Research

Researchers at NU work on projects of societal relevance that attract funding by government agencies and research bodies. A few of the sponsored projects our researchers are working on is given below:

Project Title: Genetic Diversity of Organophosphorus Hydrolases and in situ Bioremediation of Chlorpyrifos contaminated sites

Funding Agency: Department of Biotechnology

Duration: 2009 - 2012

Objectives:

1. Development of DNA probes to study the diversity of the organophosphate pesticides degrading enzyme system.
2. Development of microbial systems using these probes, capable of degrading organophosphorus pesticide Chlorpyrifos.
3. Bioremediation studies with the most efficient microbial system for the degradation of chlorpyrifos.

Executive Summary

1. Five aerobic consortia (AC, BC, DC, Q1 and Q2) were developed by enrichment from chlorpyrifos contaminated soil. Among these five consortia capable of degrading chlorpyrifos, consortia Q1 and Q2 were the best with degradation efficiency of 72 and 70% respectively. Consortia DC had the degradation ability of 60% while consortia AC and BC showed degradation of 54% and 46% respectively after 21 days, as compared to uninoculated control, which had abiotic degradation of 15%.
2. Eficient isolates from these consortia were identified as Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis, Bacillus cereus, Klebsiella species, Serratia marcescnes and Pseudomonas aeroginosa based on the 16s rDNA profile.
3. CODEHOP probes were designed on the basis of opd, opdA and mpd gene sequences. Genomic DNA of the bacteria was PCR amplified using the OPD probes. Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis gave amplification of ~1.5 Kb whereas Pseudomonas aeruginosa showed 900 bp and 300 bp with oligos A1F and D3R. Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis gave amplification of ~1.2 Kb whereas Pseudomonas aeruginosa showed 700 bp and 200 bp with oligos A3F and D2R.
4. The selected partial opd gene fragments from Pseudomonas fluorescence and Brucella melitensis were cloned into pGEM/T vector and sequencing was done with SP6 and T7 primers. Phylogenetic analysis showed that Pseudomonas fluorescence gene segment showed 81% similarity to opd A gene of Agrobacterium tumefaciens where as Brucella melitensis was 61% similar to the known opd sequences in the database. However, the specific primers based on either opd, opdA or mpd gene did not yield any amplification which indicated that our isolates may harbor novel chlorpyrifos degrading genes. Thus these bacterial isolates were used for In situ bioremediation of chlorpyrifos contaminated soil.
5. Microcosm studies were conducted with chlorpyrifos contaminated soil, inoculated with cultures of consortia DC, Q1 and Q2, defined consortia MPQ (with equivalent proportions of Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis and Pseudomonas aeruginosa) and Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis and Pseudomonas aeruginosa. Consortia Q2 and MPQ degraded 93 & 98% of chlorpyrifos at 60days while consortia Q1 and DC showed 95 and 89% respectively after 60 days as compared to 28% in uninoculated control. Among the isolates, Pseudomonas aeruginosa and Brucella melitensis showed 99% and 98% degradation while Pseudomonas fluorescence and Bacillus subtilis showed 94 & 96% degradation respectively after 60 days as compared to 26% degradation in uninoculated control.
6. Population was maintained concomitant with chlorpyrifos degradation and there was just a slight decrease from inoculated population of 3.72 x 108, 1.99 x 109, 4.89x 108 and 5.75 x 108 (log value 8.57, 9.3, 8.69 and 8.76) for consortia Q1, Q2, DC and MPQ to 1.35 x 108, 1.18 x 108, 3.63 x 107 and 1.51 x 108 (log value = 8.13, 8.07, 7.56 and 8.18) cfu/gm soil respectively and 3.8 x 108, 3.63 x 108, 3.72 x 108 and 3.55 x 108 (log value = 8.58, 8..56, 8.57, 8.55) cfu/gm soil to 2.45 x 108, 2.45 x 108, 2.39 x 108 and 2.45 x 108 (log value = 8.39, 8.39, 8.38 and 8.39) cfu/gm soil respectively after 60 days for Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis and Pseudomonas aeruginosa respectively. REP-PCR was carried out to ascertain the actual population of the introduced degraders in soil. Antibiotic plating gave a higher estimation of population (64-68%) survival as compared to molecular fingerprinting (34-46%).
7. Metabolite analysis during in situ degradation of chlorpyrifos in plots inoculated with Pseudomonas aeruginosa indicated the formation of TCP (2.5mg/L) after 20 days which disappeared to negligible amounts after 30 days. No other metabolite peak was obtained from any of the other samples.

