Study on the Growth Law of Photosynthetic Bacteria in Wastewater
Treatment
Qiuhua Shen
a
Departments of Coast Engineering, Naval Logistical Academy, Tianjin, 300450, China
Keywords: Photosynthetic Bacteria (PSB), Medium Optimization, Wastewater Treatment.
Abstract: There are many methods to measure microbial growth. In this study, we took photosynthetic bacteria (PSB)
as the main body and optimized its culture medium formula. Our study displayed that the optimal medium
formula for PSB was sodium acetate 3.3 g/L, NH4Cl 0.6 g/L, KH2PO4 0.9 g/L, MgSO4 0.5 g/L, and beef
extract 1.5 g/L. The optimized medium could effectively facilitate the growth of PSB.
1 INTRODUCTION
1
1.1 Photosynthetic Bacteria
Photosynthetic Bacteria (PSB), a type of bacteria that
can produce photosynthesis, are one of the earliest
bacteria on earth. They utilize various amino acids,
sugars, fatty acids or ethanol in the environment as
hydrogen donors to synthesize organic compounds
under photosynthesis. They can adapt to different
ecological environments and are mainly distributed in
the soil and the silt of rivers, ponds, sewage ditches,
oxidation ponds, and oceans. PSB are widely
distributed and have many kinds. According to
whether they generate oxygen during photosynthesis,
they can be divided into oxygen-producing PSB
represented by cyanobacteria and non-oxygen-
producing PSB represented by rhodopseudomonas.
Generally, non-oxygen-producing PSB include four
groups of green non-sulfur bacteria, purple sulfur
bacteria, green sulfur bacteria, and purple non-sulfur
bacteria. Oxygen-producing PSB, usually called
aerobic photosynthetic bacteria, are divided into
cyanobacteria and prochlorophyte.
PSB wastewater treatment technology is a newly
emerging wastewater treatment method in recent
years. Compared with the conventional activated
sludge wastewater treatment technology, it has the
advantages of recovering single-cell protein and no
secondary pollutants, hence it has attracted wide
attention. The PSB wastewater treatment technology
a
https://orcid.org/0000-0002-9143-2153
can recycle and reuse biological resources while
reducing pollutants and protecting the environment,
so as to achieve the unity of environmental and
economic benefits. Wastewater contains a lot of
organic solids, the water body is dark brown, and it
can cause irreversible pollution to groundwater. PSB
have a variety of metabolic pathways and can
efficiently treat organic wastewater. Their unique
light-energy heterotrophic characteristics enable
them to survive in high-concentration organic
wastewater, and finally convert the organic matter in
the wastewater into nutrients needed for the growth
of aquatic organisms through metabolism to achieve
the purpose of purifying water quality. PSB have
strong vitality and are widely distributed, playing a
vital role in wastewater treatment, biological feed,
and biological hydrogen refining. Nevertheless, due
to the low biomass and high cost of cultivation during
the cultivation process, the application prospects of
PSB are restricted.
2 METHODS TO MEASURE THE
GROWTH OF PSB
Methods to determine the number of PSB contain
direct microscope counting, dilution plate counting,
dilution culture counting, and turbidimetry.
Shen, Q.
Study on the Growth Law of Photosynthetic Bacteria in Wastewater Treatment.
DOI: 10.5220/0011382500003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1201-1205
ISBN: 978-989-758-595-1
Copyright
c
2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
1201
2.1 Direct Microscope Counting
Method
Direct microscope counting method is a quick and
convenient method that place an appropriate amount
of microbial sample suspension to be tested on a
special counting plate with a fixed area and volume,
and then count the microorganisms directly under the
microscope. At present, there are mainly two types of
counting plates in the laboratory. Bacterial counters
can be utilized for general bacteria while
hemocytometers can be used for larger yeast or mold
spores. The principles and methods of use of these
two types of counting plates are the same, except that
the thinner bacteria counting plates are better for
observation with oil mirrors.
2.2 Dilute Plate Counting Method
The dilute plate counting method is a method of
diluting the tested sample solution by an appropriate
multiple to which the microorganisms are dispersed
into single cells, and then measuring the number of
microorganisms by the number of single colonies
formed on the solid medium under suitable growth
conditions. In actual operation, firstly, the sample
solution needs to be diluted gradually. Then, a certain
amount of sample solution is uniformly spread on the
solid medium with appropriate growth conditions and
cultivated upside down for a certain time. Finally, the
colonies on the plate are counted. The most
significant step of this method is dilution. Choosing
an appropriate dilution factor can decrease errors and
improve the accuracy of the determination.
