Heavy Metal Content and Bacterial Diversity in Heavily Polluted Baotou Region of Yellow River

Materials Express

Heavy metal content and bacterial diversity in heavily polluted Baotou region of yellow river

Yuehong Wu1,†, Tong Wen1,†, Jing Yin2, Jianqing Liu1, Yanling Xing1, Zhongwei Yang2, Meirong Wang3,LingLu1, and Yong Ma1,∗ 1Department of Biological Science and Technology, Baotou Teachers’ College, Baotou 014030, Inner Mongolia, P. R. China 2Department of Environment and Health, Tianjin Institute of Environmental & Operational Medicine, Tianjin 300050, P. R. China 3Department of Chemistry, Baotou Teachers’ College, Baotou 014030, Inner Mongolia, P. R. China

ABSTRACT Article The Yellow River is the main water source for both local industrial and agricultural use, and the main source of local drinking water as well.IP: 192.168.39.211 In this study, eight On: Tue, water 28 samples Sep 2021 of 17:35:39 Baotou region were analyzed, in which the heavy metal (Cd, Zn, Cu, Pb,Copyright: Cr, Hg and American As) content Scientific were detectedPublishers using plasma-mass spectrometry and Delivered by Ingenta reduction gasiﬁcation-atomic ﬂuorescence spectrometry. Genus of water bacteria in eight water samples were investigated by means of high-throughput sequencing. Results showed that all the contents of heavy metals exceed GB (GuoBiao) standard of China, which implied the water of the Yellow River in Baotou region are at risk of being polluted. Furthermore, a large number of conditioned pathogens (Flavobacterium, Pseudomonas, Acinetobacter and Chryseobacterium) were found, some bacteria (such as Methanobacteria) can break down harmful substance and organic matter. In conclusion, according to this study, we try to put forward an important reference for the pollution control of Yellow River in Baotou region. Keywords: Yellow River, Pollution, Heavy Metal, High-Throughput Sequencing, Bacterial Diversity.

1. INTRODUCTION local agriculture and manufacturing industries. In recent The Yellow River plays an important strategic position in years, the economic overgrowth in China has led to seri- the social and national economic development of China. ous polluted in many rivers, including the Yellow River. Over the past years, with the rapid development of indus- On June 26, 2004, a large-scale water pollution incident try and agriculture on both sides of the Yellow River, the occurred in the Baotou region of the Yellow River, result- ecosystem was threatened by loading excessive pollutants. ing in the death of about 70 to 80% of wild fish in the The deterioration of the water environment in the Yellow water, with direct economic losses exceeding 200 million River makes the purification of the Yellow River an immi- yuan. What is worse, more than 1.3 million urban residents nent action [1]. were deprived regular water supply [2]. Baotou is the main industrial city in northwest China Heavy metal pollutants in rivers endanger the environ- and located in the Western part of Inner Mongolia, were ment and health. Thus, the investigation of the concentra- the Yellow River flows through. It not only provides drink- tion of heavy metals [3] is an important method to assess ing and domestic water for residents on both sides of the the pollution level of rivers. Many related researches have Yellow River, but also is the main source of water for been done on the accumulation of heavy metals in the sediments of rivers, lakes, marine, and even in coastal ani- ∗Author to whom correspondence should be addressed. mals and plants. Many reports [4–13] assessed the poten- †These two authors contributed equally to this work. tial ecological risks through the pollution trends of certain

Mater. Express, Vol. 10, No. 10, 2020 1661 Materials Express Heavy metal content and bacterial diversity in heavily polluted Baotou region Wu et al.

heavy metals. In addition, the concentrations of heavy met- Table I. Description of sampling sites. als such as copper, lead, zinc and cadmium at limited Sampling location Longitude Latitude Altitude sampling points in different periods of the Yellow River in Baotou section have also been reported [14]. How- 1#. Zhaojundao 4032.00 N 10939.46 E1040M ever, some reports have focused on the determination of 2#. Kunhe 40 30.15 N 109 47.55 E1010M 3#. Madi 4030.80 N 10949.55 E1010M heavy metals in the sediments of Yellow River [15–17]. 4#. Xiaobaihe 4030.75 N 10950.60 E1006M Thus, more sampling points and more heavy metal indica- 5#. Longcheng 4030.95 N 10952.50 E1010M tors should be determined, especially the determination of 6#. Daqiaoxi 4031.94 N 10955.00 E1010M 7#. Daqiaodong 4031.95 N 10955.80 E1003M heavy metals in the Yellow River water. Bacteria play an important role in organic decom- 8#. Wangdahan 40 31.33 N 109 58.50 E 999.5 M position and biogeochemical cycles. Modern molecular biology provides the key technologies to determine the upstream (Zhaojundao) to downstream (Wangdahan) were bacterial genus in environmental sample. To ﬁnd out the named as points 1∼8. bacterial composition and their diversity for drinking water Samples were taken on November 3, 2018. Water sam- safety in the region, the analysis of 16S rDNA directly ples were randomly collected with a sterile sampler each from environmental samples can be very effective [18–21]. In recent years, some molecular sequencing technologies, especially high-throughput sequencing, have been (A) (B) widely used to study bacterial populations in different samples [22–29]. Therefore, the assessment of water pollution and the investigation of microbial ecology in the Yellow River in Baotou region are of great signiﬁcance. Although some studies have been done before but the heavy metals and bacterial diversity in the Yellow River are dynamic indi- cators that will change with theIP: surrounding 192.168.39.211 environment. On: Tue, 28 Sep 2021 17:35:39 Therefore, latest researches about theCopyright: heavy metal American content Scientific Publishers (C) (D) and the diversity and abundance of aquatic bacteriaDelivered within by Ingenta 30 kilometers upstream and downstream of Baotou to eval- uate its pollution level are necessary. This study provides Article important systematic analysis data of bacterial diversity in this area and provides latest references for the local pollution control of the Yellow River.

