Screening and Characterization of Polyacrylamide Degrading Fungi

Yujie Cao

, Qingfang Zhang

, Minmin Zhao

, Chengxiang Bai

and Tianfeng Wang

1,2,*

School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China

College of Resources and Environment, Jiujiang University, Jiujiang, Jiangxi, China

Keywords: Polyacrylamide, Degrading Fungi, Growth Characteristics, Degradation Conditions.

Abstract: In the study, soil samples around the oil concentration station of Yanchang Oilfield were selected for the

experiment. With 10

-2

as the soil dilution, the strains were screened by the plate stress enrichment culture

method. Finally, three strains of high polyacrylamide degrading fungi were determined, which were

respectively recorded as HPAMF1, HPAMF2 and HPAMF9. The rDNA-ITS assay showed that strains

HPAMF1, HPAMF2 and HPAMF9 were Trichoderma alba, Fusarium and Merimbla, respectively. The

growth characteristics and degradation conditions of the polyacrylamide degrading fungi were investigated

and optimized. It was found that the degradation capacity of the three strains reached the strongest when the

polyacrylamide was used as the sole nitrogen source.

1 INTRODUCTION

Polyacrylamide has flocculation

(Haveroen, 2005);

(Owen, 2002), adsorption

(Szögi, 2007); (Sojka,

2006), thickening, resistance reduction and other

properties, which has been widely used in petroleum

exploitation, water treatment, papermaking, textile

printing and dyeing, agriculture, medicine, sugar,

building materials, aquaculture, construction and

other fields (Shu, 2021); (Panova, 2021); (Yang,

2020).

Polyacrylamide is non-toxic, but it will undergo

physical, chemical, and biological degradation in

natural environment, and monomer acrylamide will

be produced after degradation. Acrylamide may

cause neurotoxicity, reproductive toxicity,

genotoxicity and carcinogenicity

(Huang, 2018).

Therefore, the degradation of polyacrylamide

wastewater has become a focus of attention in recent

years.

Biodegradation technology is low cost, non-

polluting and mature, which could be used in the

degradation of polyacrylamide

(Wijngaarden, 2016).

In the exploration of polyacrylamide degrading

microorganism, the study of bacteria is much more

than that of fungi. Because the species of known

polyacrylamide degrading fungus are few and the

research is not in-depth, this will become a new

direction of microbial polyacrylamide degrading.

2 MATERIAL AND METHODS

2.1 Sampling

The soil samples were taken from the surrounding

soil samples of an oil selection station in Yanchang

Oilfield.

Polyacrylamide purchased from Xi'an Lanxiang

Chemical Co., LTD., which is anion polyacrylamide,

with molecular weight of 5×10

2.2 Medium

2.2.1 Martin Medium

·3H

O 1 g, MgSO

·7H

O 0.5 g, peptone 5 g,

glucose 10 g, constant volume to 1000 mL. Prepare

rose-bengal with a concentration of 1% (m/m), and

add 3.3 mL to the culture solution. Stir thoroughly

until the mixture is evenly mixed, then weigh 15~20

g agar in the culture medium and heat to melt at

natural pH.

2.2.2 Basal Culture Medium

Sucrose 30 g, KH

·3H

O 0.4 g, K

HPO

·3H

1.6 g, MgSO

·7H

O 0.06 g, NaCl 0.5 g, Fe

(SO

)

0.03 g, CaCl

0.01 g, CuSO

0.05 g, ZnSO

0.05 g,

with distilled water to 1000 mL at natural pH.

1296

Cao, Y., Zhang, Q., Zhao, M., Bai, C. and Wang, T.

Screening and Characterization of Polyacrylamide Degrading Fungi.

DOI: 10.5220/0011508800003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1296-1301

ISBN: 978-989-758-595-1

2.3 Screening of Degrading Fungi

2.3.1 Selection of Soil Dilution

Soil samples around the oil concentration station of

Yanchang Oilfield were taken and stored at 4 ℃. 2.0

g of soil sample was weighed and put into a conical

flask, which was placed in a constant temperature gas

bath oscillator at 34 ℃ and 140 rpm for 3 h. Then

dilute the soil suspension to 10

-1

, 10

-2

, 10

-3

, 10

-4

, and

-5

, and spread them evenly on Martin medium.

Make three parallel samples for each dilution. The

test was repeated three times for each strain. Observe

the growth of the colony in the culture medium and

select the appropriate soil dilution for subsequent

experiments.

