quality of lakes worldwide. Daniel J. et al. state that runoff from agriculture and development, pollution from the septic systems and sewers, sewage sludge spreading, and other human-related activities increase the flow of nutrients, phosphate, and organic substances into the aquatic ecosystem, resulting in an algal bloom. ^[1]
Smith and Schindler state that reducing eutrophication should be a key concern when considering future policy, and a sustainable solution for everyone, including farmers and ranchers, seems feasible.^[2] Bryan M. et al. point out that the methods of minimizing non-point pollution, such as dredging and geoengineering in lakes, can be used to significantly reduce harmful nitrate and phosphate leaching. However, the process of de-eutrophication is expensive. ^[3]

1. Serious Threat to Biodiversity

Yann Hautier, Pascal A. Niklaus, Andy Hector state that eutrophication is a serious issue, and the pH value is the most common indicator of algal blooms. Algal blooms limit the sunlight available to bottom-dwelling organisms and cause wide swings in the amount of dissolved oxygen in the water, increasing the water’s alkalinity and pH. Under eutrophic conditions, dissolved oxygen greatly increases during the day, but decreases after dark due to the respiring algae and microorganisms that feed on the increasing mass of dead algae. When dissolved oxygen levels decline to hypoxic levels, fish and other marine animals suffocate. As a result, creatures such as fish, shrimp, and especially immobile bottom dwellers die off as anaerobic conditions ensue, promoting the growth of anaerobic bacteria. This is the mechanism of the dead zone formation. ^[4]
In literature studies, many species are unable to reproduce and currently, more than half of lakes worldwide are under eutrophication.  As pH value increases, the rate of freshwater algal blooms increases, and the entire biodiversity of lakes changes.^[1][2][3][5]

1. Remediation by Ecosystemized multi-Probiotics

Val H. Smith’s current research on eutrophication is focused on the extent of the problem and its implications, emphasizing remediation methods of internal nutrient release budget as a major factor to control eutrophication.^[2] Miltiadis Zamparas and Ierotheos Zacharias note that the physical and chemical lake restoration method is one of the methods, but it is not a panacea. It is necessary for connection with an assessment of the potential adverse effects on humans, livestock, biotic and abiotic factors. ^[5] The technique of inducing restoration of micro-ecosystems would be a new aquatic ecology engineering method used to treat eutrophication, that would avoid the disadvantages of physical and chemical methods.
Method and Materials
Water Samples: 
Water used in this experiment was collected from three open-air long-term monitoring sites, as shown in Table 1.
Table 1 the parameter of three open-air long-term monitoring ponds before the test.

Site	Area (m2)	Depth (m)	capacity(m3)	water source	RPI
Pond 1	4000	1	4000	rainwater	6.2 (Heavy pollution)
Pond 2	40	2.5	100	rainwater and tap water	5 (Mid pollution)
Pond 3	3	0.4	1.2	rainwater	6.7 (Heavy pollution)

The monitoring sites: Pond 1, Pond 2 and Pond 3 are shown in Figure 1, Figure 2 and Figure 3, respectively.
Figure 1. Pond 1, HsinChu, Taiwan, October 2018

