Isolation and Identification of Cadmium-Reducing Bacteria from Contaminated Coastal Sediment in The Northern Coast of Indramayu, Indonesia

doi:10.21203/rs.3.rs-1966691/v1

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Isolation and Identification of Cadmium-Reducing Bacteria from Contaminated Coastal Sediment in The Northern Coast of Indramayu, Indonesia

https://doi.org/10.21203/rs.3.rs-1966691/v1

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Cadmium (Cd) is one of the heavy metals pollutants and its accumulation impacted the sustainability of marine organisms. The aim of current research was to isolate and identify the cadmium-reducing bacteria from contaminated coastal sediment in Karangsong port, Indramayu, Indonesia. The isolates were investigated for its potential to reduce cadmium showed the cadmium reduction drastically up to 50% at 6 hours treated under different cadmium concentration of 0,5, 1, and 1,5 ppm, respectively. Morphological characteristics were observed in most of isolates. Out of 8 isolates, two selected strains such as Karangsong Cd3 and Cd7 were identified by 16S rRNA sequencing as Pseudoalteromonas issachenkonii strain KMM 3549 (Acc. No. NR 025139.1) and Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Acc. No. NR 041787.1), respectively. The cadmium resistance profile showed that the selected isolates were resistant to various concentration of cadmium (Cd). These results suggest that the Karangsong Cd 3 and Cd 7 could facilitate the new references of future microbial application for bioremediations efforts.

Cadmium

Karangsong

Pseudoalteromonas sp

Bacteria

Water pollution

Cd 3 and Cd 7 strains were isolated from Karangsong Port sediments.
Cd 3 and Cd 7 were closely related to Pseudoalteromonas sp species.
Indigenous Cd 3 and Cd 7 were potentially reducing cadmium.

The rapid development of industries impacted on the sustainability of marine environment. Waste and hazardous pollutants from industrial waste elevated in seawater that trigger on the survival of marine ecosystems and other living things, especially humans (Suprapti et al., 2016). Marine pollution became a serious issue and received considerable attention in various scientific international communities (Faizal et al., 2022; Purba et al., 2021). Many hazardous sources of heavy metals in sea water were commonly derived from industries, mining, and agriculture activities (Bargagli et al., 2000). Industrial hazardous wastes are categorized in a grouping of hazardous and toxic materials such as Hg, Cd, Cu, Zn, and Ni, (B3) (Sanusi, 1985).

Cadmium is commonly found as a by-product of Zinc refining and a toxic pollutant to the marine organisms (Satarug et al., 2003; Shi et al., 2015). This anthropogenic pollutant is released into the coastal region from rivers and groundwater (Abe, 2007). Because of their soluble and destructive to marine ecosystems, cadmium was considered an environmental threat (Chiarelli et al., 2019). It was reported that cadmium exposure impacted the low fertilization, growth rate, and embryogenesis in the marine organisms (Memon & Schröder, 2009; Reichelt-Brushett & Harrison, 2004; Roccheri and Matranga 2010).

Bioaccumulation of cadmium in the marine environments are commonly located in industrial and agriculture activities. Such area includes Karangsong port in the northern coastal area of Indramayu, Indonesia (Fig. 1), where located in rapid industrial growth of manufacturing, long term waste oil from oil company, and fish auction waste. Furthermore, many investigators have reported the accumulation of cadmium level in Karangsong Coast area (Herawati et al., 2018). Efforts to control heavy metals by developing a consortium of indigenized bacteria as bioremediation agent for Cd metal pollution in waters contaminated with industrial waste in Karangsong Port have never been experimented.

The microorganisms were found to have a great tolerance exposed to heavily polluted areas and their characteristics included the physiological and genetic changes by secreting a soluble enzyme and resistance mechanism (Gupta & Diwan, 2016; Kanazawa & Mori, 1996; Khan et al., 2015). A comprehensive analysis of bioremediation from the contaminated coastal sediment should facilitate our knowledge for the understanding of the availability of cadmium-reducing bacteria. In this study, we aimed to isolate and identify the bacteria from the sediments at Karangsong port with the potential to reduce cadmium. Cadmium-reducing bacteria were isolated from Karangasong sediments and identified as Karangasong CD3 and CD7 and their corresponding 16s rDNA sequence analysis were aligned for phylogenetic tree with other Pseudoalteromonas sp. diverse species. Furthermore, cadmium metal tests were demonstrated for isolated bacteria from Karangasong port sediment.

2.1 Sediment collection

Sediment samples were collected from Karangsong Harbour located in the northern coastal area of Indramayu, West Java, Indonesia: L.site (S 06°56'2" E 107°46'38"). Samples were taken at the ports approximately 50 cm above sea level around the contaminated with heavy metal cadmium (Cd) immersed area (Herawati et al., 2018).