2. MATERIALS AND METHODS (E) (F) 2.1. Sample Collection and Sample Pretreatment Totally eight water samples were collected from upstream to downstream in the Baotou region of the Yellow River. As shown in Figure 1 and Table I, sample points from

(G)

Fig. 2. Comparison of heavy metal contents in 8 water samples. Fig. 1. The locations of 8 sampling sites in Baotou section of the (A) Copper content; (B) zinc content; (C) lead content; (D) chromium Yellow river. content; (E) cadmium content; (F) arsenic content; (G) mercury content.

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Table II. Diversity and bacterial richness in the samples.

Sampling location Seq. num. OTU num. Shannon index Simpson ACE index Chao1 index Coverage

1#: Zhaojundao 41,721 1947 5.177 0.023 2078.41 2052.20 0.993 2#: Kunhe 16,499 670 2.978 0.242 926.66 875.07 0.985 3#: Madi 13,387 785 3.443 0.156 1264.98 1126.36 0.974 4#: Xiaobaihe 16,126 519 2.959 0.230 703.13 654.80 0.989 5#: Longcheng 12,615 650 3.861 0.077 864.83 802.60 0.983 6#: Daqiaoxi 10,574 728 3.981 0.070 1529.46 1085.40 0.967 7#: Daqiaodong 9,922 664 3.915 0.062 953.83 855.26 0.974 8#: Wangdahan 11,379 540 3.616 0.082 646.05 598.35 0.988

location. Two liters of water filled into sterile sampling Based on the analysis of the OTU clustering, the bottles were collected at each site, and then stored in a Chao1 and Shannon index of samples were calculated refrigerator at −20 C for further use. by using QIIME software (version 2.0, Rob Knight Lab, Boulder, CO, USA) [31]. The similarity of species com- 2.2. Heavy Metal Element Content in Water Samples position of different water samples was investigated. The The filter was first washed with 100 mL of deionized data structure of bacterial community was studied with water, and then 200 mL water samples were filtered QIIME software. PCoA is the most intuitive way to com- through a 0.45-micron millipore filter. The filtered water pare differences in diversity and composition of different samples. samples were digested directly by HNO3–HF–HCl. The contents of Cd, Zn, Cu, Pb and Cr were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, 3. RESULTS AND DISCUSSION Agilent 7500, USA). The contents of arsenic and mer- 3.1. Heavy Metal Element Content in Water Samples Article cury were determined by reduction gasification-atomic flu- The average heavy metal contents are shown in IP: 192.168.39.211 On: Tue, 28 Sep 2021 17:35:39 orescence spectrometry (Lumina 3300, Canada). All the Figures 2(A) and (G). Compared with the national Class reagents used in the test were of superiorCopyright: purity. American Scientific Publishers Delivered by IngentaIII water quality standards of China [32], the heavy metal contents of most water samples are close or exceed the 2.3. DNA Extraction and PCR Amplification maximum value of the class III water quality standard, The total DNA of the Yellow River water samples indicating that most of the water samples were in dan- extracted from the filters by using the EZNA Water DNA ger of being polluted. The sample point 2 is located at Kit (Omega Bio-Tek, USA). The V3–V4 regions of the the junction of the Kundulun River and the Yellow River. 16S rDNA gene of bacteria were amplified using the The Kundulun River is the main seasonal river in Baotou. following primers: 341F: CCTACGGGNGGCWGCAG; However, the domestic sewage and industrial wastewater 805R: GACTACHVGGGTATCTAATCC. The PCR ampli- discharged into the downstream of the Kundulun River and fication was performed in 20 L mixtures, at 95 Cas then flowing into the Yellow River, [13], which may be initial denaturing for 2 min, then denaturing at 95 Cfor one important cause of contamination of sample point 2. 30 s, then annealing at 55 C for 30 s, next extension at 72 C for 30 s, 25 cycles, and final extension at 72 Cfor 5 min. Purified PCR products were delivered to Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) for further high-throughput sequencing (sequencing report number: 16S184065AH).

