Research on the Impact of Alkylbenzene Sulfonate Surfactants in Use

in Oilfields on Native Biodiversity of Mining Areas and Response to

Environmental Remediation of Native Biodiversity

Zhanyou He

1,2,a,*

, Ziqian Xu

1,2,b

, Min Zhao

1,2,c

, Yan Li

1,2,d

and Ning Liu

1,2,e

Research Institute of Oil and Gas Technology of Changqing Oilfield Company, China

National Engineering Laboratory for Exploration and Development of Low-permeability Oil and Gas Field, China

liyan001_cq@petrochina.com.cn,

liun15_cq@petrochina.com.cn

Keywords:

Alkylbenzene Sulfonate, Surfactants, Soil Bacteria, High-Throughput Sequencing.

Abstract:

In order to clarify the effect of alkylbenzene sulfonate surfactants used in oil field on native biodiversity in

mining area. In this paper, high-throughput sequencing and biodiversity analysis were performed on the soil

continuously polluted by alkylbenzene sulfonate surfactants. Clostridia, BPC102 and Bacteroidia became the

dominant bacteria in the soil environment, with strong self-repair response of environmental organisms.

Bacteria S035, Bacilli and Dothideomycetes showed negative response, indicating that alkylbenzo sulfonate

surfactants inhibited and affected the growth, development and reproduction of these native organisms. The

results showed that alkylbenzene sulfonate surfactant had obvious effects on soil biodiversity in mining area,

which provided scientific basis for environmental impact assessment and environmental management of

surfactants.

1 INTRODUCTION

Alkylbenzene sulfonate surfactants are widely used

in the field of chemical flooding in low-permeability

oilfields for enhanced oil recovery due to their

excellent oil displacement performance. As an

important oil field EOR chemical agent, the

application range of surfactants is still expanding and

the consumption is also increasing day by day. In the

process of use, a large amount of waste water and

waste residues containing surfactants are inevitably

discharged and infiltrated into the soil. Large-scale

industrial use of alkylbenzene sulfonate surfactants

urgently needs to clarify its impact on the native

biodiversity of oilfields and mining areas and the

response of native biodiversity to its environmental

bioremediation.

The environmental behavior of alkylbenzene

sulfonate surfactants in soil mainly includes

migration, adsorption and degradation. As an

important place for energy exchange of various

substances recycling machines, soil is usually the

destination of migration, retention and deposition of

pollutants in the environment. After surfactants enter

the mining environment, they will first have a certain

impact on the biodiversity of the mining area. The

impact degree is positively correlated with the impact

of local biodiversity, and negatively correlated with

the bioremediation response of local biodiversity. If

the native biodiversity has a strong response to the

environmental bioremediation of alkylbenzene

sulfonate surfactants, it indicates that the oil field has

a high tolerance of alkylbenzene sulfonate surfactants

and a large marginal safety concentration, that is, the

environmental biological reference value is large, and

the environmental biological toxicity is small. On the

other hand, if the native biodiversity responds weakly

to the environmental bioremediation of alkylbenzene

sulfonate surfactants. The microbial flora that can

degrade the surfactant cannot be enriched in a short

period of time, the tolerance of alkylbenzene

sulfonate surfactants in the oilfield will be low and

the safety marginal concentration will be small. In

other words, alkylbenzene sulfonate surfactants have

low environmental biological reference value and

high environmental biological toxicity in oilfield

mining area. Therefore, it is of great significance to

study the effects of alkylbenzene sulfonate

surfactants on native biodiversity and the response of

native biodiversity to environmental bioremediation.

He, Z., Xu, Z., Zhao, M., Li, Y. and Liu, N.

Research on the Impact of Alkylbenzene Sulfonate Surfactants in Use in Oilﬁelds on Native Biodiversity of Mining Areas and Response to Environmental Remediation of Native Biodiversity.

