Wetland Model Study of Plant Rhizosphere

Microorganism in the Treatment of Sanitary

Wastewater
Li Shen-gui
Department of Urban Construction
Huazhong University of Science and Technology Wuchang Branch
Wuhan, China
lishengui@263.net

Abstract—The degradation effect of adding rhizosphere
microorganism artificially on the COD of sanitary wastewater
based on wetland models was studied in the experiment. Two
rhizosphere bacteria isolated from the constructed wetland were
mixed with a certain proportion of wastewater and injected into
the wetland model. The COD degradation of wastewater was
examined at 12h, 24h, 36h, and 48h respectively. The experiment
lasted for two months in the spring, in which the changes of
bacteria number and clogging status of the wetland substrate
were investigated. The COD degradation ratio of the bacteria
model was significantly higher than that of the control model
(P<0.05), and the difference was even greater along with the time.
The 48h COD degradation ratio for sanitary wastewater in the
control model and the two bacteria models (hereafter model 1
and model 2) was 50.6%, 73.0% and 75.3% respectively.
Moreover, the result of substrate permeability experiment
showed that the infiltration duration of the control model is 28%
longer than that of the bacteria model .The above experiment
results implied that adding rhizosphere microorganism
artificially may improve the COD degradation ratio of sanitary
wastewater in wetland, and alleviate the wetland clogging
significantly. Based on these experiment results, further work
should be oriented to exploit the technology.
Keywords-COD degradation ratio; rhizosphere microorganism;
wetland model; clogging
I. INTRODUCTION
The wetland wastewater treatment systems have been
widely developed and applied for its low investment and
treatment cost [1]. With the tertiary coordination function of
physics, chemistry and biology of the compounding ecosystem
of substrate–microorganism- plant, the constructed wetland can
purify the wastewater efficiently and restore the water
environment by filtration, adsorption, precipitation, ion
exchange, plant absorbance and microbial decomposition.
Moreover, the constructed wetland may stimulate the growth of
green plants and yield more products㸪to realize recycling and
safe treatment of wastewater by nutrients and biogeochemical
cycles in ecosystems [2-4].
Wetland plants rhizosphere microorganisms is an important
activated component in pollutants degradation of wetland [5].
There exist some studies on the screening of wetland plants
rhizosphere microorganisms [6-7] 㸪while the research on the
application of screening strains to the wetland model is not
reported. The degradation effect of adding rhizosphere
microorganism artificially on the COD of sanitary wastewater
based on wetland models was studied in the experiment. The
COD degradation results of sanitary wastewater, both in a short
time and in a long time, are reported. The number of bacteria
and wetland substrate clogging in a long time were also
examined.
II. MATERIALS AND METHODS
A. Materials
1) Bacteria source.
Two bacteria strains which are efficient in the degradation
of COD in sanitary wastewater were isolated in the lab from
the south lake wetland and used in the experiment [7].
2) The culture medium of beef-cream and peptone.
3) Instruments.
Sterile console, Autoclave pot, Rotary shaker, the balance,
Centrifuge, Lovibond COD Instrument and Reagents.
4) Wastewater source
The wastewater used in the experiment was collected from
Shahu sewage treatment plant of Wuhan. The COD of
wastewater was 85mg/L.
5) Constructed wetland model
As shown in figure1, the parameters of the wetland model
in this experiment were 0.4m in length, 0.26m in width and
0.25m in height. The bottom layer of Wetland was underlain by
a layer of gravel with the thickness of 0.1-m and 0.03-0.05m in
diameter of the gravel. The upper was a layer of fine sand with
the thickness of 0.15-m. Four cannas were planted on the
treatment bed in two lines. The model was placed in the
greenhouse. The influent passed into the wetland models by
gravity, and the effluent sluiced out of the outlet. Discharge
valve was placed on the bottom of model.
Three models in the experiment: The control model - no
bacterium was added artificially;
Model 1 -bacteria 1 was added artificially;
Model 2 - bacteria 2 was added artificially.