2.4. Data Analysis The composition and diversity of bacterial communities were tested by means of High-throughput sequencing tech- nique. The Operational Taxonomic Units (OTU) clustering can help understanding the community distribution information in multiple sample sequences. It was performed at 97% similarity by means of UPARSE software (Version 7.1) [30]. In each OTU, the sequence with the highest abundance was placed in the NCBI database for Fig. 3. ACE-index, Chao1-index and Shannon index of the 8 samples genus classiﬁcation. with respect to their locations along the river.

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3.2. Alpha Diversity Analysis of Bacteria in Each in water samples is closely related to water pollution. More Sample Group research is needed to be done to reveal its relationship. The alpha diversity as a diversity indicator of the detected genus was estimated based on the Simpson 3.3. Beta Diversity Among Sample Groups index (Table II). The Abundance-based Coverage Estima- Beta diversity was analyzed by comparing the genus sim- tor (ACE) index and the Chao1 index were used to esti- ilarities between samples. Analysis (principal co-ordinates mate the genus richness, there graphical representation of analysis) (PCoA) is the most intuitive method to explore these along the river was shown in Figure 3. and visualize similarities or dissimilarities in data. Three It was observed that the upstream sample 1 taken two-dimensional plots were created to visualize the ﬁrst at Zhaojundao, displayed the highest bacterial diversity, three dimensions (Fig. 4). while samples 2 and 4 (from Kunhe and Xiaobaihe) had Results showed that sometimes the bacterial genus the lowest diversity of the bacterial OTUs. The diversity compositions of samples were very different when their of bacteria in the upstream river is high, while the diver- geographic locations are long distance. Such as sam- sity of bacteria in the downstream river is low. It could ple 1 (Zhaojundao), located at the upstream; sample 4 be seen that the content of heavy metals in 2, 3, 4 water (Xiaobaihe) located at middle and samples 8 (Wangdahan) samples are very high. It is possible that the presence of located at downstream. The bacterial genus compositions some heavy metals affects bacterial diversity. Compared in these three locations were very different. However, the with sample point 2 and 4 upstream, the bacterial diversity bacterial genus compositions of samples 2 and 7 were of sample point 5, 6, 7 downstream increased. The sample quite similar, although the distance between their locations point 5, 6, 7 downstream are near by the Kundulun River, are approximately 7 km. Conversely, the distance between which contains many pollutants. The diversity of bacteria sample site 6 (Daqiaoxi) and sample site 7 (Daqiaodong)

IP: 192.168.39.211 On: Tue, 28 Sep 2021 17:35:39 Copyright: American Scientific Publishers Delivered by Ingenta Article

Fig. 4. PCoA analysis of diversity for the ﬁrst three dimensions representing 22.0% (PCoA 1), 19.7% (PCoA 2) and 17.3% (PCoA 3) of the variation.

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Fig. 7. Distribution plot of the barcoded sequence reads sorted per genus for the 8 samples.

Figures 7 and 8 showed the similarities and differences of bacterial genera in eight water samples. The highest contents in the eight water samples were Flavobacterium, Pseudomonas, Massilia, Janthinobacterium, Acinetobac- ter, Psychrobacter and Chryseobacterium. Among them, Fig. 5. OTU Venn diagram between samples. the genus Flavobacterium, Pseudomonas Acinetobac- ter and Chryseobacterium belong to the conditional are less than 1 km far, but the bacterial genus composi- pathogens, which mainly cause human lung, brain and blood infections. This information is very important for tions of samples 6 and 7 were quite different. These data the health of the people living along the Yellow River. It Article indicated that location of the sampling sites along the river is also crucial for the residents who use the Yellow River did not correlate with bacterial genus composition. IP: 192.168.39.211 On: Tue, 28as aSep source 2021 of 17:35:39 drinking water. In addition.it is worth not- Copyright: American Scientificing that Publishers the water sample also contains a large number of 3.4. Analysis of Eight Sampling Points of BacterialDelivered by Ingenta Massilia. They have strong degradation of phosphate and Composition at the Genus Level phenanthrene in water. OTU is an artiﬁcial taxonomic unit for systematic analysis The number of conditional pathogens in sample 1 or population genetics research. The obtained OTUs were (Zhaojundao) in the upstream is much less than that in the grouped at the level of phylum and genus. Figures 5 and 6 showed that the similarity and difference of bacteria in eight water samples at genus level.

Fig. 8. Ternary plot of bacterial genera colored per phylum for three samples. Notes: The dots represent genera and are positioned towards the samples where they are signiﬁcantly enriched relative to the other two samples. Gray dots represent genera that were not enriched, and the circle size Fig. 6. Bray tree plot of bacteria at eight sampling points. represents the average relative abundance of each genus.

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Violle, C., Barnard, R.L. and Philippot, L., 2017. Effectiveness of analysis of soil heavy metal pollution from lower reaches of Yel- Article ecological rescue for altered soil microbial communities and func- low river irrigation in China. Science of the Total Environment, 633, tions. The ISME Journal, 11, pp.272–283.IP: 192.168.39.211 On: Tue, 28 Seppp.1136–1147. 2021 17:35:39 Copyright: American Scientific Publishers Delivered by IngentaReceived: 8 February 2020. Accepted: 7 April 2020.

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