The Monitoring of Nizhniy Kaban Lake by 16s Rrna Gene Amplicon Data Set-Based Bacterial Diversity

Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

The Monitoring of Nizhniy Kaban Lake by 16s

Rrna Gene Amplicon Data Set-Based Bacterial

Diversity

Ludmila L. Frolova*, Anthony Elias Sverdrup

Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia

Received: 05/08/2019

Accepted: 20/11/2019

Published: 20/12/2019

Abstract

The paper presents the results of research of bacterial diversity of Nizhniy Kaban Lake (Kazan, Russia) for 2016-2017, using

the marker gene 16S rRNA of hydrobionts, based on the method of next-generation sequencing. Nizhniy Kaban, Verhniy Kaban

and Sredniy Kaban are included in the system of Kaban Lakes. They are located in the center of a large industrial city, and suffer

anthropogenic load. According to ecological studies, Kaban Lakes are polluted. The sequences of 16S rRNA Bacteria gene

fragment of the freshwater lake Nizhniy Kaban were submitted to the international database in fastq format on the website NCBI

with the numbers SRR7510984 and SRR7516469. The comparative analysis of metagenomic data showed a significant change in

bacterial diversity over the years. A total of 103030 (2016) and 90402 (2017) high-quality reads were obtained; 76% (2016) and

70% (2017) of the bacterial population was classified to the genus level, while 0.25% (2017) was classified to the species level.

In total, 18 species of Bacteria were identified. Among them, bacteria occurring in the human gastrointestinal tract, were found

the most often. These types of bacteria can be a threat to human health. Therefore, the species composition of Bacteria

community should be taken into account when assessing the ecological state of water reservoirs.

Keywords: Gene 16S rRNA, Next-generation sequencing, Freshwater lake, Bacteria

1

phytoplankton by rbcL gene, in order to assess the

1

Introduction

ecological state of the freshwater Nizhniy Kaban lake (6-7).

The work presents the results of research of bacterial

diversity of Nizhniy Kaban lake (Kazan, Russia) for 2016-

Currently, the assessment of water quality is carried out

using various physicochemical and biological methods.

One of them is the method of isolation and identification of

microorganisms. The presence of bacterial pathogens in the

water reservoirs adversely affects human health. They can

be the agents of such diseases as cholera, diarrhea,

dysentery, hepatitis A, typhoid fever and poliomyelitis (1).

To assess the diversity of microorganisms in various

environments, for example, in human intestine, bottom

sediments of Lake Baikal or in the hot springs of

Kamchatka, the methods of next-generation sequencing are

used (2). This technology allows to accelerate the process

of determining the sequences of organisms' genomes (3).

The sequencing of 16S rRNA gene is a universal and

effective approach for taxonomic characterization, as this

gene is present in the genomes of all prokaryotes, and has

relatively low variability (4).

2017, using the marker gene 16S rRNA of hydrobionts,

based on the method of next-generation sequencing.

Nizhniy Kaban, Verhniy Kaban and Sredniy Kaban are

included in the system of Kaban Lakes. They are located in

the center of

a large industrial city, and suffer

anthropogenic load. According to ecological studies, Kaban

Lakes are polluted.

2

Methods

The sampling from Nizhniy Kaban Lake (Kazan,

Russia) was carried out during 2016-2017, in accordance

with the standard hydrobiological methods (8).

DNA was isolated from the pellet, using FAST DNA

Kit (MP biomedicals), according to the manufacturer's

protocol. The pellet was obtained by centrifuging of 50 ml

of the sample, at a speed of 10,000 g for 15 minutes. The

amplification of the isolated DNA was carried out using

PhusionHigh-Fidelity DNA polemerase (ThermoFisher),

and primers (Table 1). After this, the second PCR cycle

was performed in order to index the samples (Nextera XT

indices). Purification of PCR products was performed using

Metagenomics can provide valuable information on the

functional ecology of environmental communities (5). We

previously used metagenomic DNA sequencing for the

identification of zooplankton by СО1 gene, and

Corresponding author: Ludmila L. Frolova, Kazan

Federal University. Email: Lucie.Frolova@gmail.com.