2.2 Cadmium Concentration Reduction assay (Cd).

To determine Cd concentration reduction assay, seawater was filtered with a 0.22 µm filter and transferred into a 500-ml test bottle. Three levels of Cd (0.5, 1, and 1.5 mg/L) were prepared and added to the filter seawater solutions. The source of bacteria amounting to about 10 ml was also added and gently swirled. Then, each test medium was mixed into an 80-ml vial bottle, and monitored starting for 0, 12, 24, and 48 hours, respectively (citation).

2.3. Bacterial Isolation

To isolate the bacteria reducing cadmium, Karangsong sediment collected from Karangsong beach by using piston core of 60 cm depth, and the sediment immediately stored in flask and kept at 4°C. Sediments were dispersed in distilled water with serial dilution and the pore water collected. Aliquots suspension (1 mL) of 10^− 5 dilution were spread onto Petri dishes using NA medium dissolved with 100% natural sea water for the isolation bacteria. The agar plates were incubated at 37°C for 24 h. Growing colonies of bacteria obtained from this step were separately streaked on separated NA medium plates to maintain the inoculum of pure cultures.

2.4 Molecular Identification

The genomic DNA of cadmium-reducing isolates was extracted using a Trisure Bioline Extraction kit (Bioline, United Kingdom) and the 16S rRNA was amplified via polymerase chain reaction (PCR). The universal primers 9F (5’-GAGTTTGATCCTGGCTCAG-3’), and 1542R (5’-AGAAAGGAGGTGATCCAGCC-3’) were used. The reaction was performed in 60 µL with Master Mix GoTaq G2 (Promega, Wisconsin, USA). PCR product with amplicon size of 1500 bp was sent to First Base company using Illumina MiSeq, NovaSeq 6000 sequencer. The sequences were analyzed using BioeditTM software and submitted to the gene bank database.

2.5 Phylogenetic Analysis

MEGA X software was used to generated the phylogenetic tree using the neighbour-joining tree method. A bootstrap value of 1,000 replicates was used to estimate the confidence level with values of 70% or higher indicates a reliable clustering.

2.6 Testing of Cadmium-resistant Isolates

The cadmium resistance study was carried out to identify the cadmium-resistant isolates. A loop of isolates was inoculated in Nutrient Broth and incubated at 37°C for 18 h. The isolates were exposed to 1 mg/L, 1.5 mg/L¹, and 2 mg/L concentration of Cd for 18 h. The cells density was measured using spectrophotometer (Agieleant technologies, california, USA).

3.1 Analysis of Cadmium-Reduction Bacteria

The isolates were carried out for its potential to reduce cadmium in nutrient broth having initial cadmium concentration of 0.5, 1, and 1,5 ppm (Fig. 2a), respectively. It was observed that the isolates reduced the concentration of cadmium drastically up to 50% at 6 h. However, the isolates had stable growth cultured at different intervals and at different levels of cadmium concentrations. These results suggesting that some bacteria possible to remove the cadmium from seawater containing cadmium.

3.2 Morphology and Molecular Identification of Bacterial Strains

The morphological characteristics of Karangasong Cd colonies were irregular, round, and spindle as shown in Table 1. Bacterial strains were isolated from Karangasong port were mainly off-white and yellowish in color as examined by the light microscopy (Table 1). The gram staining observation showed that bacterial colonies were diplobacilli and coccobacillus (Supp Fig. 1). The genomic DNA isolation and 16s rDNA PCR were performed for the cadmium-reducing isolates and the sample were analyzed for sequencing. The universal primer of 16s rDNA was designated and subjected to PCR amplification showed a clear target band of about 1500 bp (Figure. 3). Results of the BLAST analysis showed that the Cd 3 isolate was 77.28% similarity index with that Pseudoalteromonas issachenkonii strain KMM 3549 (Accession number NR 025139.1). The Cd 7 bacterial isolate was identical to Pseudoalteromonas spiralis strain Te-2-2 (Accession number NR 114801.1) with an identity value of 95.88%. Our result showed motphological and 16s rDNA sequence analysis, selected isolates were found identical to Pseudoalteromonas sp.

