Engineering, 2013, 5, 459-462
http://dx.doi.org/10.4236/eng.2013.510B094 Published Online October 2013 (http://www.scirp.org/journal/eng)
Copyright © 2013 SciRes. ENG
Restoration Study of Microorganisms in
Lake Water Purification
Fang Yang, Rui Zhu
Hebei Electric Power Design & Research Institute, Shijiazhuang, China
Email: z hurui 423@163.com, bdsunjinxu@163.com
Received 2013
ABSTRACT
We screened the bacteria with restoration and purification functions in lake water and found that, Bacillus subtilis had
the highest water purification ability. Photosynthetic bacteria, lactobacillus bacteria, nitrifying bacter ia and oligotrophic
bacteria also showed different levels of lake water purification and restoration. L1873 orthogonal experiments were de-
signed to optimize the ratio of complex agents to get the best ratio of various bacteria as 10 g/L Bacillus subtilis, 2 g/L
photosynthetic bacteria , 0.8 g/L lactobacillus , 0.6 g/L nitrifying b acteria 1, 0.4 g/L nitrifying bacteria 2 and 0.6 g /L oli-
gotrophic bacteria. When lake water was purified for 30 d under this ratio, total phosphorus content decr eased 85.90%,
total nitrogen content decreased 70.96%, and COD value decreased 81.19%.
Keywords: Water Purification Microorganisms; COD; Total Phosphorus; Total Nitrogen
1. Introduction
Due to the impacts of human life and pollution, nitrogen,
large amounts of phosphorus and other nutrients entered
into the lake [1]. This causes rapid growth of algae and
other plankton, decreased dissolved oxygen in water bo-
dies, deteriorated water quality, and massive death of fish
and other organisms; this phenomenon is known as water
bloom of lake water. The major components that cause
lake water bloom were phosphorus, nitrogen and organic
carbon [2]. The microbial water purification and restora-
tion rely mainly on microbial metabolism for th e purpose
of deaminase, dephosphorization and carbon transfer to
purify and r e stor e water bodies [3].
Lake water is generally hydrostatic water and more
prone to eutrophication, which is further exacerbated by
the plant nutrients imputs from industrial wastewater,
domestic sewage and agricultural runoff. It is hard to
solve the eutrophication of lake solely re ly ing on the self-
purification of lake water, and lake eutrophication is ur-
gent to be solved [4,5].
In this study, we screened the bacteria strains with
restoration and purification functions in lake water and
studied the ratio of bacteria agents in order to obtain the
microbial agents with significant purification and resto-
ration effects on lake water.
2. Materials and Equipment
2.1. Materials and Strains
Lake water was obtained from Hengshui Lake at Nation-
al Wetland Nature Reserve; Bacillus subtilis, Photo-
synthetic bacteria, Lac tob acillus bacteria, Nitrifying bac-
teria and Oligotrophic bacteria were preserved by our
laboratory.
2.2. Instruments
Multi-parameter water quality analyzer was purchased
from Hualian Technology Co., Ltd. (5b-6c).
3. Methods
3.1. Determination of the Total Number of
Bacteria
The number of bacteria was determined by plate count-
ing method.
3.2. Determination of Total Nitrogen
Total nitrogen was determined as described in “Alkaline
potassium persulfate digestion ultraviolet spectrophoto-
metry determination of water quality total nitrogen
(GB11894-89).
3.3. Determination of Total Phosphorus
Total phosphorus was determined as described in “Am-
monium molybdate spectrophotometry determination of
water quality total phosphorus (GB 11893-89).
3.4. Determination of COD
CODMn was determined as described in “protein-per-
F. YANG, R. ZHU
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460
manganate index determination” (GB11892-89).
3.5. Microorganism Culture
1) Seed culture media
Seed culture media for Bacillus subtilis, photosynthet-
ic bacteria, lactobacillus bacteria, nitrifying bacteria and
were beef extract peptone medium.
2) Proliferation mediu m (g/L): glucose 1, yeast extract
0.7, peptone 1, (NH4)2SO4 0.2, MgSO4·7H2O 0.2, KH2PO4
1, and agar 18 g, pH 7.2.
3.6. Screening of Lake Wat er Rest oration and
Purification Bacteria
According to information reported in literature, microor-
ganisms with w ater restoration and purification functions,
Bacillus subtilis, photosynthetic bacteria, lactobacillus
bacteria, nitrifying bacteria 1, nitrifying bacteria 2 and
oligotrophic bacteria, w ere sel ected, and total phosphorus,
total nitrogen and COD value in lake water were meas-
ured. Lake water from same position was selected and
aliquoted into 7 1L-flasks. The adding amount of micro-
organisms were: 6 g /L Bacillus subtilis, 2 g/L photosyn-
thetic bacteria, 0.4 g/L lactobacillus, 0.2 g/L nitrifying
bacteria 1, 0.2 g/L nitrifying bacteria 2, and 0.4 g/L oli-
gotrophic bacteria, with plain water as control. Different
water indicators in lake wa ter wer e determined 20 d after
adding agents, a nd the measure temperature was 20˚C.