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Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

Agencourt AMPure XP beads (Beckman Coulter). The

obtained DNA libraries were sequenced on the device

Illumina MiSeq (MiSeq Reagent kit v3).

The software Krona chart (10) and GraphPad (11) were

used to build charts.

3

Results and Discussion

In 2016-2017, the next-generation sequencing method

Table 1: Primers for PCR of 16S rRNA gene

Primers

6SF_I

Sequences

'-

was applied with the aim to identify Bacteria from Nizhniy

Kaban Lake.

5

1

tcgtcggcagcgtcagatgtgtataagagacagcctacggg

nggcwgcag-3'

3.1 Krona chart of the bacteria represented by 16S rRNA

(

forward)

gene amplicon-based bacterial diversity

5

'-

16SR_I

The percentage distribution of Bacteria of Nizhniy

Kaban Lake by species diversity and reads for 2016 is

shown in Fig. 1-2. The percentage distribution of Bacteria

of Nizhniy Kaban Lake by species diversity and reads for

gtctcgtgggctcggagatgtgtataagagacaggactach

vgggtatctaatcc-3'

(

reverse)

Metagenomic data were entered into the international

2017 is shown in Fig. 3-4. Each circle represents the

SRA database on the website NCBI with numbers:

SRR7510984 and SRR7516469 (9). After filtering the

reads by quality, trimming of sequences and removing of

chimeric sequences, the obtained nucleotide sequences of

phylum, class, order, family, genus, and species from the

inside to the outside of the circle, respectively, indicated by

the percent diversity, based on the absolute number of

representative bacteria.

1

6S rRNA Bacteria gene were aligned, using the software

BLAST, in order to establish the taxonomic composition.

Figure 1: The percentage of species diversity of 16S rRNA Bacteria of Nizhniy Kaban Lake (2016)

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Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

Figure 2: The percentage of 16S rRNA Bacteria reads of Nizhniy Kaban Lake (2016)

Figure 3: The percentage of species diversity of 16S rRNA Bacteria of Nizhniy Kaban Lake (2017)

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Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

Figure 4: The percentage of 16S rRNA Bacteria reads of Nizhniy Kaban Lake (2017)

3.2 The percentage of 16S rRNA Bacteria of Nizhniy

Kaban Lake by phylum

The percentage of species diversity of 16S rRNA

Bacteria of Nizhniy Kaban Lake by phylum (2016-2017) is

shown in Figure 5. As can be seen from Fig. 5,

Proteobacteria Bacteroidetes

(56.7%/38.57%),

(

28.57%/9.28%) and Actinobacteria (14.29%/9.79%) are

the most numerous by species diversity at the level of

phylum, respectively by years.

Figure 6: The percentage of 16S rRNA Bacteria reads of Nizhniy

Kaban Lake by phylum (2016-2017)

3.3 The percentage of 16S rRNA Bacteria of Nizhniy

Kaban Lake by class

The percentage of species diversity of 16S rRNA

Bacteria of Nizhniy Kaban Lake by class (2016-2017) is

shown in Fig. 7, parts 1-2. As can be seen from Fig. 7,

Alphaproteobacteria (14.3%/21.1%), Betaproteobacteria

Figure 5: The percentage of species diversity of 16S rRNA

Bacteria of Nizhniy Kaban Lake by phylum (2016-2017)

(15.71%/16.49%) Gammaproteobacteria

and

(

5.71%/14.43%) are the most numerous by species

The percentage of 16S rRNA Bacteria reads of Nizhniy

Kaban Lake by phylum (2016-2017) is shown in Figure 6.

As can be seen from Fig. 6, Cyanobacteria

diversity. The percentage of 16S rRNA Bacteria reads of

Nizhniy Kaban Lake by class (2016-2017) is shown in Fig.

8

Oscillatoriophycideae (75.60%/68.15%) is the most

numerous by reads among Bacteria.

, parts 1-2. As can be seen from Fig. 8, the species

(

75.63%/75.19%) and Proteobacteria (3.68%/19%) are the

most numerous by reads.

