Open Access Repository of Indian Theses

PAVASKAR, NEELA

CHAPTER 5                                                               SUMMARY AND CONCLUSIONS 
 - 156 -
Twenty four soil samples were collected from different agricultural fields of 
Ibrahimabad, Medak district, Andhra Pradesh, India, cultivating sweet sorghum and 
many other monocot crops. All the fungal isolates were screened for ferulolyl esterases in 
two phases. Phase 1 consisted of a plate assay method and phase 2 as submerged cultures. 
The fungal isolates were screened on a minimal agar medium containing (g l-1): 
(NH4)2SO4 1.5, KH2PO4 0.5, MgSO4 0.25, CaCl2 0.05, FeCl3 0.01, yeast extract 1.0 and 
bacteriological agar 20.0. The sterile medium was supplemented with ethyl ferulate (1%) 
in 5% solution of dimethylformamide (DMF). A total of about 150 fungi were isolated 
from these 24 soil samples and further screened for their ability to produce feruloyl 
esterases 
 
Forty two feruloyl esterase producing fungi were isolated based on this screening 
procedure. The cultural and morphological characterization of these 42 isolates indicated 
that they belonged to 6 different morphotypes after dereplication. Two were only 
identified to the genus level: Aspergillus sp. and Sclerotium sp. The remaining fungi, 
identified up to the species were Aspergillus terreus, Aspergillus fumigatus, Aspergillus 
niger and Lasiodiplodia theobromae. Aspergillus terreus, Lasiodiplodia theobromae and 
Sclerotium sp., are the three new species which were new additions to the list of feruloyl 
esterase-producing fungi.  
 
The HPLC protocol was standardized for quick detection and quantification of 
both ferulic acid (product) and methyl ferulate (substrate) with retention times of 3.9 and 
8.57 minutes, respectively. The feruloyl esterase activity in the culture supernatants of the 
liquid minimal medium was found to range between 0.05 to 6.7 U ml-1. Among these 42 
isolates, the feruloyl esterase activity of Aspergillus terreus was high (6.7 U ml-1), while 
the production ability of Lasiodiplodia theobromae and Sclerotium sp. was very low 
(1.27 and 0.96 U ml-1, respectively). Aspergillus terreus GA2 identified as a promising 
feruloyl esterase producer was selected for further studies on optimization of feruloyl 
esterase production, characterization and application of ferulic acid recovery from maize 
bran as a lost-cost lignocellulosic susbtrate.  
 
CHAPTER 5                                                               SUMMARY AND CONCLUSIONS 
 - 157 -
In order to obtain maximum yields of feruloyl esterase, solid state fermentation 
(SSF) conditions for enzyme production were standardized. Effective feruloyl esterase 
production was observed with maize bran as substrate followed by wheat bran, coconut 
husk and rice husk among the tested agro-waste crop residues. Optimum particle size of 
0.71-0.3 mm and moisture content of 80% favored the enzyme production. Moreover, 
optimum feruloyl esterase production was observed at pH 6.0 and a temperature of 30°C. 
Supplementation of potato starch (0.6%) and casein (1%) as as carbon and nitrogen 
sources, respectively, favored enzyme production. Furthermore, the culture produced the 
enzyme after 7 days of incubation when the C:N ratio was 5. Optimization of the SSF 
conditions revealed that maximum enzyme activity (1162 U gds-1) was observed after 7 
days in a production medium of 80% moisture content and pH 6.0 containing maize bran 
16 g (25%, w/v) of particle size of 0.71-0.3 mm, potato starch 0.6%, casein 3.0% and 64 
ml of formulated basal salt solution [(per liter): (NH4)2SO4 (1.5 g), KH2PO4 (0.5 g), 
MgSO4 (0.25 g), CaCl2 (0.05 g), FeCl3 (0.01 g), Na2HPO4 (1.5 g)]. Overall, the enzyme 
production was enhanced by 3.2 fold as compared to the un-optimized conditions. 
 