2.3 Dilution Culture Counting Method
The dilution culture counting method, also known as
the maximum probability method, is based on
mathematical probability and statistics. In this
method, a series of dilutions of the bacterial culture
solution are carried out until the diluted solution is
inoculated on the culture medium and no or very little
bacterial growth occurs. Based on the lowest dilution
at which growth occurs and the highest dilution at
which no growth occurs, the method relies on the
“Most probable number (MPN)” theory to calculate
the approximate number of bacteria per unit volume
of the sample.
3 MATERIALS AND METHODS
3.1 Experimental Strains
PSB obtained by separation and culture in the
laboratory of our college.
3.2 Medium
3.2.1
Liquid Culture Medium
2.5 g of sodium acetate, 2.0 g of beef extract, 0.5 g of
MgSO
4
, 1.0 g of NH
4
Cl, 0.5 g of KH
2
PO
4
and 1000
mL of sterile water.
3.2.2 Solid Medium
1.5 g of beef extract, 5.0 g of peptone, 2.5 g of NaCl,
7.0 g of agar and 500 mL of sterile water.
3.3 Experimental Equipment
MGC-300 light incubator, 752 UV-Vis
spectrophotometer and YXQ-50S11 high-pressure
steam sterilization pot.
3.4 Determination of The Standard
Curve of PSB
3.4.1 Determination of Optical Density
(OD
660
)
The bacterial concentration of PSB was expressed by
optical density (OD
660
). After the bacteria solution
was appropriately diluted, the absorbance value was
measured at a wavelength of 660 nm, with a blank
liquid medium used as a control.
The optical density value of the bacterial cell
(OD660) = OD value × dilution multiple, and the
regression equation measured was as follows:
y=0.50601x (r=0.99907) (1)
In this equation, x represents the concentration of
PSB, y represents the OD
660
value, and r represents
the correlation coefficient.
3.4.2 Dilute Plate Counting Experiment
Nine sets of sterile petri dishes were taken, and 3 sets
of 10
-5
,10
-6
, and 10
-7
were marked with markers. The
PSB suspension was diluted to 10
-1
, 10
-2
, 10
-3
, 10
-4
,
10
-5
, 10
-6
and 10
-7
in a 10-fold concentration gradient.
Then, 3 1 mL sterile straws were utilized to draw 0.1
mL of 10
-5
, 10
-6
and 10
-7
diluted bacterial suspension
which were put them into numbered sterile petri
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
1202
dishes. The petri dish was placed in a constant
temperature medium at 32 °C for cultivation. After
the bacterial suspension was cultured for 48 h, the
petri dish was taken out. Subsequently, the colonies
formed in each petri dish were counted, and the
average number of colonies on the 3 plates of the
same dilution was calculated according to the
following formula:
Colony-forming unit per milliliter (CFU) =
average number of colonies × dilution factor × 10.
3.4.3 Orthogonal Experiment
PSB can directly treat high-concentration organic
wastewater without the problem of sludge treatment.
For the moment, there are many studies on the
optimization of the growth conditions of PSB. These
conditions play a crucial part in the growth and
reproduction of PSB and have very paramount
practical significance. The growth of PSB is affected
by many factors. The experiment is designed
according to the L
16
(45) orthogonal experiment table,
and the best medium for PSB is screened out.
Table 1: Orthogonal experiment factor level table of PSB culture medium.
Level A B C D E
Sodium acetate
(g/L)
NH
4
Cl
(g/L)
KH
2
PO
4
(g/L)
MgSO
4
(g/L)
Beef
extract
(g/L)
1 2.1 0.6 0.3 0.3 0.5
2 2.5 1.0 0.5 0.5 1.0
3 2.9 1.4 0.7 0.7 1.5
4 3.3 1.8 0.9 0.9 2.0
4 EXPERIMENTAL RESULTS
4.1 Dilute Plate Counting Results
(Table 2).
Table 2 shows the number of photosynthetic bacteria
and the results of plate colony counting.
Table 2: Dilute plate counting results.