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2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

Figure 8: part 2. The percentage of 16S rRNA Bacteria reads of

Nizhniy Kaban Lake by class (2016-2017)

3

.4 The percentage of 16S rRNA Bacteria of Nizhniy

Figure 7: part 1. The percentage of species diversity of 16S rRNA

Bacteria of Nizhniy Kaban Lake by class (2016-2017)

Kaban Lake by order

The percentage of species diversity and reads of 16S

rRNA Bacteria of Nizhniy Kaban Lake by order (2016-

2017) is shown in Fig. 9-11. As can be seen from Fig. 9-11,

the following orders were of the greatest importance in

terms of species diversity and/or reads, in 2016:

Oscillatoriales

17.14%/3.79%),

(1.43%/75.60%),

Sphingobacteriales

(14.29%/2.05%),

(

Burkholderiales

Flavobacteriales (11.43%/2.69%); in 2017: Oscillatoriales

1.03%/67.63%), Burkholderiales (10.31%/4.08%),

Actinomycetales (8.25%/0.47%).

(

Figure 7: part 2. The percentage of species diversity of 16S rRNA

Bacteria of Nizhniy Kaban Lake by class (2016-2017)

Figure 10: The percentage of species diversity and reads of 16S

rRNA Bacteria of Nizhniy Kaban Lake by order (2016)

1

– Oscillatoriales (1.43%/75.60%), 2 – Frankiales

7.14%/6.25%),

– Sphingobacteriales (17.14%/3.80%)

(

3

Figure 8: part 1. The percentage of 16S rRNA Bacteria reads of

Nizhniy Kaban Lake by class (2016-2017)

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Figure 9: The percentage of species diversity and reads of 16S rRNA Bacteria of Nizhniy Kaban Lake by order (2016-2017)

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genera were unique for 2016, 19.4% genera were common

for 2016-2017, and 68.2% genera were unique for bacterial

community of 2017. Flavobacterium (1.4%/0.65%),

Fluviicola (0.32%/0.42%), Lewinella (0.4%/0.3%),

Limnobacter

(0.03%/0.15%),

Limnohabitans

(

0.11%/0.02%), Mycobacterium (0.11%/0.04%), Opitutus

0.19%/0.08%),

Phenylobacterium

(0.05%/0.01%),

Polynucleobacter

(0.03%/0.02%),

Planktothrix

Planctomyces

69.82%/42.40%),

(

(0.40%/0.06%),

Prosthecobacter (0.16%/0.03%), Rickettsia (0.08%/0.01%),

Sediminibacterium (0.22%/0.63%), Zymomonas

0.22%/0.02%) were the common genera for 2016-2017.

(

Figure 11: The percentage of species diversity and reads of 16S

rRNA Bacteria of Nizhniy Kaban Lake by order (2017)

1

–

Oscillatoriales

Pseudoanabaenales (1.54%/6.95%),

Enterobacteriales (2.06%/5.6%),

Burkholderiales (10.31%/4.08%)

(1.03%/67.63%),

2

4

–

3

–

3.5 The percentage of 16S rRNA Bacteria of Nizhniy

Kaban Lake by family

The total number of identified Bacteria by family is

2

6/93 families, respectively for 2016/2017. The families by

reads are shown in Fig. 12, among them 11% (2016), and

7% (2017) are not classified. The following families are

the most represented: Microcoleaceae - 38% (2016), 42.5%

2017), Sporichthyaceae 5.78% (2016);

Enterobacteriaceae - 9.85% (2017), Comamonadaceae -

1

(

-

5.54% (2017).

Figure 13: The percentage of 16S rRNA Bacteria of Nizhniy Kaban

3.6 The percentage of 16S rRNA Bacteria of Nizhniy

Lake by genus (2016-2017)

Kaban Lake by genus

The percentage of 16S rRNA Bacteria of Nizhniy

Kaban Lake by genus (2016-2017) is shown in Fig.13. As

can be seen from Fig. 13 - 12.4% of identified Bacteria

Figure 12: The percentage of 16S rRNA Bacteria reads of Nizhniy Kaban Lake by family (2016-2017)

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Table 2: Summary of Bacteria identified to the species level from 16S rRNA gene-based metagenomic study of freshwater from

Nizhniy Kaban Lake (2017)