3.7. Design of Orthogonal Experiment for
Complex Agent s Ratio
According to relevant experimental reports and conven -
tional value, orthogonal experiments were preformed
mainly using Bacillus subtilis with five other supple-
mental microorganisms, each containing three concentra-
tions (L1873). The level of orthogonal design factors was
shown in Table 1.
4. Results and Discussion
4.1. Screening of Lake Wat er Rest oration and
Purification Bacteria
According to relevant literature, six kinds of microor-
ganisms with lake water purification and r estoration abil -
ity were selected for pilot study. Related indicators were
measured, and the re sults were shown in Figure 1.
As shown in Figure 1, 20 d after adding agents, total
phosphorus, total nitrogen and COD values in each water
sample revealed that all six bacteria had different levels
of water pur ification and restor ation ability. Bacillus s ub-
tilis had strongest purificatio n ability of total phosphorus
purification, lactobacillus had higher ability of nitrogen
purification, and photosynthetic bacteria higher ability
Table 1. L18(37) orthogonal experimental design.
level Factors
A(g/L) B(g/L) C(g/L) D(g/L) E(g/ L) F(g/L) G
1 6 1 0.4 0.2 0.2 0.4 1
2 8 2 0.6 0.4 0.4 0.6 2
3 10 3 0.8 0.6 0.6 0.8 3
Notes: A: Bacillus subtilis; B: Photosynthetic bacteria; C: Lactic acid bac-
teria; D: Nitroba cte ria 1; E : Nitrobacteria 2; F: Oligotrophic bacteria; G: Error.
The total phosphorus
The total nitrogen
COD
The total phosphorus (mg/L). The total nitr ogen (mg/L). COD (mg/L)
B
C
D
E
F
G
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
A: The initial quality of lake water
B: The quality for lake water of adding Bacillus subtilis
C: The quality for lake water of adding Photosynthetic bacteria
D: The quality for lake water of adding Lactic acid bacteria
E: The quality for lake water of adding Nitrobacteria 1
F: The quality for lake water of adding Nitrobacteria 2
G: The quality for lake water of adding Oligotrophic bacteria
A
Figure 1. The purifying and repairing bacteria screening
for lake water.
of COD purification. Comprehensive analysis of three
measured indicators to compare to the purification ability
of each bacterium showed that Bacillus subtilis group
exhibited strongest water purification capability. Ac-
cording to the purification characteristics of each bacte-
rium, we used a complex agent based on Bacillus subtilis
with five other supplemental bacteria as purification
agents for studies.
4.2. Ratio of Complex Agent
Using COD value as L1873 orthogonal experiment results,
complex agent was added and the results were deter-
mined 20 d later, as shown in Table 2.
As shown in Tables 2 and 3, the ratio difference of
various agents had high impacts on the measured COD
values. Among each agent ratio, Bacillus subtilis and
nitrifying bacteria displayed highly significant impacts,
and lactobacillus and oligotrophic bacteria had signifi-
cant impacts. According to the F value, the order of im-
pacts of each bacteria on COD was: Bacillus subtilis >
F. YANG, R. ZHU
Copyright © 2013 SciRes. ENG
461
Table 2. Visual analysis for L18(37) orthogonal experimental
design.
Number
of test
Factors COD
(mg/L)
A B C D E F G
1 1 1 1 1 1 1 1 16.91
2 1 2 2 2 2 2 2 17.32
3 1 3 3 3 3 3 3 15.25
4 2 1 1 2 2 3 3 13.05
5 2 2 2 3 3 1 1 11.25
6 2 3 3 1 1 2 2 10.85
7 3 1 2 1 3 2 3 7.22
8 3 2 3 2 1 3 1 7.35
9 3 3 1 3 2 1 2 7.38
10 1 1 3 3 2 2 1 5.36
11 1 2 1 1 3 3 2 7.9
12 1 3 2 2 1 1 3 9.53
13 2 1 2 3 1 3 2 7.52
14 2 2 3 1 2 1 3 7.9
15 2 3 1 2 3 2 1 8.82
16 3 1 3 2 3 1 2 7.65
17 3 2 1 3 1 2 3 5.27
18 3 3 2 1 2 3 1 6.81
K1 12.05 9.62 9.89 9.60 9.57 10.10 9.42
K2 9.90 9.50 9.94 10.62 9.64 9.14 9.77
K3 6.95 9.77 9.06 8.67 9.68 9.65 9.70
R 5.10 0.28 0.88 1.95 0.11 0.96 0.35
Table 3. The variance analysis for mycelium production.