Facile and efficient chemical synthesis methodologies were developed to prepare 
the esters of acids and alcohols. The synthesis of these different aliphatic, aromatic 
saturated and unsaturated esters is based on Lewis acid catalyzed esterification 
(Compounds 1-11). The synthesis of aryl ester is based on the coupling of acid and 
phenol by DCC (Compound 12).The synthesis of aliphatic esters is based on the coupling 
of acid and diazomethane (Compound 13). The synthesis of different aryl acetates is 
based on esterification by acetic anhydride (Compounds 15-18).The synthesis of 
monoacetate from meso-diol was done enzymatically using porcine pancreatic lipase 
(PPL) (Compound 19). The compounds were synthesized to characterize and to calculate 
the catalytic efficiencies of the three purified FAEs. Hydrolytic profiling was done on the 
basis of (a) Changes in types and substitution on the benzene ring (substrates 1-11), (b) 
changing of the aliphatic region substitution group on ester bond (substrates 2, 4 and 12), 
(c) position and number of ester bond on benzene ring (substrates 13-18), and (d) 
requirement of benzene ring for ester bond cleavage (substrate 19). 
CHAPTER 5                                                               SUMMARY AND CONCLUSIONS 
 - 158 -
The enzymes from Aspergillus terreus strain GA2 responsible for the feruloyl 
esterase activity were purified to homogeneity using ion-exchange, hydrophobic 
interaction and gel filtration chromatographies, and designated as AtFae-1, AtFae-2 and 
AtFae-3. Biochemical characterization and classification of these enzymes were done. 
The enzymes were monomeric in nature having molecular masses of 74 kDa, 23 kDa and 
36 kDa. Active proteins bands were identified by a developed pH-dependent zymogram 
on native PAGE. The three enzymes also exhibited variation in pH tolerance ranging 
between pH 5-8 and temperature stability of up to 55°C. Inhibition studies revealed that 
the serine residue is essential for the FAE activity; moreover aspartyl and glutamyl 
residues were not totally involved at the active site of the enzymes. Metal ions such as 
Ca2+, K+ and Mg2+ increased and stabilized the enzyme activity for all the three feruloyl 
esterases. Kinetic data indicated that all three enzymes showed good catalytic efficiencies 
(kcat/Km) against different synthesized alkyl and aryl esters indicating their broad substrate 
specificity. The peptide mass fingerprinting by MALDI/TOF-MS analysis and enzyme 
affinity towards methoxy and hydroxy substituents on the benzene ring revealed that the 
AtFae-1 was type A and  AtFae-2, AtFae-3 were type C feruloyl esterases. Moreover our 
studies on the role of different feruloyl esterases on various types of substrates revealed 
that these enzymes not only acted on cinnamic acid esters but also on alkyl and aryl 
esters. This suggests their importance in not only releasing ferulic and/or cinnamic acids 
but also different alkyl/aryl compounds like phenolic acids from plant material. 
The Aspergillus terreus strain GA2 under study produced multiple enzymes can 
thus acted synergistically in releasing ferulic acid from agro-waste substrates. Taguchi L-
9 Orthogonal Array was adopted to study the impact of different parameters for the 
release of ferulic acid from maize bran by treating it with extracellular FAE and other 
hydrolytic enzymes from the crude enzyme extract of Aspergillus terreus GA2. Three 
factors were optimized using Taguchi statistical design, among them temperature 
contributed the maximum impact [45%] on the overall ferulic acid yield followed by the 
enzyme dosage [43%]. pH showed the least impact [8%] at the individual level. When 
these levels were applied in the experiment to release ferulic acid, there was 8% increase 
in the yield which was 13.8 mg gds-1 as compared to the conventional method of 
optimization. 70% of the alkali extractable ferulic acid was obtained on enzyme treatment 
CHAPTER 5                                                               SUMMARY AND CONCLUSIONS 
 - 159 -
and 86% ferulic acid was recovered using powdered activated charcoal. >95% pure 
ferulic acid was obtained after silica gel chromatography which was confirmed by 1H 
NMR and ESI mass spectroscopy. After optimization of the enzymatic hydrolysis 
conditions for recovery of ferulic acid, this method was scaled up and the ferulic acid 
yield obtained was 14.2 g kg-1 of dry maize bran. This indicated that a significant amount 
of ferulic acid was released from maize bran using the crude extract of Aspergillus 
terreus GA2. The recovery and purification method developed was efficient. 
Considering the different salient properties and their catalytic efficiency for 
different substrates, the feruloyl esterases from Aspergillus terreus GA2 can be attractive 
candidates for different biotechnological applications.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 


