Detection, optimization of nitrate reductase enzyme and its application in wastewater treatment
Marwa Moustafa Hassan Eltarahony;
Abstract
The main objective of this study was to employ local microorganisms to do douple function; nitrate removing from wastewater and biosynthesis of nanomaterials. To achieve this target, four strategies were following:
1. Screening, selection and characterization for NR- producing bacteria:
Screening for NR-producing bacteria was carried out. Forty three isolates were obtained from environmental samples collected from various Egyptian ecosystems with diverse habitats. Subsequently, isolates were subjected for NR assay. While, some of them recorded as NR producersothers were denitrifiers. To distinguish among them and to select the most powerful NR-producing strain, the following were performed:
1.1. To reduce the number of isolates 16S rDNA- RFLP was employed.Twenty one RFLPclusters reflecting twenty five distinct bacterial isolates were obtained.
1.2. Among the 25 isolates, the most powerful NR-producing strain was selectedon SM medium containing inverted Durham tube. All of them were able to reduce nitrate, while 11 of them were scored as putative/true denitrifiers (D) without NO2- accumulation. Isolate S5 that exhibited the highest NR activity within 12h of incubation, remove nitrate within 48h and eliminatingNO2-within 90h was selected and subjected for the following characterization scheme:
1.2.1. Strain S5designatedas MMT was identified molecularly by 16S rDNA sequence (~727 bp).NCBI/GenBank homology search revealed about 99% sequence similarity with all species of the genus Achromobacter. The DNA sequence of MMT was submitted to the GenBankwithaccession number KT735046.
1.2.2. Gram staining, physiological assays and SEM & TEM analysis of MMT cells demonstrated that MMT cells are facultative anaerobic, Gram negative non-motile rods with 0.6 μm length and 0.27 μm width.
1.2.3. MMTshowed various biochemical activitiesable to grow over a wide temperature and pH ranges with optimum at 20-30OC and 6.0–7.0, respectively.MMTgrows well upto 3% of NaCl.
1.2.4. The anti-biograme of the MMTtowards several classes of antibiotics was performed. In general, MMT was sensitive to the class of antibiotics that inhibit nucleic acid synthesis and aminoglycoside group that inhibit protein synthesis.
1.2.5. About 500 bp and 600 bp narG and napA genes were detected using Nested-PCR.Both genes were cloned, sequenced and aligned with NCBI sequence databases under accession numbers KT877347 (napA) and KT884546 (narG).
1.2.6. The crud NR complex was resolved as single bands on native PAGE gels when stained with reduced benzyle viologen. Zymograme confirms the expression of both NAR and NAP under anaerobic and aerobic conditions, respectively.
1. Screening, selection and characterization for NR- producing bacteria:
Screening for NR-producing bacteria was carried out. Forty three isolates were obtained from environmental samples collected from various Egyptian ecosystems with diverse habitats. Subsequently, isolates were subjected for NR assay. While, some of them recorded as NR producersothers were denitrifiers. To distinguish among them and to select the most powerful NR-producing strain, the following were performed:
1.1. To reduce the number of isolates 16S rDNA- RFLP was employed.Twenty one RFLPclusters reflecting twenty five distinct bacterial isolates were obtained.
1.2. Among the 25 isolates, the most powerful NR-producing strain was selectedon SM medium containing inverted Durham tube. All of them were able to reduce nitrate, while 11 of them were scored as putative/true denitrifiers (D) without NO2- accumulation. Isolate S5 that exhibited the highest NR activity within 12h of incubation, remove nitrate within 48h and eliminatingNO2-within 90h was selected and subjected for the following characterization scheme:
1.2.1. Strain S5designatedas MMT was identified molecularly by 16S rDNA sequence (~727 bp).NCBI/GenBank homology search revealed about 99% sequence similarity with all species of the genus Achromobacter. The DNA sequence of MMT was submitted to the GenBankwithaccession number KT735046.
1.2.2. Gram staining, physiological assays and SEM & TEM analysis of MMT cells demonstrated that MMT cells are facultative anaerobic, Gram negative non-motile rods with 0.6 μm length and 0.27 μm width.
1.2.3. MMTshowed various biochemical activitiesable to grow over a wide temperature and pH ranges with optimum at 20-30OC and 6.0–7.0, respectively.MMTgrows well upto 3% of NaCl.
1.2.4. The anti-biograme of the MMTtowards several classes of antibiotics was performed. In general, MMT was sensitive to the class of antibiotics that inhibit nucleic acid synthesis and aminoglycoside group that inhibit protein synthesis.
1.2.5. About 500 bp and 600 bp narG and napA genes were detected using Nested-PCR.Both genes were cloned, sequenced and aligned with NCBI sequence databases under accession numbers KT877347 (napA) and KT884546 (narG).
1.2.6. The crud NR complex was resolved as single bands on native PAGE gels when stained with reduced benzyle viologen. Zymograme confirms the expression of both NAR and NAP under anaerobic and aerobic conditions, respectively.
Other data
| Title | Detection, optimization of nitrate reductase enzyme and its application in wastewater treatment | Other Titles | " رصد و تحسين انزيم النيترات ريدكتاز وتطبيقه فى معالجه المياه الملوثه" | Authors | Marwa Moustafa Hassan Eltarahony | Issue Date | 2016 |
Attached Files
| File | Size | Format | |
|---|---|---|---|
| G12327.pdf | 312.48 kB | Adobe PDF | View/Open |
Similar Items from Core Recommender Database
Items in Ain Shams Scholar are protected by copyright, with all rights reserved, unless otherwise indicated.