This project is supported by the Research Grant of Hubei Province in

Science and Technique (2006AA305A0402)

___________________________________
978-1-61284-340-7/11/$26.00 ©2011 IEEE


 Figure.1 Structure of the wetland model
B. Methods
1) Bacteria culture
Add two preserved bacteria liquids, 0.05 ml each, which
were preserved in the refrigerator at -80Υ, into 50ml of liquid
culture medium respectively. Incubate the medium on rotary
shaker for 48 hours at 28 Υat 120r/min. Three parallel samples
were selected for each group.
Inject the well-cultured bacteria liquid into 50ml centrifuge
tubes with normal saline. Centrifuge the tubes twice at
4000rpm for 20min. Volume the precipitation (bacteria) to 5ml.
The number of bacteria ranged from 2.9 ™ 106 ~ 3.5™ 106
cfu/ml.
2) Model operation for short-time
3.0 ml of bacteria fluid was added to 3L of wastewater.
The mixture was injected into the model, and the degradation
of wastewater COD was examined at 12h, 24h, 36h, and 48h
respectively by standard Potessium Dichromate. No bacterium
was added artificially to the control model.
3) Model operation for long-time
The result of the short-time experiment showed that the
degradation differences between the bacteria models were very
little. Then model 1 and the control model were used for the
long-time experiment. After 48h of hydraulic retention time ,
the degradation of wastewater COD was examined by standard
Potessium Dichromate. At the same time, the stored
wastewater was emptied before the next cycle. The mixture of
3 Liters of wastewater and bacteria was injected into the
experimental model. No bacterium was added artificially to the
control model. The experiment lasted for 62 days.
4) The number of substrate bacteria
Collection of the upper substrate: the substrate in upper
10cm layer was collected by a specific sampling tube in situ.
The bacteria number of the substrate was examined with
dilution and plate counting method [8].
The bacteria number in the effluent of the bacteria model
was examined with the same method as above.
5) Substrate permeability
Porosity is the ratio of the filling material pore volume over
the total volume. Substrate in wetland model system trapped
pollutants in the process of wastewater treatment. Therefore,
compared with the natural state, the pore volume decreased.
The sandy porosity in natural state is called total porosity N,

Figure.2 Test tube of soil permeability
Whereas porosity of the wetland in actual state is called
effective porosity Ne [9].
In April 2009, four pieces of pipes, 0.4m in length, were
marked as 0, 0 ', 1, 1 ' respectively, where 0, 0 'were for the
control model and 1, 1' for the bacteria model. The substrate
was collected by a specific sampling tube in situ, keeping its
original shape in the process of sampling. The bottoms of the
sampling tubes were bound with gauze and rubber band, and
the infiltration duration of 300ml water was measured.
III. RESULTS AND ANALYSIS
A. The COD degradation ratio in short-time
As we can see from Figure 3, the COD degradation ratio of
the three models increased over time. The COD degradation
ratio of Model 2 was the highest at 48h, reaching to 75.3%. In
comparison, the ratio of Model 1 was 73.0% and the control
model was 50.6%.
By analyzing the variance, the COD degradation ratio had
no significant difference between model 1 and model 2 (P >
0.05). Within the first 24h, the degradation ratios of the
bacteria models were significantly higher than the control
model (P < 0.05), and the differences were even greater over
time. The differences were as high as 22.4 ~ 27.9 at 48h.
B. The COD degradation ratio in long-time
The experiment lasted for 62 days (2009.03.01- 2009.04.30)
which can be divided into 3 stages: earlier stage, middle stage
and later stage. Results of model operation are as following:
At first, the degradation ratio of the bacteria model was
significantly lower than the control model (10%-25%).
Figure.3 The COD degradation ratios of sanitary wastewater in the three
wetland models at different time

