Genetic Variability Screening of the Leptolyngbya Boryana with Expressed ChrR Gene for the Biotransformation of Cr (VI) to Cr (III) Reduction

Chromium is well known pollutant for its mutagenicity, and carcinogenicity in humans. Excessive uses of chromium in leather tanning industries, stainless-steel production, and wood preservatives have resulted as chromium contamination in soil and water. This investigation indicates the effective use of Leptolyngbya boryana as an eco-friendly option to overcome Chromium (VI) toxicity in tannery euents. The main objective of this research was to nd out ChrR gene and its variability in the context of Cr (VI) stress. This is a novel study in the relation of Leptolyngbya boryana. Industrial polluted soil samples were collected and processed according to the standard protocols for ChrR variation and 16S rRNA gene. DNA was isolated and amplied through PCR. Amplied DNA was sequenced and aligned with the known sequences. In this study a strong co-relation was established in the nucleotide sequences of ChrR and 16S rRNA genes. MIC was determined for Cr (VI) and pure strains of Leptolyngbya boryana were identied and isolated from soil. In the present study presence of ChrR gene variability was recorded in Leptolyngbya boryana which is a cyanobacterium in the soil of tannery euent under Cr (VI) stressed condition and its gene variability was conrmed by sequencing. We can conclude that Leptolyngbya boryana strain could be eco-friendly option to overcome Chromium (VI) toxicity in tannery euents.


Introduction
Wastes generated from the industries have created big crisis and it is big challenge to convert them in into the eco-friendly compounds by sustained methods. Industrial wastes (e uents) contain various toxic metals, harmful volatile compounds, along with several organic and inorganic compounds. There is a continuous demand of development of new strategies and novel sustained methods to overcome industrial waste management due to increasing urbanization (Evelyne et al., 2014). Long-term exposure of industrial e uents in the atmosphere can cause infectious diseases, neurological disorders, cancer, etc (Megharaj et al., 2003). The liberation of these toxic compounds, there is big losses in all the domains of society (Deepali, 2011).
In the industries e uents many toxic pollutants have been noticed e.g. chromium, sul des, phenolic compounds, magnesium, sodium, potassium etc. One of the important toxic compounds known as chromium is also a necessary micronutrient for the development of many microorganisms (Thacker et al., 2005). But higher amount of chromium is highly toxic in all the environment e.g. air, water and soil. In nature, soil may retain Cr concentration ranges from 10 to 50 mg/kg (Pechova et al., 2007). In a survey, Indian tannery industries alone about 2000-3000 tons of chromium emits into the environment with high chromium concentrations from 2000 and 5000 mg/l. Through, it was a safe recommended permissible discharge limits is 2 mg/l (Belay, 2010).
In the nature, some microbes (especially blue green algae) have been reported for biological reduction (Biotransformation) of Cr (VI) into Cr (III). In the several studies it has been tried to identify chromium reductant microorganisms. Some fungi were also studied for their chromium (VI) reducing capacity (Deepali, 2011;Jayalakshmi et al., 2013). The ability of microbes to survive under Cr (VI) metal exposure and perform detoxi cation mechanism into Cr (III) is being trying to rectify globally. Every microbe has its own speci c metal tolerance ability (capacity) under environmental conditions. In the literature, some mechanisms of Cr (VI) reducing strategies have been mentioned e.g. exclusion by permeability barrier, active transport e ux pumps, intra and extra cellular appropriation, enzymatic methods etc (Bruins et al., 2000). One of the bacteria which have been convert toxic Cr (VI) to nontoxic Cr (III) had been mentioned earlier (Jayalakshmi et al., 2013;Qian 2013]. Similarly, one of the fungus which has performed Cr (VI) bioabsorptive property has been explored. During biosorption Cr (VI) gets bound to the functional groups present on the surface of microbes and gets percolated inside (Noorjahan et al., 2014).
In nature vast varieties of cyanobacteria grows on soil surfaces and morphologically phylogenetically might be different (Joo et al., 2007). Well known microbe Leptolyngbya, which is a lamentous form of cyanobacterium and have characterized by the thin width of their cylindrical trichomes. Leptolyngbya have been isolated from various industrial e uents in the soil. Leptolyngbya boryana species is phylogenetically connected to Leptolyngbya sp (Anagnostidis et al., 2014).
In the present study, research was performed to search novel Cr (VI) reductant bacteria of microbes in tannery e uent soil. The nding of this research may be more suitable, effective, eco-friendly, sustainable and cost-effective biological treatment of leather industry wastewater. In the many studies Leptolyngbya boryana has been exposed in the nitrogen xation and other related genomic analysis (VI) to nontoxic Cr (III) is not mentioned anywhere. So, in the study we have focused on its potential roles for the reduction of Cr (VI) to Cr (III).