Figure 2. Pond 2, Beitou, Taipei, Taiwan, October 2018

Figure 3. Pond 3, Beitou, Taipei, Taiwan, October 2018
Yen-Chang Chen, Hui-Chung Yeh, and Chiang Wei took the River Pollution Index (RPI) as a measure of eutrophication.^[6] The water samples for this experiment were collected using the same method of RPI: water samples were filtered into clean containers using a 200-micron water filter and stored in the dark at 5 °C for no more than 24 hr to prepare the test treatments, at which time they were brought to the test room temperature (25~27 °C).
Ecosystemized multi-Probiotics Preparation:
The Ecosystemized multi-Probiotics, provided by Dr. HewDer Wu, were poured into the water body until the concentration averaged ~6 ppm based on the capacity of each Pond. The quantity and frequency of multi-Probiotics for the 3 ponds are shown as follows:
Pond 1: 30-litre multi-Probiotics twice per week; the 30-litre is diluted with water to be 300 litres, then sprayed evenly on the pond with a pump.
Pond 2: 0.6-liter multi-Probiotics per week; the 0.6-litre is diluted with water to be 6 litres, then sprayed evenly on the pond by hand.
Pond 3: 0.01-litre multi-Probiotics per week; the 0.01-litre is diluted with water to be 600ml, then poured evenly on the small pond by hand.
Ecosystemized multi-Probiotics consist of various bacteria with appropriate proportions of aerobic, anaerobic and facultatively anaerobic. All the beneficial bacteria are screened from the soil directly with non-toxic and non-pathogenic Microbes and are non-genetically modified or engineered.
Testing
The five test items (i.e. Dissolved Oxygen(DO), Biological Oxygen Demand 5(BOD₅₎, Suspended Solids, Ammonia Nitrogen/NH₄⁺ and pH value) were run for around 60-days with 3 replicates each for the three differing ponds. The unit of DO, BOD₅, Suspended Solid, and Ammonia Nitrogen/NH₄⁺ is “mg/L”. Each test vessel was purged with distilled water before being used. To prevent stagnation and bacteria build-up, the water sample was tested within 24 hr of collection from the ponds. The instructions and observations are described as follows:

1. DO: The electrode can be placed in the appropriate depth of the BOD bottle or another appropriate sampling container. After the sample is collected, the sample is slowly placed in the BOD₅ bottle and analyzed immediately on site.
2. BOD₅: The BOD₅ consists of filling with samples to overflowing, and incubating it at 20 °C for 5 d in an airtight bottle. Dissolved oxygen is measured initially and after incubation and the BOD is computed from the difference between initial and final DO.
3. Suspended Solid: The method of Suspended Solid is 500ml of the sample, passed through a prepared, pre-weighed filter paper. The filter paper is dried at 104 ± 1°C. After drying the filter is reweighed and the Suspended Solid is calculated.
4. Ammonia Nitrogen/NH₄⁺:Take 10mL of blank water sample and 10mL of water sample to be tested, respectively add 0.2mL of reagent 1, shaking well; then add 0.2mL of reagent 2, respectively, shaking well. Stand at room temperature for 10min, use a colorimetric bottle to perform colorimetric testing.
5. pH meter: Using pH 7.0 and pH 4.0 standard buffer solution calibration instrument, respectively. Then take out the electrode and temperature probe, wipe dry with lens cleaning paper, immerse in the solution to be tested, press the \”HOLD\” key to release the lock, and the value on the display is the pH value of the solution to be measured.
6. Observation: Typical observations included the appearance and transparency of the water body and water sample after the multi-Probiotics treat the water in ponds.

Results
The results of Dissolved Oxygen, Biology Oxygen Demand 5, SuspendedSolid, Ammonia Nitrogen/NH₄⁺ and pH value for Pond 1, Pond 2 and Pond 3 are shown in the following tables:

1. Dissolved oxygen (DO)：

Dissolved oxygen (DO) refers to the amount of oxygen dissolved in water. It is an important index for assessing water quality. Table 2 shows the Dissolved oxygen of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics during the testing period. It was found that the Dissolved oxygen of three ponds was non-regular with the addition of multi-Probiotics. The value of dissolved oxygen was prepared for the further study of BOD₅.
Table 2 the Dissolved oxygen of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics during the test period.