2.3.2 Isolation of Polyacrylamide-degrading

Fungi

A certain amount of polyacrylamide was prepared

and added to the sterilized Martin culture medium by

means of plate stress enrichment method, where the

concentration of polyacrylamide was 100, 200, 500,

1000 and 1500 mg·L

-1

. A drop was added to Martin

medium containing polyacrylamide and coated

evenly. Three parallel samples were made for each

concentration and incubated at 34 ℃ for 72 h. fungi

with good growth and fast growth rate were selected

and further enriched and separated on Martin medium

containing polyacrylamide until pure colonies were

obtained.

2.3.3 Screening of Polyacrylamide

Degrading Fungi

The spores on each fungal body were washed with

phosphate buffer solution, and the spore suspension

was diluted to 10

CFU· mL

-1

, and stored in the

refrigerator at 4℃ for future use. The polyacrylamide

was added to the basal culture medium at a

concentration of 500 mg·L

-1

, and the spore

suspension were added to the medium at a volume

ratio of 2%. Then the suspensions were cultured in a

shaking table at 34 ℃ and 140 rpm for 10 days, and

the uninoculated solution was used as the control.

After the culture medium was diluted to an

appropriate concentration after being treated by each

fungus for 10 days, the absorbance was measured by

starch-cadmium iodide spectrophotometry, and the

change of polyacrylamide concentration was

obtained, and the degradation rate was calculated, so

as to screen out the polyacrylamide efficient

degradation fungi.

2.4 Identification of Efficient

Degradation Strains

1µL spore suspension was added to Martin medium,

and each fungus was repeated for three times. After

uniform coating, the bacteria were cultured in 34 ℃

incubator for 5 days, and the characteristics of each

colony were observed. The morphology of fungus

was observed by scanning electron microscope. The

strains were identified by rDNA-ITS assay.

2.5 Method for Determination of

Degradability of Each Fungus

Strains

The growth of polyacrylamide degraded fungus was

determined by dry cell weight. The polyacrylamide

concentration was determined by starch-cadmium

iodide method, and its removal rate was calculated.

2.6 Growth and Degradation

Characteristics of Single Each

Fungus

2.6.1 Growth with Polyacrylamide as the

Only Carbon Source

Three basal culture medium was prepared to remove

sucrose and added 500 mg·L

-1

Cl, and then

inoculated with the three dominant strains which can

degrade polyacrylamide, respectively. Then the three

dominant strains were cultured at 34 ℃ in constant

temperature incubator, and the growth of the

degrading fungus was observed to explore whether

the three dominant strains could grow with

polyacrylamide as the only carbon source.

2.6.2 Growth with Polyacrylamide as the

Only Carbon and Nitrogen Source

Prepare three 500 mg·L

-1

basal culture medium with

polyacrylamide as the only carbon and nitrogen

source, and the three dominant fungi which can

degrade polyacrylamide isolated to basal culture

medium respectively, and then arrange them in a 34

℃ constant temperature incubator for culture, and

observe the growth of the degrading fungi. To explore

whether polyacrylamide can be used as the only

carbon and nitrogen source for the growth of the three

dominant strains.

Screening and Characterization of Polyacrylamide Degrading Fungi

1297

2.6.3 Single Growth Curve and

Polyacrylamide Degradation Rate

Curve

Three 500 mg·L

-1

basal culture medium containing

polyacrylamide were prepared and 2% (v/v)

sporospora suspensions of the three strains were

added, respectively. Three parallel groups were set in

each group, and they were placed in a 34 ℃ 140 rpm

shaking table for 10 days. The growth amount of the

fungi and the degradation rate of polyacrylamide

were measured every day.

3 RESULTS

3.1

Screening of Polyacrylamide

Degrading Fungi

After incubating in a constant temperature incubator

for 72 h, the plate with soil dilution of 10

-2

had more

types of colonies and was suitable for separation.

Different fungus was selected from the plate and

inoculated in Martin medium containing different

concentrations of polyacrylamide for stress

enrichment culture. The polyacrylamide

concentration was 100 mg·L

-1

, 200 mg·L

-1

, 500

mg·L

-1

, 1000 mg·L

-1

, 1500 mg·L

-1

. The growth of the

strains was observed in the incubator at 34 ℃ for 72

h, and the growing strains were selected for isolation

and purification. Finally, 8 strains of different

tolerance fungi were obtained.

In the basal medium containing 500 mg·L

-1

polyacrylamide, the suspensions of each sporozoa

were added at 2% (v/v). After 10 days of shaking

culture at 34 ℃ and 140 rpm, the absorbance was

measured to determine the concentration of

polyacrylamide, so as to obtain the degradation rate

of polyacrylamide by each fungus.

Table 1: Degradation ability of eight tolerant fungi to polyacrylamide.