Chemicals
All the chemicals and reagents were of analytical grade and procured from Merck (USA), Himedia (Mumbai, India), and Qiagen (Germany). The stock solution (1000 mg L −1 ) of Cr (VI) was prepared utilizing K 2 Cr 2 O 7 in the deionised water.

Collection of samples
Industrial e uent samples were collected from the nearby industries of Kanpur. Initially three cyanobacterial species had considered, but after the phylogenetically and morphological screening only Leptolyngbya boryana has included for the study. Out of 100 Samples from different places, same strains of Leptolyngbya boryana were found from 10 places used further in this study. Pathogenic and antibiotic treated Leptolyngbya boryana strains were excluded in this study.

Cyanobacteria and culture conditions
The screened Leptolyngbya boryana strains were grown in the Erlenmeyer asks containing liquid BG-II media with growth conditions: 16:8 light: dark cycle; 30 ± 2°C and the 6 irradiances of 3000-4000 lux (cool white light). Further, the isolates were routinely cultured and maintained via sub-culturing under its metabolically active state after every 20 days as mentioned previously (Yadav et al., 2021).

Morphological and Biochemical analysis
The morphology (colony morphology and colour identi cation) of isolated Leptolyngbya boryana strains were performed as described previously (Yadav et al., 2021). The biochemical estimation of Gram's stain, Catalase activity, Sucrose utility and Indole test were also performed in all strains as described (Sundari et al., 2013).

PCR ampli cation of chromium-responsive genes:
For the genomic study, total genomic DNA was isolated from isolated cells of L. boryana by using Qiagen DNA isolation kit (Germany). For PCR, reaction mixture (20 µl volume) contains 10µl master mix (Takara), 1 µl forward and reverse primer each and 30µg DNA template and nuclease free water. The PCR was performed in BIORAD T100 Thermal Cycler using the following conditions initial denaturation at 95°C for 5 min, 35 cycle consisting of 94°C for 30 s, 48°C for 30s, 72°C for 1 min and nal extension 72°C for 7 min. Ampli ed product were visualized by agarose gel electrophoresis (1% agarose in 1X TAE buffer). PCR primers for16S rRNA and ChrR gene were listed in Table 1. After the band puri cation it was submitted for the gene sequencing and following nucleotide sequences were obtained. The exact band size was found 340bp.

Statistical Analysis
For the assessment of MIC and different variables in the study SPSS software version 22 (USA) is used (Corp, 2011). Data represented as mean ±SD after three independent experiments.

Selection and identi cation of the cyanobacteria based on Morphological and biochemical analysis
The genus Leptolyngbya is a simple lamentous cyanobacterial genus with slight morphological variations between species. For the characterization of Cr reductase enzyme present in bacterium L. boryana, the optimum pH was recorded 7 and optimum temperature was found 37°C under 200 rpm in a water bath. Same bacterial strain were obtained from 10 places and subjected to optimized level of tolerance 800 mg/L of K 2 Cr 2 O 7 while other isolates were not grown well above 900 mg/L Cr (VI) concentration (Fig. 1).

