Study on the Growth Law of Photosynthetic Bacteria in Wastewater

Treatment

Qiuhua Shen

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 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

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

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

, 1.0 g of NH

Cl, 0.5 g of KH

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

-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

(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)

MgSO

(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

-5

-6

-7

1 2 3 Average 1 2 3 Average 1 2 3 Average

CFU/Plate 2182 1916 1963 2020 26

281 226 258 31 42 33 35

CFU/mL 2.02×10

2.58×10

3.53×10

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

660

of photosynthetic bacteria are shown in Table

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

1.5636

0.01174

0.7511

1.550

1.566

1.573

4.3 Orthogonal Experiment Results

Table 4: Orthogonal experiment result table.

Experimental

rou

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

1.3825 1.4263 1.4999 1.3756 1.4156

1.4951 1.5018 1.4469 1.5401 1.5086

1.4581 1.4979 1.4131 1.5299 1.6234

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

. The

optimal growth condition of the orthogonal

experiment was experimental group 13, namely

sodium acetate 3.3 g, NH

Cl l0.6 g, KH

0.9 g,

MgSO

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.

REFERENCES

Chaudhuri B K, Wiesmann U. Enhanced anaerohw

degradation of benzene by enrichment of mixed

microbial culture and optimization of the culture

medium. Appl Mierebiol Bioteehnoi, 2005, 43: 178-

187.

Chen Y F. Comparison of two Methods in the Inspection of

Bacteria [J]. Chinese Agricultural Science Bulletin,

2010, 26(17): 129-13.

Hülsen T, Barry E M, Lu Yang, et al. Domestic wastewater

treatment with purple phototrophic bacteria using a

novel continuous photo anaerobicmembranebioreactor

[J]. WaterResearch, 2016, 100: 486-495.

Kobayashi M. Microbial Energy Conversion[M]. New

York: Pergamon Press, 1977.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1204

Koku H, Eroglu I, Gunduz U, et al.Aspects of the

metabolism of hydrogen production by Rhodobacter

sphaeroides [J].Int. J.Hydrogen Energy,2002,27(11-

12):1315-1329.

Lu H F, Zhang G M, Wan T, et al. Influences of light and

oxygen conditions on photosynthetic bacteria

macromolecule degradation: Different metabolic

pathways [J]. Bioresource Technology, 2011, 102 (20):

9503-9508.

Madukasi E I, Dai X, He C, et al. Potentials of phototrophic

bacteria in treating pharmaceutical wastewater[J].

International Journal of Environmental Science &

Technology, 2010, 7(1): 165-174.

Madukasi E I, Chunhua H, Zhang G. Isolation and

application of a wild strain photosynthetic bacterium to

environmental waste management[J]. International

Journal of Environmental Science & Technology, 2011,

8(3): 513-522.

Prachanurak P, Chiemchaisri C, Chiemchaisri W, et al.

Biomass production from fermented starch wastewater

in photo-bioreactor with internal overflow

recirculation[J]. Bioresource Technolog, 2014, 165:

129-136.

Takeno K, Yamaoka Y, Sasaki K. Treatment of oil-

containing sewage wastewater using immobilized

photosynthetic bacteria [J]. World Journal of

Microbiology and Biotechnology, 2005, 21 (8/9): 1385-

1391.

Turner J A. Sustainable Hydrogen Production[J], Science,

2004, 305(5686):972-974.

TurkarsIan S, Yigit D O, Aslan K, et al.Photobio logical

hydrogen production by Rhodobacter sphaeroides

O.U.001 by utilization of waste water from milk

industry[C], 1998: 151-156.

Wang X S, Zhang T L, Hong Z L. Comparison of plate

counting method with paper slice method for detecting

total bacterial count in food [J]. Journal of Food Safety

& Quality, 2020, 11(16): 5489-5493

Yokoi H, Saiteu A, Uchid A H, et a1. Microbial hydrogen

pmduction from sweet potato starch residue. J Biosci

Bioeng, 2001, 91(1): 58-63.

Zhu Y L.Experimental Method of Bacteria Growth Curve

Determination[J].Journal of Microbiology, 2016,

36(5): 108-112.

Zhuo J S, LU Y, Yang D, et al. Clinical Value of Microbic

Growth Curve in Bacteria Cultivatingr[J].Shanxi

Journal of medical laboratory sciences,2000, 15(1) :14-

15.

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