Pond 1		Pond 2		Pond 3
BOD5 Date	Dissolved Oxygen (mg/l)	Date	Dissolved Oxygen (mg/l)	Date	Dissolved Oxygen (mg/l)
10/15	8.99	10/18	4.22	10/20	2.65
10/28	7.05	10/31	3.87	10/28	5.99
11/09	7.49	11/06	4.22	11/04	0.22
11/24	6.35	11/13	2.25	11/11	1.61
12/09	4.47	11/21	3.57	11/18	0.05
		11/27	3.81	11/24	3.15
		12/05	3.45	12/09	4.22
		12/12	4.74	12/20	4.32
		12/20	4.34

II. Biological Oxygen Demand 5 (BOD₅ )：
BOD represents the amount of organic matter which can be decomposed by aquatic organisms. It also indirectly represents the degree of organic pollution of the water. BOD is measured in a water sample during 5 days of incubation at 20℃, known as The value of BOD₅ value of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics. The BOD₅ values of Pond 1, Pond 2 and Pond 3 are 28.1, 54.3, and 32.5 mg/L, respectively, which are more than 3mg /L in initials for all three ponds. ( Figure 4) It means that the eutrophication of three ponds are all on a heavy level and severely polluted.^[6] The BOD₅ value decreased stably with the addition of multi-Probiotics for Pond 1 and Pond 2 during the test period; and 3.82 and 8.83mg / L for Pond 1 and Pond 2 at end of the test, respectively. However, Pond 3 showed an unstable trend with a BOD₅ value of 19.9 mg/L at the end. It is implied that the water quality of Pond 1 and Pond 2 improved gradually from the addition of multi-Probiotics. On the other hand, Pond 3 is a very small Pond and the water quality would be affected easily by the change of surroundings, such as rain, fallen leaves. (Figure 3)
Figure 4 The value of BOD₅ value of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics.
III. Suspended Solid content
Suspended solids refer to the organic or the inorganic particles which have not settled due to the motion of the water. The trend of Suspended Solid content of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics is shown in figure 5. The initial Suspended Solid content of Pond 1, Pond 2 and Pond 3 was 38.5, 24.9, and 15.1 mg/L, respectively. That is, Pond 1 and Pond 2 were Moderately-Polluted, and Pond 3 was Lightly-Polluted from Suspended solids initially. ^[6] In the end, suspended solid content was 10.5, 10.4, and 35.4 mg/L for Pond 1, Pond 2, and Pond 3, respectively. As well as BOD₅, the Suspended solids content decreased with the addition of multi-Probiotics for Pond 1 and Pond 2, but not for Pond 3.
Figure 5. the trend of Suspended Solid content of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics
IV. Ammonia nitrogen
The water is polluted when the value of the concentration of Ammonia nitrogen is over 0.5 mg/L. The concentration change of Ammonia nitrogen of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics is shown in figure 6. The Ammonia nitrogen of Pond 1, Pond 2 and Pond 3 was initially 1.22, 0.15, 0.85 mg/L, respectively. That is, Pond 1 was Moderately-Polluted, Pond 2 was Non-Polluted, and Pond 3 was Lightly-Polluted in initials. ^[6] In the end, the concentration of Ammonia nitrogen was 2.81, 0.57, and 4.35 mg/L for Pond 1, Pond 2, and Pond 3, respectively. The concentration of Ammonia nitrogen increased with the addition of multi-Probiotics for all three ponds.
Figure 6. concentration change of Ammonia nitrogen of Pond 1, Pond 2 and Pond 3 with the addition of the change of multi-Probiotics
V. pH value
pH refers to the negative logarithm of the concentration of hydrogen ions in the water. Natural water is generally neutral or weakly alkaline. When water is Eutrophied, the pH value can be increased substantially with the Algae bloom.
The pH value of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics is shown in figure 7. The pH value of Pond 1, Pond 2 and Pond 3 was initially 9.4, 8.05, 7.38, respectively. That is, Pond 1 was Severed-Polluted with a heavy Algae bloom, Pond 2 was Moderated-Polluted with slight Algae bloom and Pond 3 was close to neutral, initially.^[6] In the end, the pH value was 7.32, 7.29 and 7.31 for Pond 1, Pond 2, and Pond 3, respectively. The pH value approached neutral for all three ponds with the addition of multi-Probiotics.

Figure 7 the pH value of Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics.
According to the results above, the RPI value for the three ponds has been reduced to be slight pollution and no pollution, as shown in Table 3.
Table 3 the RPI value for the monitoring ponds after the test.