Phylum

Class

Order

Family

Species

%

Agromyces mediolanus¹

Candidatus Aquiluna rubra²

Micrococcus luteus³

Propionibacterium acnes⁴

Collinsella aerofaciens⁵

Bacteroides uniformis⁶

0.0379

0.0263

0.0032

0.0042

0.0221

0.0126

0.0074

0.0021

0.0042

0.0274

0.0011

0.0358

0.0158

0.0021

0.0116

0.0074

0.0011

0.0284

Microbacteriaceae

Actinobacteria

Actinomycetales

Actinobacteria

Micrococcaceae

Propionibacteriaceae

Coriobacteriaceae

Bacteroidaceae

Coriobacteriia

Bacteroidia

Bacilli

Coriobacteriales

Bacteroidales

Bacillales

Bacteroidetes

Porphyromonadaceae Parabacteroides distasonis⁷

Staphylococcaceae

Peptococcaceae

Staphylococcus epidermidis⁸

Desulfosporosinus meridiei⁹

Faecalibacterium prausnitzii¹⁰

Cetobacterium somerae¹¹

Brevundimonas diminuta¹²

Variovorax paradoxus¹³

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae

Fusobacteriaceae

Caulobacteraceae

Comamonadaceae

Moraxellaceae

Fusobacteria

Fusobacteriia

Fusobacteriales

Caulobacterales

Burkholderiales

Alphaproteobacteria

Betaproteobacteria

Acinetobacter rhizosphaerae¹⁴

Pseudomonas veronii¹⁵

Proteobacteria

Pseudomonadales

Gammaproteobacteria

Pseudomonadaceae

Xanthomonadaceae

Helicobacteraceae

Xanthomonadales

Campylobacterales

Pseudoxanthomonas mexicana¹⁶

Sulfuricurvum kujiense¹⁷

Epsilonproteobacteria

Verrucomicrobia Verrucomicrobiae

Verrucomicrobiales Verrucomicrobiaceae Prosthecobacter debontii¹⁸

1

Aniline-assimilating bacteria. They occur in the soil; there are cases of human infection (12).

It can be found in fresh and salt water (13).

It is an obligate aerobe, widely distributed in the environment. It can be found in soils, dust, water and air. It is also a part of the normal

microflora of the skin surface of humans and mammals (14).

It occurs on the skin and in the gastrointestinal tract of humans and animals. It can cause the skin diseases in humans (15).

Collinsella aerofaciens, a rod-shaped nonmotile obligate anaerobe, is the most abundant actinobacterium in the gastrointestinal tract of

healthy humans. An altered abundance of C. aerofaciens may be linked with several health disorders, including irritable bowel

syndrome (16).

Bacteroides spp are the non-spore forming gram-negative bacilli, which are the part of the human resident flora (17).

Parabacteroides distasonis belong to the main intestinal microbiota of healthy people. At the same time, these bacteria can cause some

infections (18).

2

3

4

5

6

7

8

It is a gram-positive bacteria, one of more than 40 species of the genus Staphylococcus (19). It is a part of the normal microflora of human

skin, and mucous membranes (less often) (20).

9

1

Desulfosporosinus meridiei sp. nov., is a spore-forming sulfate-reducing bacterium, isolated from gasolene-contaminated groundwater (21).

0

It is one of the most common and important commensal bacteria of human intestinal microbiota (22).

1

Cetobacterium somerae is

a gram-negative, microaerotolerant, non-spore-forming and rod-shaped bacterium from the genus

Cetobacterium, which has been isolated from human feces (23).

It was isolated from clinical samples of patients with mucoviscidosis. It is used as a potential bioremediator of marine oil pollution (24).

It can be found everywhere. It is abundantly present in environments, which are contaminated with organic compounds or heavy metals

1

2

3

(25).

1

4

Gram-negative bacteria; they are chemorganotrophs with oxidative metabolism. They are saprophytes, and universal in occurrence. They

may be the cause of many infectious processes, including meningitis, septic disease in humans, and septicemia, abortion in animals. In

February 2017, WHO ranked acinetobacteria among the most dangerous bacteria, due to their resistance to existing antibacterial drugs

(

26, 38, 39).

1

5

6

7

8

Pseudomonas veronii is a gram-negative, rod-shaped, fluorescent, motile bacterium, isolated from natural springs in France. It may be used

for bioremediation of contaminated soils, as it has been shown to degrade a variety of simple aromatic organic compounds (27, 37).