English

ELECTRICAL AND PHOTOCONDUCTING PROPERTIES OF CHEMICALLY DEPOSITED THIN FILMS OF Cds

PAKNIKAR, UDAY

Chemical Investigation on Teroenes and Related Compounds

NAYAR, M.S. BALACHANDRAN

MASS SPECTROMETRY OF ORGANIC IONS

PATIL, BHASKAR

 ABSTRACT 
  
1,3-Propanediol (1,3-PDO) is the promising bulk chemical which has attracted worldwide 
attention due to its enormous applications in polymers, cosmetics, foods, adhesives, 
lubricants, laminates, solvents, antifreezing agents and medicines. In the present study, 
1,3-PDO production was carried out from glycerol using an isolated Klebsiella 
pneumoniae 141B. Initial experiments were carried out for the enrichment, isolation and 
identification of 1,3-PDO producing microorganism. A total of 40 bacterial isolates were 
isolated and screened for 1,3-PDO production. Among these strains, the strain 141B-APJ-
IICT had shown the maximum 1,3-PDO production at 7.41 and 5.94 g/L under aerobic 
and anaerobic conditions respectively. Hence, the above potent strain was further 
identified based on morphological, physiological, biochemical and phylogenetic studies 
as Klebsiella pneumoniae. The comparative 16S rRNA gene sequence evaluation of the 
isolate 141B-APJ-IICT with literature cited bacterial strains revealed that this isolate 
showed 98% degree of homology with 16S rRNA gene of Klebsiella sp. The EMBL 
accession number of the present strain was found to be FN820293. It was deposited in 
IMTECH, Chandigarh with the accession No. MTCC 9751. In the present study, the 
microbe was referred as K. pneumoniae 141B. The identification of the 1,3-PDO 
produced by K. pneumoniae 141B was carried out initially using HPLC and further 
confirmed by GC-MS (gas chromatography mass spectrometry).  
 Various fermentation strategies such as optimization studies, cofermentation 
studies, whole cell immobilization, strain improvement and scale up studies were adopted 
to maximize the production of 1,3-PDO by the isolated K. pneumoniae 141B. A two step 
methodology was employed for optimization of 1,3-PDO production. In the first stage, 
 the conventional single variables optimization methodology was employed to select the 
suitable fermentation parameters. In the second stage, Taguchi methodology was 
employed to fine tune the levels of the critical parameters those noticed to influence the 
production of 1,3-PDO drastically during optimization studies carried out via single 
variables optimization methodology. 
For any microbial strain, optimum levels of fermentation process parameters such 
as pH, temperature, aeration, carbon and nitrogen sources vary for effective and 
economic productivities. All these parameters have two tire influences at individual and 
interactive levels elicit a cumulative effect on the growth of microorganism as well as 
subsequent metabolically related product production. In the present study, the production 
of 1,3-PDO in shake flask studies was maximized by optimizing the physical parameters 
such as incubation time, temperature, pH, aeration and inoculum level and nutritional 
parameters such as carbon, nitrogen and calcium carbonate concentration using single 
variables optimization methodology. 1,3-PDO production was increased from 7.41 to 
11.50 g/L in shake flask using the optimized conditions such as glycerol-2% (w/v), yeast 
extract-0.75% (w/v), calcium carbonate-0.5% (w/v), pH-6, temperature-37°C, agitation 
speed-150 rpm, medium volume-75 ml, inoculum level-2% (v/v) and incubation time-8 h.  
One interesting phenomenon was observed that 1,3-PDO production was not stimulated 
by vitamin B12 supplementation, which acts as a coenzyme for glycerol dehydratase 
(GDHt). GDHt is the rate limiting enzyme involved in the synthesis of 1,3-PDO. Along 