Selective Hydrogenation of CO2 by Ionic Liquid Supported Ru on TiO2

Funding Agency: Department of Science and Technology

Duration: 2013 - 2016

Objectives:

1. Synthesis of NR2 (R=H and CH3) functionalized imidazolium, ammonium and pyridinium based ionis liquids. Complete characterization of functionalized ionic liquids using NMT and IR.
2. Synthesis of the Ru-nanoparticles by the decomposition of [Ru(COD)COD)] or hydrogen reduction of [Ru(COD)(2-methylallyl)2] (COD = 1,5-cyclooctadiene and COT= 1,3,5-cylooctatriene).
3. Grafting of metal nano particles on TiO2 using functionalized ionic liquids.
4. Complete characterization of the nanocomposites by means of a series of techniques including SEM, FT-IR, XRD, BET etc.

Executive Summary:

1. The reduction of CO2 emission into the atmosphere is an urgent necessity since this gas is as a major factor responsible for the greenhouse effect. However, the levels of CO2 are increasing with increasing in the world’s population and improvement in living standards. Various physical and chemical techniques have been proposed for the fixation of exhausted CO2, such as fixation in carbonates, geological or ocean storage or afforestation. However, these approaches have severe drawbacks in terms of economic factors, safety, efficiency, and reliability of their immediate application. The catalytic transformation of CO2 in other chemicals is an important alternative for CO2 fixation. The hydrogenation of CO2, (methanation reaction), is possible a suitable alternative for the fixation of CO2; since it converts exhausted CO2 into methane (CH4), which can be recycled for use as a fuel or a chemical. However, temperatures in the range of 300–400 ºC are generally applied to this reaction and additional CO2 is generated for producing the energy required to maintain much temperatures. To overcome this drawback, a catalyst capable of affecting the methanation reaction at lower temperatures is required. However, the temperature of the methanation can be reduced down to 160ºC by the use of ruthenium nanoparticles supported in TiO2 prepared by the wet impregnation method. However, the properties of these materials may be improved by controlling the size, shape and distribution of the Ru nanoparticles on the surface. It is also known that Ru (II) compounds can promote the partial hydrogenation of CO2 to formic acid in the presence of functionalized ionic liquid that capture the acid formed and thus shift the equilibrium.

2. The structural organization of ionic liquids (ILs) can be used as “entropic drivers” for spontaneous, well-defined, and extended ordering of nanoscale structures. Indeed, we have used imidazolium ionic liquids as a template, stabilizer, and solvent for the synthesis of a plethora of transition-metal nanoparticles. The transition-metal nanoparticles dispersed in these fluids are stable and active catalysts for some reactions in multiphase conditions. The catalytic properties (activity and selectivity) of these soluble metal nanoparticles indicate that they possess a pronounced surface like (multi-site) rather than single-site-like catalytic properties. In other cases the metal nanoparticles are not stable and tend to aggregate/agglomerate or serve as simple reservoirs for mononuclear catalytically active species. The main goal of this project is to develop supported functionalized ionic liquid ruthenium nanoparticles in TiO2 (nanoparticles and nanotubes) for the hydrogenation of CO2. 3. The strategy is based on the use of ionic liquids containing amino groups that may capture partial CO2 hydrogenated products and thus control the selectivity of this reaction. For this purpose a series of NR2 (R=H and CH3) containing imidazolium, ammonium, pyridenium based ionic liquids will be prepared and used for the preparation of Ru nanoparticles in the presence of TiO2 supports. The Ru nanoparticles will be prepared by the decomposition of [Ru(COD)COD)] or hydrogen reduction of [Ru(COD)(2-methylallyl)2] (COD = 1,5-cyclooctadiene and COT= 1,3,5-cylooctatriene) dissolved in the functionalized ionic liquids. The TiO2 ionic liquid supported metal nanoparticles (MNPs) will be characterized by TEM, SEM. The CO2 hydrogenation will be followed by CG apparatus.