Pseudoxanthomonas mexicana is a species of mesophilic, motile, strictly aerobic, gram-negative, non-spore-forming, rod-shaped bacteria

with one polar flagellum, first isolated from human urine, riverside urban soil and anaerobic digester (28, 31, 35, 36).

It is a facultative anaerobe, chemolithoautotrophic sulfur-oxidizing bacterium, typical representative of the genus. The cells have the shape

of curved rods, they are mobile, and have a single polar flagellum (29, 33, 34).

It was isolated from fresh water (30, 32).

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2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

3

.7 The percentage of 16S rRNA Bacteria of Nizhniy

S.Malanin and E.Boulygina, the scientists of Kazan Federal

University, for their assistance in experimental work.

Kaban Lake by species

The species diversity of Bacteria in 2017 was 0.25% of

the total number of organisms by reads. Table 2 shows the

classification of bacterial organisms in Nizhniy Kaban

Lake. The percentage of species diversity by reads of 16S

rRNA Bacteria of Nizhniy Kaban Lake for 2017 is shown

in Fig.14.

References

1. World Health Organization

– http://www.who.int/ru/news-

room/fact-sheets/detail/drinking-water

2. Biomolecule - http://biomolecula.ru

3

. Ansorge WJ. Next-generation DNA sequencing techniques.

New biotechnology. 2009 Apr 1;25(4):195-203.

4

. Zhang J, Ding X, Guan R, Zhu C, Xu C, Zhu B, Zhang H, Xiong

Z, Xue Y, Tu J, Lu Z. Evaluation of different 16S rRNA gene

V regions for exploring bacterial diversity in a eutrophic

freshwater lake. Science of the Total Environment. 2018 Mar

1

5;618:1254-67.

5

6

. Raes J, Letunic I, Yamada T, Jensen LJ, Bork P. Toward

molecular trait‐based ecology through integration of

biogeochemical, geographical and metagenomic data.

Molecular systems biology. 2011 Jan 1;7(1).

. Frolova LL, Sverdrup AE. The Monitoring of Nizhniy Kaban

Lake by Rbcl Gene of Fresh Water Ater Organisms Using

Next-Generation Sequencing. RESEARCH JOURNAL OF

PHARMACEUTICAL BIOLOGICAL AND CHEMICAL

SCIENCES. 2018 Nov 1;9(6):262-71.

7

. Khusainov AM, Frolova LL. The Evaluation Of Water Quality

Of Nizhniy Kaban Lakeusing Next-Generation Sequencing.

Figure 14: The percentage of species diversity by reads of 16S

rRNA Bacteria of Nizhniy Kaban Lake (2017)

RESEARCH

BIOLOGICAL AND CHEMICAL SCIENCES. 2018 Nov

;9(6):272-9.

JOURNAL

OF

PHARMACEUTICAL

1

4

Summary

8

.

Bul’on V.V. “Methodological recommendations for the

collection and processing of materials in the process of

hydrobiological studies of the freshwater bodies.

Phytoplankton and its products”, L.: State Research Institute

on Lake and River Fisheries. 1981; 32.

According to the results of the study, using the modern

method of next-generation sequencing, the bacterial profile

of Nizhniy Kaban Lake for 2016-2017 was characterized.

The comparative analysis of metagenomic data showed a

significant change in bacterial diversity over the years. A

total of 103030 (2016) and 90402 (2017) high-quality reads

were obtained; 97.8% (2016) and 98.4% (2017) of the

bacterial population were classified to the phylum, while

9. National Center for Biotechnology Information

http://www.ncbi.nlm.nih.gov/

1

–

0. Krona homepage – http://github.com/marbl/Krona/wiki

1. GraphPad Software – https://www.graphpad.com/

2. Sridhar S, Wang AY, Chan JF, Yip CC, Lau SK, Woo PC,

Yuen KY. First report of human infection by Agromyces

mediolanus, a gram-positive organism found in soil. Journal of

clinical microbiology. 2015 Oct 1;53(10):3377-9.