with 1,3-PDO production, GDHt productions was also not increased in presence of 
vitamin B12. Thus, 1,3-PDO production in  vitamin B12-free medium allows the 
 development of an economical biological fermentation process as vitamin B12 is an 
expensive compound.  
The statistical experimental designs help in obtaining valuable information by 
running few experimental trials with consistency as these are based on fundamental 
principles of statistics such as replication and randomization. Hence, Taguchi 
methodology with L18 orthogonal array was used to improve the production of 1,3-PDO. 
Six critical fermentation factors such as pH, glycerol, yeast extract, calcium carbonate, 
inoculum level and temperature were selected for statistical optimization. The design of 
experimental methodology aided in the identification of most significant parameters that 
effected 1,3-PDO production at interactive and individual levels. The data suggested that 
production of 1,3-PDO was mostly influenced by the variation in concentration of 
glycerol. Approximately a 9.0% improvement was observed in 1,3-PDO yields upon 
optimization of significant parameters using Taguchi methodology.  
The most important step for the development of an economical microbial 
production process for 1,3-PDO was to access other carbon sources than glycerol. 
Particularly, glucose is generally a preferred feedstock to build up the fermentation 
process. An attempt has been made for the production of 1,3-PDO from fermentable 
sugars such as glucose and fructose using K. pneumoniae 141B. Among these two sugars, 
fructose hasd shown the maximum production of 4.94 g/L followed by glucose at 3.47 
g/L with an optimum fermentation time of 9 h. 1,3-PDO yield obtained from fructose and 
glucose was found to be 2-3 folds lower than the yield obtained from  glycerol. Hence, 
further studies were carried out to evaluate the different sugars such as glucose, fructose 
and sucrose as cosubstrates in glycerol fermentation at a molar ratio of 0.1 to 0.5 mol/mol 
 of glycerol. There was a sharp increase in the yield of 1,3-PDO from control (0.70 
mol/mol) having glycerol alone to glycerol-sucrose mixture (0.98 mol/mol) followed by 
glycerol-fructose mixture (0.90 mol/mol) and glycerol-glucose mixture (0.84 mol/mol). 
Among all the cosubstrates studied, glycerol-sucrose mixture had shown the maximum 
production of 1,3-PDO at 16.63 g/L with a fermentation time of 10 h at 0.3 mol/mol of 
sucrose-glycerol ratio.  
In general 1,3-PDO production with raw glycerol increases the profitability of 
overall process as the price of raw glycerol has fallen to about $0.05/lb, primarily because 
of the increased production of biodiesel. Hence, in the present study, 1,3-PDO production 
by K. pneumoniae 141B using raw glycerol as a substrate has been investigated. The 
maximum 1,3-PDO production from raw glycerol was observed to be 8.55 g/L with a 
molar yield of 0.52 mol/mol. To further improve the 1,3-PDO production from raw 
glycerol, Taguchi L18 orthogonal array was adopted to optimize the nutritional (raw 
glycerol, yeast extract and calcium carbonate), physical (incubation temperature and 
medium pH) and microbial (inoculum level) fermentation variables. The data revealed an 
overall yield of 10.2 g/L of 1,3-PDO, which accounted nearly 19% improvement in 
production. The molar yield of 1,3-PDO has increased from 0.52 to 0.62 mol/mol.  
Immobilization offers several potential advantages to the fermentation systems 
over free cell systems from the process engineering standpoint, which include ease of 
handling and cell separation. The rationale for choosing whole cell immobilization is that 
it provides higher yields after immobilization, higher operational stability, greater 
resistance to environmental disturbances and lower effective operational costs. Whole 