Project Title: Molecular Diversity of expansin like proteins involved in hydrolysis of cellulose

Funding Agency: Department of Science and Technology

Duration: 2013 - 2016

Objectives:

1. Development and In silico validation of molecular probes for expansin like protein coding gene/s
2. Study the diversity of expansin like protein coding gene fragments and profile of the bacterial community
3. Molecular cloning and characterization of the expansin like protein coding gene

Executive Summary:

1.The effect of crystalline cellulose on the bacterial community structure of an enriched culture from semi-desert soil by comparing the 16S rRNA gene libraries constructed from unamended soil (UN) as well as from soil which was amended with crystalline cellulose (CP) showed showed that clones from unamended soil were distributed throughout the Alpha-, Beta-, and Gamma-proteobacteria, the Firmicutes, and the Planctomycetia with the Betaproteobacteria dominating the semi-desert soil with 40% contribution to the overall bacterial diversity followed by the Firmicutes which contributed 22%. The other major contributions were from Alphaproteobacteria (15%) which was followed by the Gammaproteobacteria (8%) and the Planctomycetia (5%).

2. In the crystalline cellulose amended soil, the Flavobacteria dominated the overall bacterial diversity with contribution of 20% closely followed by the Bacteroidetes which contributed 18%. The other major groups which contributed 12% and 9% were the Planctomycetia and the Sphingobacteria respectively.

3. Screening of the bacterial isolates isolated from agricultural soil (22), infected plant leaves (86), stem (12), and rotten tomato (18) with specific primers based on expansin gene of Bacillus sp. along with the degenerate primers after Restriction fragment length polymorphism (RFLP) and sequencing showed 99% and 97% homology with the known sequence of expansin proteins . EX-1 and EX-2 were 80% identical at protein level also.

4. Metagenomic library of 5.78 X 104 clones per μg of DNA with an average size of 3.5 Kb revealed its prokaryotic origin on blast analysis with a complete ORF of 786 bp long cellulase gene (C18). A signal peptide sequence of 28 amino acids from the catalytic domain of C18 showed a high level of similarity with endoglucanase protein sequences from several other species of microorganisms. 3D structure of C18 displayed open active centre groove grafted upon their β-jelly roll fold.

5. To address the problem of poor activity of C18 towards filter paper, role of expansin was investigated. PCR based screening for expansin gene showed that among the 138 bacterial isolates 99 bacteria gave positive result. Based on sequence and phylogenetic analysis, four different variants of expansin were identified which showed 95-99% similarity to other known expansin protein sequences. These four bacteria (E11CB, M18, T11 and L35) were identified as Bacillus sp. based on 16S rRNA analysis. CODEHOP approach based primers successfully retrieved the diverse expansin gene fragments from other Bacillus spp. which showed negative result with YoaJ (EXLX1) specific primers.

6. Based on critical residues identified in both expansin and cellulase proteins, a 3D synergy model is proposed to hypothesize the individual as well as collective action of these two proteins on cellulose microfibril. EXLX1 structure (Protein Data Bank code 3D30 and 2jeN) reveals that the side chains of W-125, W-126, and Y-157 are slightly right handedly twisted, suggesting that D2 binds to the slightly twisted region of a glucan chain that bridges two cellulose microfibrils rather than a flat, highly crystalline cellulose surface. These disordered glucans provides strength to the filter paper, a synthetic network of cellulose fibers that is weakened by the action of expansin. These D2 residues facilitate expansin binding to the surface layer thereby loosening it and then making it available for interaction with residues of active site of cellulase and subsequently its hydrolysis. Computational modeling may help in understanding the effect of modifications of critical amino acid residues on the overall structure of these proteins and thus predict the interaction between these macromolecules.

Project Title: Metagenomic Approach For The Degradation Of Polyaromatics Hydrocarbons - Pyrene

Funding Agency: Department of Biotechnology

Duration: 2013 - 2016

Objectives:

1. Study of microbial communities of polyaromatic hydrocarbons [pyrene] contaminated sites by culture-independent and culture-dependent methods.
2. Metagenomic library construction and screening for pyrene degradative clones.
3. Identification and sequencing of regulator genes involved in pyrene degradation.

Executive Summary:

1.Most efficient cultureable pyrene degraders degraded 65-95% in 30 days at 50 ppm and were classified as Kocuria sp, Rhodococcus sp , Pseudomonas sp and Bacillus sp.