9

7.5% (2016) and 95.9% (2017) were classified to the class

level, 92.4% (2016) and 95.4% (2017) were classified to

the order level, 89.2% (2016) and 87.1% (2017) were

classified to the family level, 76.4% (2016) and 70.1%

13. Hahn MW. Description of seven candidate species affiliated

with the phylum Actinobacteria, representing planktonic

freshwater bacteria. International journal of systematic and

evolutionary microbiology. 2009 Jan;59(0 1):112.

1

(

2017) were classified to the genus level, and 0.25% (2017)

was classified to the species level. In total, 18 species of

Bacteria were identified. Among them, bacteria occurring

in the human gastrointestinal tract, were found the most

often. These types of bacteria can be a threat to human

health. Therefore, the species composition of Bacterial

community should be taken into account when assessing

the ecological state of water reservoirs.

4. Micrococcus luteus - www.gastroscan.ru/handbook/118/7095

5. Bhatia A, Maisonneuve JF, Persing DH. Propionibacterium

acnes and chronic diseases. InThe Infectious Etiology of

Chronic Diseases: Defining the Relationship, Enhancing the

Research, and Mitigating the Effects: Workshop Summary.,

Knobler, SL et al.(eds.) 2004 Jun 16 (pp. 74-80).

1

6. Bag S, Ghosh TS, Das B. Complete genome sequence of

Collinsella aerofaciens Isolated from the gut of a healthy

5

Conclusions

Indian

subject.

Genome

Announc..

2017

Nov

The results obtained are of great practical interest in the

2

2;5(47):e01361-17.

field of monitoring of water reservoirs. The method of

next-generation sequencing can be successfully used for the

control of water reservoirs, in particular, and for the

assessment of ecological state of water reservoirs, in

general.

7. Finegold SM, Sutter VL, Mathisen GE. Normal indigenous

intestinal flora. Human intestinal microflora in health and

disease. 1983 Jan 1;1:3-1.

18.

Parabacteroides

distasonis

-

http://www.gastroscan.ru/handbook/118/8882

9. Schleifer KH, Kloos WE. Isolation and characterization of

Staphylococci from human skin I. Amended descriptions of

1

Acknowledgements

The work is performed according to the Russian

Government Program of Competitive Growth of Kazan

Federal University. The authors would like to thank Dr.

Staphylococcus

epidermidis

and

Staphylococcus

saprophyticus and descriptions of three new species:

Staphylococcus cohnii, Staphylococcus haemolyticus, and

Staphylococcus xylosus. International Journal of Systematic

1014

Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 1006-1015

and Evolutionary Microbiology. 1975 Jan 1;25(1):50-61.

Nano-Formulation: A Review. Journal of Chemical Reviews.

2019 Mar 1;1(2. pp. 78-153):130-8.

2

0. Fey PD, Olson ME. Current concepts in biofilm formation of

Staphylococcus epidermidis. Future microbiology. 2010

Jun;5(6):917-33.

36. Nabati M, Bodaghi-Namileh V, Sarshar S. Molecular Modeling

of the antagonist compound esketamine and its molecular

docking study with non-competitive N-methyl-D-aspartate

(NMDA) receptors NR1, NR2A, NR2B and NR2D. Progress

in Chemical and Biochemical Research. 2019 Sep 1;2(3):108-

19.

37. Noorizadeh H, Farmany A. Adsorptive Removal of Arsenic

from Aqueous Solutions Using Amino Functionalized Super

paramagnetic Iron Oxide Nanoparticle/SiO2.

38. Nabati M, Kermanian MA, Mohammadnejad-Mehrabani H,

Rahbar Kafshboran H, Mehmannavaz M, Sarshar S.

Theoretical Investigation on the Antitumor Drug: ThioTEPA

and its Interaction with S-donor Biomolecules and DNA

Purine Bases. Chemical Methodologies. 2018 Apr 1;2(2. pp.

83-180):128-40.

39. Shafiekhani H, Barjoizadeh R. Modification of activated

carbon by ZnCl2, CaCl2, MgCl2 and their applications in

removal of nitrate ion from drinking water. Asian Journal of

Green Chemistry. 2019 Jan 1;3(1. pp. 1-124):1-2.