Genetic screening of selected strain
The genomic and plasmid DNA were obtained from the morphologically and biochemically con rmed strain L. boryana. In this study bacterial genotypical con rmation was performed by gene speci c (16S rRNA). The obtained 16S rRNA sequence of L. boryana was homology compared with the sequences available in NCBI database. Sequence's homology has showed 98% of identity with known sequences (Fig. 3).
Further, ChrR gene was isolated and ampli ed from L. boryana. During the study ve isolates were con rmed to have ChrR gene (Fig. 2a-c). Four isolates tolerant of Cr (VI) were found in a concentration of 600 mg/L and one ChrR gene was detected at the 800 mg/L concentration of Cr (VI).
The band size of ChrR gene was found around 340bp (con rmed by using DNA ladder) as shown in the (Fig. 4). The sequenced ChrR gene was aligned with known sequences (Fig. 5; Fig. 6). There are limited genetic studies have been mentioned in the relation of Cr (VI) reduction by analyzing 16S rRNA and ChrR genes in the microbes. In this study both 16S rRNA and ChrR genes were found out partially and recorded 98% homology identity in 16S rRNA gene and 98% homology identity with ChrR gene at the Cr (VI) stress state in the L. boryana (Fig. 3; Fig. 6). Similar study had been performed by (Baldiris et al., 2018) which shown the presence of ChrR gene in the cyanobacteria and had worked in the reduction of Cr (VI). In another study which has been performed with S. maltophilia had demonstrated crucial property for binding of metals like Hg, Co, Zn and Cd in tannery e uents (Rocco et al., 2009). The 16S rRNA (Fig. 2c) and ChrR partial genes sequenced homology has obtained by compared with available sequences in NCBI databases. Similar nding has been obtained by (Rathnayake et al., 2013) for the Cr (VI) bio-transformation in Phormidesmis molle with the presence of 16S rRNA and ChrR genes. The study had been conducted by (Sundar et al., 2010) also support the presence of 16S rRNA and ChrR genes with 99 % homology in Bacillus Cereus strain for Cr (VI) reduction.

Discussion
In the literature, partial (268 bp) chromate reductase gene had been identi ed in three Gram positive bacterial isolates from soil of Cr contaminated tannery e uents. In this study we have obtained 340 bp (Fig. 5) partial gene sequences in L. boryana. Similarly, (Deng et al., 2015) have been obtained 321 bp (partial) Cr reductase gene in the Gram positive bacteria. This con rms the presence of chromium reductase gene in the DNA sequences of these two bacteria rea rming their chromium reducing property. Thus, our ndings con rm the presence of the chromate reductase gene in L. boryana and strengthen the ability to reduce Cr (VI) to Cr (III) (Tang et al., 2000). This study also has strong co-relation with the nding of (Deshpande et al., 2005) in the relation of reduction of Cr (VI) to Cr (III).
In the Cr (VI) resistant bacteria, Cr reductases gene (ChrR) catalyze the reduction of Cr (VI) to Cr (III) with the transfer of electrons from electron donor NADPH to Cr (VI) and resulting the production of reactive oxygen species (ROS) in reactions (Thatoi et al., 2014). Cr reductase gene belongs to the chromate ion transport (ChrR) super-family and has been acknowledged vastly in Archaea, Bacteria and Eukarya (Pimentel et al., 2000). In this study (L. boryana) the ChrR gene was obtained in both plasmid and genomic DNA (Fig. 2a, and Fig. 2b). Similar nding has been mentioned in the literature by some researchers. While some researchers have been mentioned that Cr (VI) resistance gene is deferent from Cr (VI) reduction and location of both genes may be different in the microbes (Juhnke et al., 2002). For getting exact location of these genes there is need of few more studies in the microbes to explore the authentic mechanism Cr (VI) reduction.

Conclusion
To our acquaintance, this may be the rst study to report on 16S rRNA and ChrR genes co-relation with Cr (VI) reduction in L. boryana. The obtained ChrR gene in this study has 98% homology with known sequence of NCBI and genetic variability has been observed in stressed L. boryana. There is a need of identi cation and characterization of the enzymes (ChrR protein) and to nd out location of this gene in the microbes. Study can be enhanced with total genome sequencing methods, comparative genomic approaches to obtain Cr (VI) reductase activities. Additionally, such type of studies is required to know the strength and exact nature of the gene functions to determine the role of Cr (VI) biotransformation in the L. boryana.

Declarations
Ethics approval and consent to participate In this manuscript there was no any ethical clearance required.

Consent for publication
Yes Availability of data and materials Data and materials will be available on demand to authorized person

Competing interests
The authors declare no con ict of interest.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial or notfor-pro t sectors.

Authors' contributions
Ajit Pratap Singh Yadav: Conceived and designed the experiments, Performed the experiments, Analyzed and interpreted the data, Contributed reagents, materials, analysis tools or data, Wrote the paper.
Vinay Dwivedi: Conceived and designed the experiments, Analyzed and interpreted the data, Contributed reagents, materials, analysis tools or data.
Satyendra Kumar: Conceived and designed the experiments, Contributed reagents, materials, analysis tools or data.      boryana.