DOI: 10.5220/0011211200003443

In Proceedings of the 4th Inter national Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 379-384

ISBN: 978-989-758-595-1

379

In this paper, different types of bioreactors with

alkylbenzene sulfonate surfactants as the only

pollution source have been designed and

continuously operated. The second-generation

Qualcomm sequencing technology (Illumina MiSeq)

was used to conduct high-throughput sequencing of

16S rDNA V3~V4 regions and ITS1 regions on the

soil which was continuously polluted by

alkylbenzene sulfonate surfactants. The sequencing

results were evaluated by OTU cluster analysis,

Alpha diversity, species composition and abundance

analysis methods, which provided a theoretical basis

for the ecological and environmental protection in the

oilfields and mining areas.

2 METERIALS AND METHODS

2.1 Experimental Materials

Experimental target material: alkylbenzene sulfonate

surfactant used in an oilfield

Experimental soil: Fresh soil randomly collected

from a domestic oilfield chemical flooding enhanced

oil recovery test mining area, remove surface rocks

and other impurities, mix well and pass through a

2mm sieve.

Experimental equipment: In order to obtain the

influence of alkylbenzene sulfonate surfactants on

local biodiversity after entering the soil environment

and the response of local biodiversity to

bioremediation of characteristic pollution sources, a

bioreactor was designed as shown in Figure.1. The

prepared surfactant solution is continuously

introduced into the reactor soil from the top of the

bioreactor. The bottom is provided with an outlet

from which the solution can seep out. The

surrounding sampling holes are used to collect soil

samples at different contamination times.

Figure 1: Schematic diagram of bioreactor.

2.2 Experimental Methods

A peristaltic pump was used to add the prepared

surfactant solution at a certain flow rate (calculated

based on the actual leakage) from the top of the

reactor, and keep the experimental temperature

relatively constant. Samples were taken on day 7

(represented by D), day 30 (represented by E) and day

60 (represented by F), and set a group of blank

samples for control (represented by V) at the same

time. The obtained soil samples were stored in

sterilized sealed bags at −80 °C and microbial

sequencing was performed as soon as possible. The

whole experiment was carried out under dark

conditions.

3 RESULTS AND ANALYSIS

3.1 OTU Cluster Analysis

In order to facilitate analysis in the study, a single

marker is artificially set for a Taxonomic unit, namely

OTU (Operational Taxonomic Units). In order to

understand the number of species and genera in the

sequencing results of a sample, it is necessary to

classify the sequence. Through the classification

operation, sequences are divided into many groups

according to their similarity, and one group is an

OTU.

Figure.2 is a Venn diagram of the number of

bacterial OTU in soil sample. As shown in the figure,

a total of 5276 OTU were obtained in group D, 4951

OTU in group E, 5078 OTU in group F, and 4600

OTU in group V of control group. The sequence of

OTU numbers in the four soil samples is D > F > E >

V. The richness of bacterial groups was the highest in

the day7, and the lowest in the blank group. The

number of OTUs in the four groups was compared in

pairs: there were 2694 OTUs shared by group V and

Group D, 2413 OTUs shared by group V and Group

E, 2204 OTUs shared by group V and group F, 2815

OTUs shared by group D and group E and 2573

OTUs shared by group E and group F. It can be seen

that the bacterial groups in the soil samples of day7

and day30 had the highest consistency and the

smallest difference, while day60 had the lowest

consistency and the largest difference. This indicated

that as the pollution time of the alkylbenzene

sulfonate on the soil is prolonged, the difference of

the bacterial groups in the soil is greater.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

380

Figure 2: The Venn diagram of the number of bacterial

OTUs in soil samples.

Figure 3: The Venn diagram of the number of fungal OTUs

in soil samples.

As shown in the figure, there were 1114 OTUs in

the control group, 1207 OTUs in the D group, 1188

OTUs in the E group and 805 OTUs in the F group.