1. Robertson WJ, Bowman JP, Franzmann PD, Mee BJ.

Desulfosporosinus meridiei sp. nov., a spore-forming sulfate-

reducing bacterium isolated from gasolene-contaminated

groundwater. International Journal of Systematic and

Evolutionary Microbiology. 2001 Jan 1;51(1):133-40.

2. Bag S, Ghosh TS, Das B. Complete genome sequence of

Faecalibacterium prausnitzii isolated from the gut of a healthy

Indian adult. Genome Announc.. 2017 Nov 16;5(46):e01286-

2

1

7.

3. Finegold SM, Vaisanen ML, Molitoris DR, Tomzynski TJ,

Song Y, Liu C, Collins MD, Lawson PA. Cetobacterium

somerae sp. nov. from human feces and emended description

of the genus Cetobacterium. Systematic and applied

microbiology. 2003 Jan 1;26(2):177-81.

4. Ryan MP, Pembroke JT. Brevundimonas spp: emerging global

opportunistic pathogens. Virulence. 2018 Dec 31;9(1):480-93.

5. Satola B, Wübbeler JH, Steinbüchel A. Metabolic

characteristics of the species Variovorax paradoxus. Applied

microbiology and biotechnology. 2013 Jan 1;97(2):541-60.

6. World Health Organization – http://www.who.int/en/news-

room/detail/27-02-2017-who-publishes-list-of-bacteria-for-

which-new-antibiotics-are-urgently-needed

2

7. Onaca C, Kieninger M, Engesser KH, Altenbuchner J.

Degradation of alkyl methyl ketones by Pseudomonas veronii

MEK700.

Journal

of

bacteriology.

2007

May

1

5;189(10):3759-67.

2

8. Thierry S, Macarie H, Iizuka T, Geißdörfer W, Assih EA,

Spanevello M, Verhe F, Thomas P, Fudou R, Monroy O,

Labat M. Pseudoxanthomonas mexicana sp. nov. and

Pseudoxanthomonas japonensis sp. nov., isolated from diverse

environments, and emended descriptions of the genus

Pseudoxanthomonas Finkmann et al. 2000 and of its type

species. International journal of systematic and evolutionary

microbiology. 2004 Nov 1;54(6):2245-55.

2

3

9. Kodama Y, Watanabe K. Sulfuricurvum kujiense gen. nov., sp.

nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-

oxidizing bacterium isolated from an underground crude-oil

storage cavity. International journal of systematic and

evolutionary microbiology. 2004 Nov 1;54(6):2297-300.

0. Whitman WB. Bergey's Manual of Systematic Bacteriology,

Volume 4: The Bacteroidetes, Spirochaetes, Tenericutes

(Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria,

Dictyoglomi, Gemmatimonadetes, Lentisphaerae,

Verrucomicrobia, Chlamydiae, and Planctomycetes.

1. Moghimi A, Yari M. Review of procedures involving

separation and Solid Phase Extraction for the determination of

cadmium using spectrometric techniques. Journal of Chemical

Reviews. 2019 Jan 1;1(1, pp. 1-77.):1-8.

3

2. Gomaa EG, Berghout MA, Moustafa MR, El Taweel FM, Farid

HM. Thermodynamic and Theoretical solvation parameters for

2

-amino-4, 5-dimethylthiophene-3-carboxamide (ADTC) in

Ethanol and Mixed EtOH-H2O solvents. Progress in Chemical

and Biochemical Research. 2018 Oct 1;1(1, pp. 1-80):19-28.

3. Mohammadi F, Yousefi M, Ghahremanzadeh R. Green

synthesis, characterization and antimicrobial activity of silver

nanoparticles (AgNPs) using leaves and stems extract of some

3

plants. Advanced Journal of Chemistry-Section

Theoretical, Engineering and Applied Chemistry). 2019 Apr

;2(4, pp. 266-385):266-75.

A

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1

3

4. Gomaa EA, Mahmoud MH, Mousa MG, El-Dahshan EM.

Cyclic Voltammetry for the Interaction between Bismuth

Nitrate and Methyl Red in Potassium Nitrate Solutions.

Chemical Methodologies. 2019 Jan 1;3(1. pp. 1-144):1-1.

5. Ugbede Itodo H. Controlled Release of Herbicides Using

1015