Site	Area (m2)	Depth (m)	capacity(m3)	water source	RPI
Pond 1	4000	1	4000	rainwater	3.25 (Mid pollution)
Pond 2	40	2.5	100	rainwater and tap water	2.5 (slight pollution)
Pond 3	3	0.4	1.2	rainwater	5.5 (mid pollution)

Discussion
The results illustrate that the addition of multi-Probiotics to water with eutrophication can improve water quality significantly, regardless of water body size. The water quality continued to improve as probiotics were added.
The restoration of the micro-ecosystem of the lake utilizes multi-Probiotics to induce ecological restoration. The appropriate ratio of Probiotics (Ecosystemized multi-Probiotics) was induced into an ecological environment, and as a result, the eutrophication of single species in the monotonous water was converted into multiple balanced and stable ecological systems. This strategy combines various probiotics strains, containing abundant functions and enzymes, to collectively accomplish a complex task and to undergo the self-cleaning function. As a result, a healthful and stable food chain is expected to establish gradually, and the ecological environment is expected to return to normal.
I. Biological Oxygen Demand 5 (BOD₅)
With regards to Biological Oxygen Demand 5 (BOD₅), the addition of multi-Probiotics to the Ponds had a significant effect on decreasing the Biological Oxygen Demand 5 and it was pond size independent. Additionally, the addition of multi-Probiotics did increase the transparency of the ponds as well as the decrease of BOD₅ for the three ponds. This effect was significant, regardless of the pond size. (Figure 8 and Figure 9)
Regarding the factor of Pond size, the BOD₅ decreased significantly in a few days when the multi-Probiotics was added to Pond 3, but reversed and increased after mid-Nov. It is inferred that the fallen leaves could have increased the amount of organic matter in Pond 3, and Pond 3 is easily affected by external factors due to it being a very small pond.

Figure 8. The addition of multi-Probiotics did increase the transparency of Pond 1. The upper photoed on 10/15, the lower photoed onn 12/09.