Strains 1# 2# 3# 5# 6# 9# 10# 11#

Degradatio

n efficiency

(

)

27.36 27.99 14.75 18.30 22.76 27.58 9.51 23.77

As can be seen from Table 1, the degradation rate

of polyacrylamide of each degradation fungus were

different after 10 days, and 1#, 2# and 9# with higher

degradation rate than other strains were selected as

the dominant degradation fungus, which were

denoted as HPAMF1, HPAMF2 and HPAMF9.

3.2

Morphological Observation and

Identification of Polyacrylamide

Degrading Fungi

3.2.1 Colony Characteristics of

Polyacrylamide Degrading Fungi

The colony morphology was observed, and the

colony morphology was shown in Fig. 1. Observation

showed that HPAMF1 mycelia grew rapidly on the

medium, and the surface color of the colony changed

from white to yellowish green and then to dark green.

The HPAMF2 colony was grapefruit red, with

abundant velour shaped aerial mycelia, and the

mycelia were white, while the mycelia of HPAMF9

colony was light yellow, and the surface was nearly

smooth.

Figure 1: Cultural characteristic of HPAMF1, HPAMF2

and HPAMF9.

3.2.2 Characteristics of

Polyacrylamide-degraded Fungi

Three strains of fungi were placed under scanning

electron microscopy for observation, and the

scanning electron microscopy results of each strain

were shown in Fig. 2.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1298

Figure 2 SEM image of HPAMF1, HPAMF2 and

HPAMF9(×5000).

Under the scanning electron microscope, the

strain HPAMF1 was oblate with depressions; the

strain HPAMF2 was rod-shaped; and the strain

HPAMF9 was ellipsoidal with fine wrinkles on the

surface.

3.2.3 rDNA-ITS Determination

Through BLAST comparison, it is found that the

similarity between HPAMF1 and Trichoderma aspen

had reached 100%, the similarity between HPAMF2

and Fusarium sequence had reached 99.82%, and the

similarity between HPAMF3 and Merimbla sequence

had reached 99.48%. Therefore, HPAMF1 was

preliminarily identified as Trichoderma aspen,

HPAMF2 was preliminarily identified as Fusarium,

and HPAMF9 was preliminarily identified as

Merimbla through the determination of the colony

morphology, cell morphology and rDNA-ITS of

polyacrylamide-degrading fungi.

3.3

Growth and Degradation

Characteristics of Single Fungus

3.3.1 Growth of Degrading Fungi using

Polyacrylamide as the Sole Carbon

Source

HPAMF1, HPAMF2 and HPAMF9 were cultured in

a constant temperature incubator at 34 °C for 5 days.

It could be seen from Fig. 3 that HPAMF1 and

HPAMF9 have hyphae growth but HPAMF2 didn’t

grow. From this we could conclude that HPAMF1

and HPAMF9 grow with polyacrylamide as the sole

carbon source, but HPAMF2 didn’t.

Figure 3: HPAMF1, HPAMF2 and HPAMF9 grew with

polyacrylamide as the only carbon source.

Prepare basal culture medium with different

initial concentrations of polyacrylamide, in which

sucrose is removed and NH

Cl was added, and then

connected to the HPAMF1 and HPAMF9 spore

fungus suspension respectively, and placed in a

shaker at 34 ℃ 140 rpm for 10 days. After the

measurement, the degradation rate of polyacrylamide

is shown in Fig. 4.

Figure 4: Degradation curve of HPAMF1 and HPAMF9.

It can be seen from Fig. 4 that within the range of

the initial polyacrylamide concentration of 100~500

mg·L

-1

, the degradation rate of HPAMF1 and

HPAMF9 on polyacrylamide will increase rapidly as

the concentration increases, and both are at a

concentration of the maximum value is reached at

500 mg·L

-1

: 19.16%, 20.91%. When it exceeds 500

mg·L

-1

, with the continuous increase of the

concentration, the degradation rate presents an

obvious decreasing trend.

3.3.2 Growth of Degrading Fungi using

Polyacrylamide as the Only Carbon

and Nitrogen Source

Strains HPAMF1, HPAMF2 and HPAMF9 were

placed in a 34 ℃ constant temperature incubator for

5 days, and the experimental results were shown in

Fig. 5. It was observed that HPAMF1 and HPAMF9

could grow with polyacrylamide as the only carbon

and nitrogen source, while HPAMF2 could not.