2.Most of the clone families obtained through metagenomic approach belong to Actinobacteria (31%) followed by 30% of Gamma proteobacteria while Acidobacteria comprised 12% of the clone families followed by 9% of Alphaproteobacteria. Firmicutes (4%) and Epsilonbacteria (1%) were among the other groups detected in the coal tar contaminated soil.

3.16S rDNA sequence of bacterial isolates obtained through cultureable method showed the dominance of Bacillus sp, Kocuria sp, Rhodococcus sp and Pseudomonas sp which are in agreement with the dominant clone families ie Actinobacteria, Gamma proteobacteria and Firmicutes, respectively obtained through metagenomic approach.

4.Analysis of the bacterial ring hydroxylating dioxygenase alpha subunit showed a signature as C-x-H-X17 –CX which is shared with other terminal dioxygenases, the overall identity of these sequences with those of other dioxygenases was moderate (30-50%).

5. β-subunit of dioxygenase showed 17 unique sequences which were highly conserved.

6. The study indicates that further sequencing of 16S rDNA clone library may yield new bacterial families as the percentage coverage for the 16S rDNA library was only 45%. However, species accumulation curves of Rf1 and β subunit clone libraries achieved saturation of sampling, the percent coverage of Rf1 and β subunit clone libraries being 77.77% and 77.63% respectively.

Ecotoxicological Assessment Of Degraded Residues Of Commercialized Tio2 Based Nanomaterials On Bacteria

Funding Agency: Department of Biotechnology

Duration: 2013 - 2016

Objectives:

1.To study the interaction of TiO2-NP and RDNs with bacteria and effect of environmental factors like pH, osmotic pressure, salinity and cation valence on this interaction.

2. To evaluate the toxic effects of TiO2-NP and RDNs on bacteria. Toxicity will be assessed on individual level like bacterial growth inhibition, membrane alteration, viability assay as well as on the community level like bacterial population shift after exposure.

Executive Summary:

The toxicity of titanium dioxide nanoparticles (TiO2) was evaluated against bacteria. Two different forms of nanoparticles were selected, P25 size ~21nm and nanocomposite Eusolex T-2000 purchased from Merck, used extensively in sunscreen. Two different bacteria were selected one was E.coli and others was Pseudomonas. Results indicate that both types of nanoparticles were toxic towards E.coli but Pseudomonas was resistant. The reason behind this variation was Pseudomonas is an environmental stain which produces exopolysacharide (EPS) in the medium which act as a physical barrier between nanoparticle and bacteria and result into less toxicity. This indicates that biological properties of bacteria also affect the nanoparticles toxicity. Bacteria release different polysaccharides from their surface like siderophore in the case of Pseudomonas which decrease the interaction between nanoparticles and bacteria and results into less toxicity. Therefore, in the environment EPS producing strains will be more resistant towards nanoparticles. All these data provide fundamental knowledge essential to a better assessment of nanoparticle toxicity. Similarly, the surface properties of bacteria also change with the growth of bacteria which ultimately affects the toxicity. Toxicity of nanoparticles not only depends upon the physiochemical properties of nanoparticles but also on the surface properties of bacteria.

Project Title: Modeling the snow properties for their classification and identification

Funding Agency: Department of Science and Technology

Duration: 2016 - 2019

Objectives:

1. Modeling the snow and ice properties for snow and glacier classification and identification
2. Up scaling of ground data to aerial and space borne Hyperspectral data

Executive Summary:

This project has been accepted in “Big Data Analytics – HSRS Data scheme (BDA-HSRS)”, a cluster based multi-institutional networked project consisting of 37 R&D projects from premiere institutions, 4 Region-wise Central Laboratory Facility, HSRS Group Trainings, Data Acquisition (through Hyperspectral Airborne Sensor with collaboration of NASA, JPL and ISRO), Data Portal and Project Coordination Cell. The main objective of his project is to develop classification algorithm for snow and glacier Identification based on as Reflectance / Albedo, Snow grain size, Moisture content in snow, Snow mixed objects study and Snow surface temperature. The success of Hyperspectral remote sensing mainly depends on the understanding of spectroscopy of various targets of interest in the reflective (0.4-2.5) and emissive domain (3-14), therefore the second objective is to to identify and classify different snow/ice classes to model them in both domains (i.e. reflective and emissive).