cell immobilization of K. pneumoniae 141B was attempted using various entrapment and 
 adsorption matrices. Both the entrapment and adsorption matrices supported 1,3-PDO  
production. The order of 1,3-PDO production under the influence of various entrapment 
matrices was polyacrylamide > k-carrageenan > gelatin > alginate > agar. The order of 
1,3-PDO production by different adsorption matrices was polypropylene scourer > 
absorbent cotton > scrubber pad > polyurethane > polystyrene. Comparative evaluation 
of different supports, suggested that growth of the microorganism should not be restricted 
and free diffusion of gases and nutrients should be facilitated. Of all the supports that 
were screened, polypropylene scourer was found to be the ideal matrix for the whole cell 
immobilization of K. pneumoniae 141B cells. The polypropylene scourer adsorbed cells 
had shown the maximum 1,3-PDO production of 12.3 g/L during the 3
rd
 production cycle 
and they exhibited a consistent production upto 20
th
 cycle (10.71 g/L). Under 
immobilized conditions process parameters such as substrate weight and inoculum level 
were investigated. 1.0 g of polypropylene scourer and 2.0% (v/v) inoculum size were 
found to support the production of 1,3-PDO. Further reactor studies were carried out in 
fluidized bed reactor (FBR) and stirred tank reactor (STR) using polypropylene scourer 
adsorbed K. pneumoniae 141B cells. The FBR was made of glass with 38 cm height and 
5.3 cm diameter, with total volume of 1.0 L capacity. The working volume of the FBR 
was 600 ml. The total volume of STR was 1.0 L with working volume of 600 ml. The 
results from FBR and STR suggested that the polypropylene scourer adsorbed cells of K. 
pneumoniae 141B cells could be effectively used upto > 20 cycles in repeated batch 
fermentation for 1,3-PDO production. However, the yield of 1,3-PDO observed in FBR 
was approximately 30% higher than the yield obtained from the STR. Thus, FBR is the 
most advanced reactor for fermentation products. It has better features when compared to 
 STR, like good mixing, higher oxygen mass transfer rates, easier CO2 removal, easier cell 
regeneration and more uniform cell population. Overall, 1,3-PDO yield exhibited by 
polypropylene scourer adsorbed K. pneumoniae 141B cells was observed to be 7.0% 
higher than that of free suspended cells.  
The industrial potential of 1,3-PDO has stimulated research in the development of 
methods to improve strains, since the production level of 1,3-PDO in naturally occurring 
strains is sometimes low for commercial exploitation. For the development of a 
commercially feasible fermentation process, improvement in yield and overall 
productivity are essential. Keeping this in view, the isolated K. pneumoniae 141B was 
treated with physical mutagens such as UV rays and with different chemical mutagens 
such as sodium bromide (NaBr), ethidium bromide (EtBr) and hydroxylamine (NH2OH) 
to obtain a mutant with enhanced production of 1,3-PDO. The results revealed that, 
among all the UV mutants, UV-10 mutant produced the maximum 1,3-PDO production at 
12.9 g/L, among NaBr mutants, NaBr-4 had shown the maximum production at 13.4 g/L. 
Among EtBr mutants, EtBr-4 yielded a maximum titer of 13.6 g/L, while NH2OH mutant 
(NH2OH-3) recorded 13.9 g/L. The production of 1,3-PDO by these mutants followed the 
order of NH2OH > EtBr > NaBr > UV. The fermentation time of the NH2OH-3 mutant 
had decreased to 7 h, when compared to the 8 h fermentation time of parent strain. K. 
pneumoniae NH2OH-3 mutant strain exhibited 16% improvement in 1,3-PDO production 
compared to the parent strain. Hence, it was deposited in IMTECH Chandigarh with the 
accession No. Klebsiella pneumoniae MTCC 10309. A central
 