0 500 1000 1500 2000 2500

HPAM

remova

ffi

ency

(%)

Initial concentration of HPAM (mg·L

-1

)

HPAMF1

HPAMF9

Screening and Characterization of Polyacrylamide Degrading Fungi

1299

Figure 5: HPAMF1, HPAMF2 and HPAMF9 grow with

polyacrylamide as the only carbon and nitrogen source.

In the basic culture medium with different initial

concentrations of polyacrylamide and sucrose

removed, two spore suspensions, HPAMF1 and

HPAMF9, were added, respectively, and placed in a

shaking table at 34

℃ and 140 rpm for culture for 10

days. During the experiment, the degradation rate of

polyacrylamide changed as shown in Fig. 6.

Figure 6: Degradation curve of HPAMF1 and HPAMF9.

As can be seen from Fig. 6, when the initial

concentration of polyacrylamide is in the range of

100~500 mg·L

-1

, the degradation rate of HPAMF1

and HPAMF9 on polyacrylamide will increase with

the increase of the concentration, and the degradation

rate of HPAMF1 will increase rapidly in the range of

100 mg·L

-1

to 200 mg·L

-1

. The degradation rate of

HPAMF9 increased rapidly from 200 mg·L

-1

to 500

mg·L

-1

, and both strains reached the maximum

degradation rate of 8.36% and 13.21% at 500mg·L

-1

When the initial concentration of polyacrylamide is

greater than 500 mg·L

-1

, the degradation rate

decreases with the increasing of the concentration.

3.3.3 Based on the above Experimental

Results

The degradation rates of HPAMF1 and HPAMF9

strains were 27.36% and 27.58%, respectively, when

the initial concentration of polyacrylamide was 500

mg·L

-1

as the only nitrogen source. When

polyacrylamide was used as the only carbon source,

the corresponding degradation rates were 19.16% and

20.91%, respectively. When polyacrylamide was

used as the only carbon and nitrogen source, the

corresponding degradation rates were 8.36% and

13.21%, respectively.

It was found that polyacrylamide was the only

nitrogen source for HPAMF2 growth. For HPAMF1

and HPAMF9 strains, polyacrylamide can be used as

the only nitrogen source, the only carbon source and

the only carbon and nitrogen source. According to the

data analysis, the degradation capacity of these three

strains can reach the strongest when they grow with

polyacrylamide as the only nitrogen source.

Therefore, in the subsequent experiments,

polyacrylamide was set as the only nitrogen source to

study the growth and degradation characteristics of

HPAMF1, HPAMF2 and HPAMF9.

3.3.4 Growth Curve and Degradation Rate

Curve of Single Fungus

Three strains of HPAMF1, HPAMF2 and HPAMF9

were cultured. The changes of thallus growth and

polyacrylamide degradation rate are shown in Fig. 7

and Fig. 8.

0246810

100

120

140

160

Biomass (mg)

Time (d)

HPAMF1

HPAMF2

HPAMF9

Figure 7: Growth curve of HPAMF1, HPAMF2 and

HPAMF9.

0246810

HPAM removal efficiency (%)

Time (d)

HPAMF1

HPAMF2

HPAMF9

Figure 8: Degradation rate curves of HPAMF1, HPAMF2

and HPAMF9.

0 500 1000 1500 2000 2500

HPAM removal efficiency (%)

Initial concentration of HPAM (mg·L

-1

)

HPAMF1

HPAMF9

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1300

As shown in the Fig. 8, the growth curve of single

fungus HPAMF1, HPAMF2 and HPAMF9 was s-

type, in line with the microbial growth law. The strain

first passed a relatively short retardation period and

continuously adjusted itself to adapt to the new

environment. Then it entered the logarithmic growth

period, the fungus weight will show exponential

growth, at this time the degradation rate of

polyacrylamide will also increase; Then the strain

continued to grow into a stable phase, at which the

number of new cells and the number of dead cells

were in a dynamic balance, and the polyacrylamide

degradation rate tended to be stable. The maximum

removal rates of HPAMF1, HPAMF2 and HPAMF9

were 27.36%, 27.99% and 27.58%, respectively.

4 CONCLUSION

8 strains of polyacrylamide degrading fungi were

isolated from soil in this experiment. Moreover, the

dominant degradation fungi were selected through

the degradation effect of each strain on polyamide,

which were respectively recorded as HPAMF1,

HPAMF2 and HPAMF9. By observing the colony

morphology and cell characteristics of the three

fungus and identifying the three fungi, it was finally

concluded that strains HPAMF1, HPAMF2 and

HPAMF9 were Trichoderma alba, Fusarium and

Merimbla, respectively. In addition, in the growth

environment with polyacrylamide as the only

nitrogen source, the degradation ability of the three

fungi growth showed the strongest.

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