The number of OTU in the four groups of soil

samples was D > E >V > F. Fungal species richness

was highest in soil samples after 7 days of

contamination, and lowest in soil samples after 60

days of contamination. The pairwise comparison

results show that the number of OTUs shared by

group V and D is 525, the number of OTUs shared by

group D and E is 579, the number of OTUs shared by

group E and F is 418, and the number of OTUs shared

by group V and F is 418. The total number of OTUs

is 470, the number of OTUs shared by groups V and

F is 330, and the number of OTUs shared by the 4

groups of soil samples is 256. After comparison, it

was found that the consistency of fine fungal groups

was the highest and the difference was small between

the soil samples at day7 and day30. While the

consistency was the lowest and the difference was the

largest between the soil samples at day60 and the

control group. It can be seen that with the extension

of time, the difference of fungal groups in soil

contaminated by alkylbenzene sulfonate surfactant

gradually increased.

3.2 Alpha Diversity Index Analysis

MiSeq platform was used to perform high-throughput

sequencing on four groups of soil samples by

sequencing while synthesizing. The sequencing

results are shown in Table1. Shannon index and

Simpson index are usually used to estimate the

diversity of OTU species in microbial communities.

They are also commonly used to estimate the

diversity of microorganisms in sample. The larger the

Shannon index value, the higher the community

diversity. The smaller the Simpson index value, the

higher the community diversity. It can be seen from

Table1 that the order of the species diversity of

bacteria and fungi in the four groups of soil samples

is V> D> E> F. This shows that as time goes by, the

microbial diversity of the soil contaminated by

surfactants has declined, and the surfactants have an

inhibitory effect on the growth of microorganisms.

The coverage of each sample was more than 99.90%,

indicating that the sequencing effect was ideal, and

the diversity analysis results fully reflected the

information of micro

bial species in the area.

Table 1: Diversity analysis of bacterial microbial index of soil samples.

Group

Bacteria Fungi

Shannon Simpson

Coverage,

Shannon Simpson

Coverage

7.12±

0.115

0.0018±

0.00035

99.91 4.82 ±0.285 0.0236 ±0.00890 99.95

6.90±

0.393

0.0042±

0.00483

99.93 4.52 ±0.586 0.0410 ±0.04184 99.94

6.71±

0.904

0.0093±

0.01571

99.92 3.68 ±1.320 0.1398 ±0.17308 99.96

7.18±

0.085

0.0014±

0.00018

99.91 4.96 ±0.193 0.0180 ±0.00412 99.94

Research on the Impact of Alkylbenzene Sulfonate Surfactants in Use in Oilﬁelds on Native Biodiversity of Mining Areas and Response to

Environmental Remediation of Native Biodiversity

381

Random sampling of sequencing sequences is

used to construct a curve based on the number of

extracted sequences and the number of OTU

represented by them, that is the dilution curve.

Generally, when the curve tends to be flat, it indicates

that the number of samples is reasonable.

Figure.4(a)and Figure.4(b) are the dilution curves of

soil samples. It can be seen that the dilution curves of

bacteria and fungi of the samples are basically flat,

indicating that the sequencing and sampling are

reasonable and can truly reflect the microorganisms

in the soil samples. Combined with the coverage of

each sample, it shows that most of the microbial

groups are included in the sequencing results, which

can truly reflect the composition of the microbial

community in the soil in this area.

(a) Bacteria

(b) Fungi

Figure 4: Dilution curve of the sample.

3.3 Analysis of Community Structure

As shown in Figure.5, more than 70 types of bacteria

were detected in the four groups of soil samples.

Among them, β-Proteobacteria, α-Proteobacteria,

Acidobacteria, Δ-Proteobacteria and γ-Proteobacteria

are the absolute dominant species. The proportions of

the five dominant bacteria in the blank group were

9.04%, 8.55%, 9.62%, 7.16%, and 4.96%,

respectively. By day 7, the proportions of these five

bacteria were 11.04%, 8.35%, 9.97%, 7.01% and

6.41%. By day 30, the proportions of the five

dominant bacteria were 10.95%, 10.14%, 7.90%,

8.93%, and 7.13%. By day 60, the proportions of the

five bacteria were 12.46%, 10.50%, 8.24%, 9.24%

and 6.92%, respectively. In general, the relative

content of the five dominant bacteria did not change

much with the prolongation of pollution time, and

their growth and reproduction in the soil were

relatively stable, and they were not greatly affected

by alkylbenzene sulfonate surfactants.