3.3 Phylogenetic analysis

The evolutionary relationship distances were constructed using neighbour-joining tree method, and statistically tested using the bootstrap method with 1,000 replications (Fig. 4). The phylogenetic tree that Cd 3 sequence is closely related to Pseudoalteromonas issachenkonii strain KMM 3549 (Fig. 4a). The phylogeny tree also shows a kinship relationship between the two high sequences with a bootstrap value of 100% and an identity of 77%. Furthermore, Cd 7 sequence is related to Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Fig. 4b). Kinship relationship is also found in the phylogeny tree showing two high sequences with a bootstrap value of 99% and an identity of 97%, indicating that, from 1000 reconstructed phylogeny trees, Cd 7 sequence is 99% related to Pseudoalteromonas tetraodonis GFC strain IAM 14160.

3.4 Test of Cadmium-reduction on selected bacterial isolates

To observe the potential of Karangsong Cd bacterial isolates on growth treated upon cadmium, Karangsong bacterial Cd 2, Cd3, Cd6, Cd7 isolates were chosen for further analysis based on their ability to grow on the LB agar containing Cd. The sensitivity of selected Cd bacterial isolates to cadmium were assessed. The selected bacteria isolates, Cd2, Cd3, Cd6, and Cd7, exhibited the 1.27 x 10⁹. ,1.62 x 10⁹,1. 45 x 10⁹,1.55 x 10⁹, respectively. Resistant efficiency of the four selected isolates was higher in Cd3 and Cd 7 (Table. 3).

In this study, some of the isolates of Karangsong Port were found to remove the cadmium from seawater containing cadmium (Fig. 4). It was reported that the bonding of heavy metals in a solution occurs with the exchange process of the ions on the cell wall where they are replaced by heavy metal ions (Martins et al., 2006). Presumably, several factors play an essential role in the reduction and absorption processes of heavy metals, comprising phenotype factors, biomass factors, and medium factors.

The isolation of novel isolates from Karangsong Port showed 77% and 97% identity values observed by using BLAST search. Two bacterial species were confirmed identical of which homology level shows at least 97% (Stackebrandt & Goebel, 1994). The selected two isolates were identified by 16S rDNA gene sequencing. Chosen isolates were identified closely related to Pseudoalteromonas issachenkonii strain KMM 3549 and Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Fig. 5). The bacteria species of Pseudoalteromonas issachenkonii strain KMM 3549 is classified as part of the genus Pseudoalteromonas bacteria, the order Alteromonadales, the Gammaproteobacteria class, and the proteobacteria phylum. This species has both heterotrophic and aerobic properties (Gauthier et al., 1995), and it is one of the proteobacteria groups that are abundant and widely distributed in the marine environment. This species grows at temperatures ranging 4 − 37^oC with pH level of 6.0 − 10.0. The optimum growth is at the temperatures between 28 − 30^oC with an optimum pH of 7.5 − 8.0. Species cannot grow at temperatures over 40^oC. This species is a gram-negative bacterium, spindle-transparent colony form, with rod-shaped cells, non-endospore, 0.7 − 0.9 µm in diameter, and 1 − 1.2 µm in length. The species Pseudoalteromonas issachenkonii strain KMM 2549 (Accession Number NR 025139.1) was first isolated from the brown algal thallus (Fucus evanescens) collected in Kraternaya Bay, Kurile Islands in the Pacific Ocean (Ivanova et al 2002). Based on the isolation results obtained during the previous study (Salafy 2018), the Cd 3 bacterial isolate has morphological characteristics in the form of bacilli (rods), with the colony in the form of spindles, transparent white in color, and possesses gram-negative properties. These morphological characteristics are in accordance with the characteristics of Pseudoalteromonas issachenkonii strain KMM 2549 (Accession Number NR 025139.1).

The bacterial species Pseudoalteromonas tetraodonis GFC strain IAM 14160 belongs to the genus Pseudoalteromonas bacteria, the order Alteromonadales, the Gammaproteobacteria class, and the proteobacteria phylum. This species grows at temperatures ranging from 4 − 35^oC with pH levels 5.5 − 9.5. The optimum growth is at a temperature between 25 − 30^oC supported by an optimum pH of 7.5 − 8.0. Species cannot grow at temperatures over 40^oC. These species are gram-negative, aerobic, rod-shaped, non-endospore, with flagellated cells. It was reported that this bacterium was first isolated from puffer fish mucus (Fugu poecilonotus) to show tetrodoxin prevalent in toxin-carrying species, in association with biota (Ivanova et al., 2001). Pseudoalteromonas was classified as Alteromonas tetraodonia in 1990, but was reclassified in 2001 to the genus Pseudoalteromona. Based on the isolation results obtained during the previous study (Hall, 2018), the Cd 7 bacterial isolate has rod-shaped morphological characteristics with gram-negative properties, and exactly these morphological characteristics are in accordance with the characteristics of Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Accession Number NR 041787.1).