Dilatability
10
-5
10
-6
10
-7
1 2 3 Average 1 2 3 Average 1 2 3 Average
CFU/Plate 2182 1916 1963 2020 26
7
281 226 258 31 42 33 35
CFU/mL 2.02×10
9
2.58×10
9
3.53×10
9
4.2 Experimental Results of OD
660
Value of PSB Suspension (Table 3)
To investigate the growth of photosynthetic bacteria,
the experimental results of bacterial concentration
OD
660
of photosynthetic bacteria are shown in Table
3.
Table 3: OD
660
value results of PSB suspension.
Sample Concentrati
on (g/L)
Mean
concentration
(g/L)
Average
(Abs)
Standard
deviation (SD)
Relative Standard
Deviation%(RSD)
Graduation
(Abs)
3 parallel samples 3.1
3.1
3.1
3.1
1.5636
0.01174
0.7511
1.550
1.566
1.573
4.3 Orthogonal Experiment Results
Table 4: Orthogonal experiment result table.
Experimental
g
rou
p
A B C D E OD
660
1 1 1 1 1 1 1.1532
Study on the Growth Law of Photosynthetic Bacteria in Wastewater Treatment
1203
2 1 2 2 2 2 1.4331
3 1 3 3 3 3 1.5001
4 1 4 4 4 4 1.4435
5 2 1 2 3 4 1.3505
6 2 2 1 4 3 1.6746
7 2 3 4 1 2 1.5173
8 2 4 3 2 1 1.4378
9 3 1 3 4 2 1.3607
10 3 2 4 3 1 1.5455
11 3 3 1 2 4 1.4484
12 3 4 2 1 3 1.4779
13 4 1 4 2 3 1.8409
14 4 2 3 1 4 1.3539
15 4 3 2 4 1 1.5259
16 4 4 1 3 2 1.7233
k
1
1.3825 1.4263 1.4999 1.3756 1.4156
k
2
1.4951 1.5018 1.4469 1.5401 1.5086
K
3
1.4581 1.4979 1.4131 1.5299 1.6234
K
4
1.6110 1.5206 1.5868 1.5012 1.3991
R 0.2285 0.0943 0.1737 0.1645 0.2243
According to the optimization experiment of the
proliferation medium formula and from the visual
analysis of Table 4, it could be concluded that the
order of each factor affecting the OD
660
value of PSB
growth was A>E>C>D>B. The factor A sodium
acetate had the most evident effect on the growth of
PSB while the factor B ammonium chloride had the
least effect on the growth of PSB. The best medium
formula combination for PSB was A
4
B
4
C
4
D
3
E
3
. The
optimal growth condition of the orthogonal
experiment was experimental group 13, namely
sodium acetate 3.3 g, NH
4
Cl l0.6 g, KH
2
PO
4
0.9 g,
MgSO
4
0.5 g, beef extract 1.5 g.
5 RESEARCH PROGRESS OF
PSB IN WASTEWATER
TREATMENT
PSB have different physiological and biochemical
functions like nitrogen fixation, carbon fixation,
dehydrogenation, and sulfide oxidation, playing a
crucial part in the natural carbon, nitrogen, and sulfur
cycles. As a result, it has significant scientific
research value. The wastewater treatment technology
of PSB is established on the basis of ecological
succession laws and principles. In the 1960s,
Kobayash et al. unveiled the role and principle of PSB
in the self-purification process of natural wastewater.
In the 1970s, the application of PSB in the
purification and treatment of organic wastewater and
the research as a source of feed protein were carried
out abroad extensively. At present, hydrogen has
become an efficient clean fuel. In the process of
various biological hydrogen production, the
application of PSB to produce hydrogen under light
is the current research hotspot. The PSB hydrogen
production process can be combined with the organic
wastewater treatment process. The utilization of small
molecular organic acids to produce clean energy
under light can degrade organic matter in wastewater
while producing hydrogen, hence, it is considered the
most environmentally friendly hydrogen production
method. For instance, Turkarslan et al.
employed
rhodobacter sphaeroides to produce hydrogen by
photosynthesis based on wastewater produced in
dairy plants. At the beginning of the experiment, the
bacteria could not grow, but after adding a proper
amount of malate to the wastewater, the bacteria
began to grow and produce hydrogen.
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