Factors The square
of deviations Free
degree F The critical
value of F Significance
A 78.627 2 2125.054 19.000 ****
B 0.228 2 6.162 19.000
C 2.933 2 79.270 19.000 *
D 11.397 2 308.027 19.000 **
E 0.037 2 1.000 19.000
F 2.787 2 75.324 19.000 *
G 0.423 2 11.432 19.000
Error 0.04 2
nitrifying bacteria 1 > lactobacillus > oligotrophic bacte-
ria > photosynthetic bacteria > nitrifying bacteria 2. Ac-
cording to the K value, the best bacteria combination was:
A3B2C3D3E2F2, i.e. 10 g/L Bacillus subtilis, 2 g/L photo-
synthetic bacteria, 0 .8 g/L lactobacillus, 0.6 g/L nitrify-
ing bacteria 1, 0.4 g/L nitrifying bacteria 2, and 0.6 g/L
oligotrophic bacteria. It is proved by experiments that
under th is condition, the COD value at 20 d after adding
complex agents was 5.13 mg/L, which was lower than
the minimal value in orthogonal experimental design.
Comparing with the COD value in original lake water
sample, the number decreased 81.15%.
4.3. The Impact of Compl ex Agent on Total
Phosphorus Reduction
Complex agen t in optimal ratio was added into lake wa-
ter, total phosphorus content in lake water was measured
every 5 d. The results were shown in Fi gure 2.
As shown in Figure 2, aft e r complex agent in optimal
ratio was added into lake water, the total phosphorus
content in lake water quickly declined in 10 d. Between
10 d and 30 d, the total phosphorus content decreased,
but in a slow rate. Within the experimental period, the
lowest total phosphorus content was observed at 30 d,
with the minimal value of 0.11 mg/L and the relative
reduction of 85.90%, indicating that addition of complex
agent in this ratio can effectively reduce the phosphorus
content in lake water.
4.4. The Impact of Complex Agent on Total
Nitrogen Reduct ion
Complex agent in optimal ratio was added into lake wa-
ter, total nitrogen content in lake water was measured
every 5 d. The results were shown in Figure 3.
As shown in Figure 3, af ter co mplex agent in optimal
ratio was add ed in to lake w ater , the total nitrogen content
in lake water quickly declined in 20 d then slowly de-
creased. Within the experimental period, the lowest total
nitrogen content was observed at 30 d, with the minimal
value of 4.03 mg/L and the relative reduction of 70.96%,
The total phosphorus (mg/L)
0.0
0
The total phosphorus
5
10
15
20
25
30
Days
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Figure 2. The complex agent effect to total phosphorus of
lake water.
F. YANG, R. ZHU
Copyright © 2013 SciRes. ENG
462
indicating that the complex agent in this ratio had good
effects on reducing nitrogen content in lake water.
4.5. The Impact of Complex Agent on COD
Value
Complex agen t in optimal ratio was added into lake wa-
ter, COD value in lake water was measured every 5 d.
The results were shown in Figure 4.
As shown in Figure 4, when adding complex agent in
optimal ratio, the COD value showed highest reduction
extent in the first 20 d, which may be because the better
effects and higher activity of complex agent during this
period. After 20 d, the COD valu e in lake water decreased
slightly, but remained similar. A t 30 d, the COD value in
lake wat er was lowest, with the minimal value of 5.13
mg/L and the relative reduction of 81.89% comparing to
The total ni t rogen (mg/L)
0
0
The total nitrogen
5
10
15
20
25
30
Days
2
4
6
8
10
12
14
16
Figure 3. The complex agent effect to total nitrogen of lake
water.
COD (mg/L)
0
0
COD
5
10
15
20
25
30
Days
5
10
15
20
25
30
Figure 4. The complex agent effect to COD of lake water.
no complex agents control, indicating that addition of
complex agent in this ratio can effectively reduce the
COD value in lake water.
5. Conclusion
Eutrophication can cause deteriorated water quality and
reduced level of dissolved oxygen in water, wh ich af-
fected the living of water creatures. Application of mi-
croorganisms for water purification and restoration can
effectively purify water. Through the screening of bacte-
ria with water restora tion and purification capacity show-
ed that Bacillus subtilis had strongest water purification
ability, and other bacteria had different levels of impacts
on water purification and restoration. Using L1873 or-
thogonal experimental design to optimize the ratio of
complex agents, we got the optimal ratio of each bacteria
was: 10 g/L Bacillus subtilis, 2 g/L photosynthetic bacte-
ria, 0.8 g/L lactobacillus, 0.6 g/L nitrifying bacteria 1, 0.4
g/L nitrifying bacter ia 2 and 0.6 g/L oligotrophic b acteria.
When lake water was purified for 30 d under this ratio,
total phosphorus content decreased 85.90%, total nitro-
gen content decreased 70.96%, and COD value decreased
81.19%. These indicated that complex agent in this ratio
can effectively reduce the total phosphorus, total nitrogen
and COD value in lake water with good purification ef-
fect on lake wate r.
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