Figure 9 the addition of multi-Probiotics did increase the transparency of Pond 3. The left was photographed on 10/20. The right was photographed on 12/20.
II. Suspended Solid content
The suspended solid content decreased with the addition of multi-Probiotics in Pond 1 and Pond 2. However, it increased in Pond 3. (Figure 5) The decrease of suspended solid content in Pond 1 and Pond 2 is possibly due to the fact that the organic particles, plankton and algae have been consumed by the addition of multi-Probiotics. The suspended solid content in Pond 3 increased, possibly because the organic particles from decomposing materials also contributed to the concentration of total suspended solids. As algae, plants and animals decay, the decomposition process allows small organic particles to break away and enter the water column as suspended solids. Pond 3 is a very small pond and is easily affected by fallen leaves and other factors.
Overall, the transparency of the three ponds increased, and the algae disappeared gradually with the addition of multi-Probiotics (Figure 8 and Figure 9).
 III. Ammonia nitrogen
There was no obvious change in the concentration of Ammonia nitrogen in Pond 1, Pond 2 and Pond 3 with the addition of multi-Probiotics. Ammonia nitrogen comes mainly from the decomposition of feces, animal and plant remains. Thus, there would have been a constant source of feces, animal and plant remains to decompose into Ammonia nitrogen during the period of the experiment. That could be the reason why the concentration of Ammonia nitrogen did not decrease during the period of the experiment. (Figure 6)
IV. pH value
The pH value of water with eutrophication is rather complicated. The mechanism of lowering the pH value is described as following:
a. Inorganic Chemicals stem and pH
The introduction of nitric and phosphoric acids makes the environment a temporary nutrient source for an algae bloom.
b. Algal Blooms and pH
Photosynthesizing algae cover the lake or pond surface, competing for light. The chemical byproducts of this photosynthesis process increase the pH of the water, making it more alkaline. Delicate organisms that cannot survive under these chemical conditions will die, while more resistantanimals that feed on algae will experience population growth.
c. Organic Matter and pH
As time passes, the algae begin to die. The dying algae drop to the bottom of the lake and decompose. Bacteria, which decompose this organic matter, consume oxygen from the water and produce alkaline byproducts. Bottom-feeding animals that cannot handle the low oxygen content and high pH would die, decreasing the biodiversity of the environment.
The pH value of all the three ponds reached neutral after adding the multi-Probiotics. ( in Figure 7).
V. Summary
In conducting this experiment, there were several possible factors of uncertainty. It should be noted that these results were not collected in a completely controlled environment. The ponds would have been subjected to other stressors of rain, fallen leaves, sunny and winds which would have altered the results in terms of DO, BOD₅, suspended solids content, Ammonia nitrogen and pH value, etc. Nevertheless, the water quality improved with the addition of multi-Probiotics in Pond 1 and Pond 2. This result could be due to the fact that Pond 1 and Pond 2 (over 100 m3) are larger and thus more tolerant to the above impacts. On the contrary, Pond 3 is too small to tolerate the above environmental factors.
This experiment’s use of multi-Probiotics and the results obtained are significant given that globally billions of metric tons of eutrophied water resources have become an increasing problem due to the sheer volume and mass of water pollution. In this experiment, the appropriate ratio of Probiotics was induced into a small-scale ecological environment and could be extended to larger bodies of water. Furthermore, the multi-Probiotics could also be used in marine protected areas to reduce eutrophication and mitigate the dead zones of the ocean.
The associated costs, dependent on the method of application, must be taken into consideration. For accessible lakes, the multi-Probiotics could be applied by boating the watershed or lakeside. Furthermore, the dosage of multi-Probiotics would need to be calculated taking into consideration the volume of the lake. 3~10 ppm of the volume of lake per week is the most economical. Constant monitoring of the lake eutrophication level would be required to determine if and when additional applications of multi-Probiotics would be required.
Although the results of this experiment were promising, this topic required more study and experimentation to determine if remediation using multi-Probiotics has any long-term effects or impacts on other lake organisms. The potential impacts on aquatic organisms in ponds warrants further research. A bottom mud treatment of the lake could be employed to see if leaching from the bottom mud can be reduced using a multi-Probiotic method.
This approach of using multi-Probiotics is preferable to the current method due to high cost or deleterious environmental effects. Multi-Probiotics are a readily available and usable resource. Therefore, the use of multi-Probiotics to help combat environmental problems such as eutrophication, in turn, helps to alleviate the growing issue of water pollution and water scarcity. Finally, the multi-Probiotics may have minimal side effects on the aquatic ecosystem, allowing the water body to recover to a balanced state.
Conclusion
This work suggests that the addition of multi-Probiotics can help mitigate the eutrophication of ponds. Therefore, this method could be effective for the remediation of lake micro-ecosystems, which is beneficial for the recovery of aquatic organisms and water quality.
The multi-Probiotics application does not require costly equipment to treat the mass volume water with eutrophication. For example, in an accessible lake, the multi-Probiotics could be applied by boating the watershed or lakeside. The dosage of multi-Probiotics required is dependent on the volume of the lake, and there is no observed negative side effect. As a result, the use of multi-Probiotics has very few deleterious environmental impacts. Furthermore, the multi-Probiotics can also be used as a preventative measure in lakes with low organic pollutants to protect the lake from eutrophication, thus helping maintain the existing ecosystem. Accordingly, this experiment identifies a method to remediate eutrophied lakes and has the potential to be beneficial in maintaining lake ecosystems. This in turn helps protect lake biodiversity and mitigate algae blooms while at the same time contributes toward addressing the global issue of water scarcity.
Acknowledgements:
The author would like to thank Dr. HewDer Wu for his helpful advice on various technical issues examined in this paper. The author also thanks Taiwan Greendevice Co. Ltd. for providing the instrument for the test of water quality.
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