The bacteria were successfully isolated and coded Cd 3 and Cd 7 from Karangsong port sediments. To the best of our knowledge, this is the first report on isolation of isolates from Karangsong Port sediments and identification of their associated bacteria species. Cadmium reducing mechanisms in bacteria was separated into two phase such as binding phase and active transport. The binding phase depends on the cell metabolism, where absorption occurs through the cell wall or external surface, followed by an active transport, that is depending on the cell metabolism. In metabolic processes, heavy metals can accumulate in cell membranes (extracellular) and within the cytoplasm (intracellular) (Aminah & Nur, 2018). Our results showed two novel isolates from Karangsong Port sediments implies the potential basis of using indigenous Cadmium (Cd) reducing bacteria for bioremediation application (Table. 2). It should be interesting to inspect the efficiency of bioremediation agent by using these novel indigenized bacteria for removing Cd toxic pollution in contaminated waters from different polluted areas.

Two novel bacteria were isolated from from Karangsong port sediment that showed reduced the cadmium (Cd). Cd 3 and Cd 7 is closely related to Pseudoalteromonas issachenkonii strain KMM 3549 (Acc. No. NR 025139.1) and Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Acc. NR 041787.1), respectively. These results suggested that novel bacterial isolates could facilitate the necessary approaches to be applied to aquatic ecosystems that are known to be heavily polluted by metal cadmium (Cd).

Acknowledgements - This research was supported by Universitas Padjadjaran and Directorate General of Higher Education, the Ministry of Education and Culture, Indonesia with internal grant contract No.603/UN6.M/LT/2018.

Funding - All sources of funding for the research supported by Universitas Padjadjaran and Directorate General of Higher Education, the Ministry of Education and Culture, Indonesia with internal grant contract No.603/UN6.M/LT/2018.

Conflicts of interests - The authors whose names are listed above and involved in this study certify that they have NO affiliations with or involvement in any organization or entity with any financial interest and non-financial interest in the subject matter or materials discussed in this manuscript.

Availability of Data and Materials - The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Code Availability – not applicable

Author Contribution - All authors contributed on this paper. Conceptualization was performed by Yudi N Ihsan and Jiang Mingguo. Supervision by Yudi N Ihsan, Jiang Mingguo and Tri Dewi K Pribadi. Methodology was performed by Yudi N Ihsan, Jiang Mingguo and Azimah Ismail. Project administration and writing – draft preparation by Kalysta Fellatami. Writing – review and editing was by Tri Dewi K Pribadi, Azimah Ismail, and Buntora Pasaribu.

Ethics Approval – not applicable

Consent to participate – not applicable

Consent for publications – not applicable

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Table 1. Colony morphology of isolated bacteria

Isolate Codes	Colony Features
Isolate Codes	Size	Elevation	Shape	Color
A1	Large	Plateau	Irregular	Yellowish white
A2	Moderate	Flat	Round	Yellowish white
A3	Small	Flat	Spindle	Transparent white
A4	Moderate	Flat	Round	Transparent white
A5	Small	Convex	Spindle	Yellowish white

Table 2. Optical Density (OD) Value at t = 0 and t – 18

	Bacteria Code	Optical Density (OD)	Bacterial Density
Initial	Control	0	0
	Cd2	0.0773	6.18 x 10⁷
	Cd 3	0.0915	7.32 x 10⁷
	Cd 6	0.0905	7,24 x 10⁷
	Cd 7	0.0725	5.8 x 10⁷
End	Control	0	0
	Cd 2	1.5863	1,27 x 10⁹
	Cd 3	2.02253	1.62 x 10⁹
	Cd 6	1.8187	1,45 x 10⁹
	Cd 7	1.94043	1.55 x 10⁹
End-Initial	Control	0	0
	Cd 2	1.509	1,21 x 10⁹
	Cd 3	1.931	1.54 x 10⁹
	Cd 6	1.728	1,38 x 10⁹
	Cd 7	1.868	1.49 x 10⁹

No competing interests reported.

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Isolation and Identification of Cadmium-Reducing Bacteria from Contaminated Coastal Sediment in The Northern Coast of Indramayu, Indonesia

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Abstract

Figures

Highlights

1. Introduction

2. Materials And Methods

2.1 Sediment collection

2.2 Cadmium Concentration Reduction assay (Cd).

2.3. Bacterial Isolation

2.4 Molecular Identification

2.5 Phylogenetic Analysis

2.6 Testing of Cadmium-resistant Isolates

3. Results

3.1 Analysis of Cadmium-Reduction Bacteria

3.2 Morphology and Molecular Identification of Bacterial Strains

3.3 Phylogenetic analysis

3.4 Test of Cadmium-reduction on selected bacterial isolates

4. Discussion

5. Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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