Figure.6 is a Heatmap based on the genus level.

The Heatmap can reflect the similarity and difference

in species composition of all samples at a specific

taxonomic level. The more similar species or samples

are, the closer they are to each other in the cluster tree,

which can also indicate that certain bacterial groups

may have specific distributions.

The relative abundance of Clostridia in soil

samples at day30 and day60 was higher than day7 and

black group. Similarly, the relative abundances of

bacteria BPC102 and Bacteroidia were higher in the

soil samples on day60 than in other soil samples,

indicating that under the continuous pollution of

alkylbenzene sulfonate, Clostridia, BPC102 and

Bacteroidia gradually became the dominant bacteria

in the soil. However, the relative abundance of

bacteria S035 and Bacilli decreases with the

extension of the experiment time, indicating that the

growth and propagation of bacteria S035 and Bacilli

are inhibited by alkylbenzene sulfonate.

Figure 5: Histogram of bacterial community composition at

class classification level.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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Figure 6: Heatmap at class classification level.

Figure 7 is a histogram of fungal community

composition based on class classification level. More

than 20 types of fungi were detected in the four

groups. Among them, the relative proportions of

Sordariomycetes, Mortierellomycetes and

Agaricomycetes were 21.49%~28.03%,

10.81%~27.95%, 13.29~19.62%, which were

relatively high in soil samples, and were the absolute

dominant fungal species. The relative content of

Dothideomycetes in the blank group was 10.15%,

which decreased to 2.02% on day 60, indicating that

alkylbenzene sulfonate would inhibit the growth and

reproduction of Dothideomycetes.

Figure 7: Histogram of bacterial community composition at

class classification level.

Figure 8: Heatmap at class classification level.

4 CONCLUSION

In this paper, the oil field soil continuously polluted

by alkylbenzene sulfonate surfactants was taken as

the research object. The results of high-throughput

sequencing and biodiversity analysis were as follows:

(1) As the soil is continuously polluted by

alkylbenzene sulfonate surfactants for a longer time,

the abundance and diversity of bacteria and fungi in

the soil are decreasing, and the group differences of

microorganisms are gradually increasing. It shows

that alkylbenzene sulfonate surfactants have a

significant impact on the native biodiversity in the

soil environment of mining areas.

(2) Microbial sequencing showed that

alkylbenzene sulfonate contaminated soil had

dominant bacteria, They were Betaproteobacteria,

Alphaproteobacteria, Alphaproteobacteria,

Acidobacteria, Deltaproteobacteria,

Gammaproteobacteria, Clostridia, BPC102 and

Bacteroidia.

(3) Clostridia, BPC102 and Bacteroidia had

positive response to alkylbenzene sulfonates, and

gradually became the dominant bacteria in the soil

environment of the oil field. It showed that the native

biodiversity of the oilfield mining area had a strong

environmental biological self-repair response to

alkylbenzene sulfonate surfactants.

(4) The relative content of bacteria S035, Bacilli,

and fungus Dothideomycetes (Polycystomycetes)

decreased with the extension of the contamination

time, that is, it showed a negative response to alkyl

Research on the Impact of Alkylbenzene Sulfonate Surfactants in Use in Oilﬁelds on Native Biodiversity of Mining Areas and Response to

Environmental Remediation of Native Biodiversity

383

benzene sulfonates. The results showed that

alkylbenzenesulfonate surfactants inhibited and

affected the growth, development and reproduction of

these native organisms.

It can be seen that alkylbenzene sulfonate

surfactant have an obvious impact on the diversity of

soil biodiversity in mining areas. Therefore, their use

and discharge must consider environmental capacity,

fundamentally reduce their direct discharge to the

environment, and increase the treatment of

wastewater and waste residue containing surfactants.

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