composite design (CCD) 
of response surface methodology was applied to determine the optimal conditions for 1,3-
PDO production using K. pneumoniae NH2OH-3 mutant. The data revealed that glycerol 
 was the most important factor affecting the production of 1,3-PDO with a P value of 
1.55E-09. After optimization, 1,3-PDO production by K. pneumoniae NH2OH-3 mutant 
had increased to 25%, when compared to the parent strain. 
Scale up research reveals key parameters, controlling steps and indicates process 
routes and constraints in the light of process economics. Hence, scale up studies for the 
production of 1,3-PDO were carried out using 1.0 L STR by K. pneumoniae 141B. the 
results showed that, 1,3-PDO production was increased in STR, when compared to 1,3-
PDO production in shake flask, which corresponds to 5.0%, 10%, 9.0%, 7.0% and 3.0% 
enhancement at 20, 40, 60, 80 and 100 g/L glycerol concentrations respectively. The 
reason for increased production in STR might be due to the increase in vessel volume and 
also supply of external aeration, which helps in uptake of nutrients freely by cells. The 
optimum process parameters for 1,3-PDO production in STR were observed to be 
glycerol-40 g/L, inoculum level-10% (v/v), agitation-200 rpm, aeration-1.0 vvm, 
temperature-37°C and yeast extract-7.5 g/L. Further, 1,3-PDO production was carried out 
by K. pneumoniae NH2OH-3 mutant strain in STR at the above optimized conditions. 
The production of 1,3-PDO by mutant strain was improved from 23.8 g/L to 28.2 g/L 
from shake flask to STR respectively at 40 g/L glycerol content. Overall, the mutant 
strain had shown approximately 16% improvement in 1,3-PDO production compared to 
the parent strain in STR.  
For economical biological production of 1,3-PDO, an efficient and energy saving 
strategy for product separation and purification is essential. Since the product shall be 
used mainly in polymer chemistry, the grade of purification has to be from 95% up to 
over 99%, depending on the type of impurities and the demanded product properties. In 
 the present study, the separation of 1,3-PDO from fermentation broth was achieved by 
four subsequent steps such as removal of biomass, removal of proteins, concentration of 
broth and fractional distillation of the concentrated broth. Biomass was removed from the 
fermentation broth by centrifugation. The proteins, colored impurities and residual salts 
of medium were removed by isopropyl alcohol and charcoal treatment. The charcoal 
treated broth was further concentrated in rota vapor and the concentrated clarified broth 
was finally separated using the fractional distillation method. The overall yield of 1,3-
PDO recovery was calculated to be 60.65% with 95.21% of purity.  
The results of this work indicated that the present isolated strain i.e. K. 
pneumoniae 141B was a potent strain, which posses the potential to produce 1,3-PDO. 
The investigations suggest that the production of 1,3-PDO by sucrose-glycerol 
cofermentation, strain improvement and whole cell immobilization strategies were more 
beneficial as they supported high titers of 1,3-PDO production at 0.98, 0.84 and 0.78 
mol/mol of glycerol respectively, from 20 g/L glycerol concentration.  
 