Figure.4 The COD degradation ratio of sanitary wastewater in two
wetland models in long-time in spring
However, the degradation ratio of the bacteria model
increased steadily over time, from 39.6% to 75%. While within
this time the ratio of the control model was constant at the level
of about 60 %. If stopping adding bacteria to the bacteria
model for the first time, the degradation ratio obviously
decreased to 48.1%.Keeping the initial bacteria amount and
with the applying of bacteria again, the degradation ratio of the
bacteria model increased steadily and reached to 85.9%.When
stopping adding bacteria for the second time, the degradation
ratio of the bacteria model was higher than the control model in
the earlier two times, but lower than the control model in the
later two times. These results imply that the treatment effect of
the bacteria model would guarantee for four days in short-time
after stopping adding bacteria. Starting to add bacteria for the
third time while keeping initial bacteria amount, the
degradation ratio continued to rise.
The experiment results can be summarized as follows:
Under stable operation condition, biotic community with
relatively stable microorganism biomass and microbial activity
was gradually developed in wetland substrate. The bacteria of
upper soil also increased in the continuous and intermittent
process of adding bacteria.
With the growth of the bacteria number, the degradation
ratios of the control model and the bacteria models improved
steadily, which implies that the COD removal of wastewater
relied on the amount of microorganisms in the constructed
wetland [5].
The number of bacteria in the effluent of the bacteria model
ranged from 102-103 /ml. Hence, the effluent was worthless for
reusing due to the low concentrations of bacteria
D. The clogging of the wetland substrate
Experiment result: the infiltration time of 300mL water for
the control model was 3'33", and the time for the bacteria
model was 2'30".
The total packing volume of the constructed wetland:
VT = 0.40 ™ 0.26 ™0.25= 0.026 m3
The water volume which could be hold in the packing
under natural condition is 0.0035 m3. After a long-time
operation, the water volume changed to 0.0026 m3 in the
control model and 0.0030 m3 in the bacteria model.

When the degradation ratio was stable, the bacteria amount
was reduced to half the initial bacteria amount. Seen from
Figure 4, the degradation ratio decreased to 38.1%. The result
indicates that the effect of the bacteria model depends on the
initial bacteria amount. Adding bacteria for the fourth time
with initial amount unchanged, the degradation ratio was on the
rise again.
The results imply artificially adding bacteria may
significantly improve the degradation ratio of sanitary
wastewater in wetland model. However, if stopping adding the
bacteria or reducing the amount of bacteria added, the
treatment effect will be weakened rapidly. A mature biofilm
did not form during the experiment, therefore, the amount of
bacteria added should not be reduced.
N= V0 / VT = 0.0035 / 0.026 = 13.5%.
The control model:
Ne = Ve/ VT = 0.0026/0.026 = 10%
The bacteria model:
Ne = Ve/ VT = 0.0030 / 0.026 = 11.5 %
After the operation of several months, the porosity of the
control model decreased from 13.5% to 10.0% whereas the
porosity of the bacteria model decreased from 13.5% to 11.5%.
And the infiltration duration of the control model was 28%
longer than that of the bacteria model. The results indicated
that artificially adding rhizosphere microorganism may have a
certain alleviation effect on the wetland clogging.

C. The number of substrate bacteria IV. CONCLUSIONS

Artificially adding bacteria can effectively degrade the
TABLE I. CHANGE OF BACTERIA NUMBER IN THE MODELS complex organic compounds in wastewater, reduce organic
The The matters in the substrate and improve the substrate permeability.
The The bacteria In the short time, artificially adding bacteria can improve the
degradati bacteria
degradation number of
Time ratio of
on ratio number of
controlled
degradation ratio of organic matter. And in a long time, it can
of controlled increase substrate permeability and alleviate the wetland
controlled model
bacteria model clogging.
model% cfu/ml
model% cfu/ml
09.03.17 61% 48% 0.8h106 1.4h106 The degradation effect of the bacteria model greatly
6 6
depends on the bacteria amount. The treatment effect will
09.04.06 67% 58% 2.8h10 5.0h10
decrease once stopping using the bacteria or reducing the
09.04.28 70% 85% 3.0h10 6
9.2h107 bacteria amount, which will push the project cost.
Further work lies in: firstly, how to keep microorganisms
in good activity in a cold climate to ensure the operation of


wetland in cold regions. Secondly, screen microorganisms with
spores are screened so that they can be made into powder to
reduce operation and transportation cost in practice, which
makes the application of microorganism more feasible.
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