  
 
 
 
 
  


PROPERTIES AND FUNCTIONS OF ESTERASES (Toxic organophosphorus esterase Inhibitors and their effect on the N and K content of mouse liver tissue)

PATIL, VISHVANATH

V 
 
 
Abstract: 
The Alphaproteobacteria represent a biologically diverse group of bacteria 
with members like Brucella, Bartonella, Agrobacterium and Ochrobactrum that 
are capable of interacting with eukaryotic cells. The genus Ochrobactrum  is a 
Gram-negative, capsulating, aerobic bacilli having closest phylogenticgenetic 
relative ogenus Brucella as evidenced by protein profiling, western blot, 
immunoelectrophoresis, amplified fragment length polymorphism, 16S rRNA 
gene and RecA gene sequence based studies. In earlier study, several 
bacteria other than Helicobacter pylori have been detected earlier in gastric 
biopsies in individuls diagnosed with non-ulcer dyspepsia. A unique 
observation was the presence of severe fibrosis in the lamina propria of the 
gastric mucosa revealed during histological examination of the gastric antral 
biopsy. Whether this fibrosis was caused either partially or totally by 
Ochrobactrum was not clear. O. anthropi have also been associated along 
with H. pylori with mild gastritis in squirrel monkeys. Importantly, both H. pylori 
and Ochrobactrum produce urease, and thus the detection of H. pylori by 
urease test in the presence of Ochrobactrum may be confounded. The role of 
Ochrobactrum in gastric pathology remains uncertain and requires detailed 
pathologic, microbiological and genetic investigations in order to evaluate the 
link between H. pylori and Ochrobactrum in the gastric niche. This thesis 
attempts to determine the population structure of Ochrobactrum sp. By 
development of new multilocus sequence analysis scheme and to gain whole-
genome based insights into the putative gene determinants of Ochrobactrum 
for survival in the highly acidic stomach lumen environment. 
In all, 75 Ochrobactrum spp. strains were recovered from  patients suffering 
with gastroduodenal diseases. These isolates were identified by biochemical 
characteristics and 16S rRNA gene sequencing and phylogenetic affiliation 
studies indicated that they indeed belong to genus Ochrobactrum. The new 
multilocus sequence analysis method comprised of four different gene loci 
VI 
 
from each chromosome was developed for and applied to characterize the 
genomic relatedness of Ochrobactrum sp. All sequences were aligned by 
Clustal W software. Phylogenetic analysis was carried out in Molecular 
Evolutionary Genetic analysis (MEGA) version 5.  The results reveals 
formation of two large groups, the first group included O. intermedium specific 
clade and the second group included strains related to O. anthropi. The 
multilocus sequence analysis involving seven genes (dnaK, recA, rpoB, trpE, 
aroC, and gap) was conducted to determine the population structure of 
Ochrobactrum sp. of clinical and environmental origin. 
 The draft genome sequence of strain M86 and strain 229E was determined 
by Ion Torrent Personal Genome Machine (PGM™) sequencer using a 316 
chip with 200-bp single-end shotgun sequencing. A total of 2,602,696 reads 
were obtained. PGM sequencing resulted in about 67X genome coverage with 
148 contigs. The de novo approach was applied to finalize the unclosed draft 
genome using MIRA 3.4.0 version using default parameters. Prediction and 
annotation of genes were done using RAST server with SEED database and 
ISGA pipeline. The data were further validated using gene prediction tools 
such as Glimmer. Functional annotation was also performed by PGAAP using 
public database of National Centre for Biotechnology Information (NCBI).The 
Ochrobactrum intermedium strain M86 whole genome shotgun (WGS) project 
was submitted to the GenBank and has the project accession 
AOGE00000000 and consists of sequences AOGE01000001-
AOGE01000148. ).The Ochrobactrum intermedium strain 229E whole 
genome shotgun (WGS) project was submitted to the GenBank and has the 
project accession ASXJ00000000. 
Genome of O. intermedium strain M86 was sequenced on the IonTorrent 
Personal Genome Machine (PGM™) using 316 chip that resulted in 
2,602,696 total reads with a mean read length of 155 bp. de-novo assembly 
using the MIRA assembler v3.4.0 with default parameters yielded ~67X 
coverage. A total of 148 contigs with >500 bp length were obtained. The 
unclosed draft genome sequence of strain M86 is of 5,188,688 bps and 5043 
VII 
 
predicted coding DNA sequences (CDSs) and 66 RNA genes with mean G + 
C content of 57.9 %. RAST server based annotation of the whole genome, 
showed the presence of 437 subsystems.Genome of O. intermedium strain 
229E was sequenced on the IonTorrent Personal Genome Machine (PGM™) 
using 316 chip that resulted 7,50,279  total reads with a mean read length of 
185 bp. de-novo assembly using the MIRA assembler v3.4.0 with default 
parameters yielded ~18X coverage. A total of 128 contigs with >500 bp length 
were obtained. The unclosed draft genome sequence of strain M86 is of 
4,808,223 bps and 5641 predicted coding DNA sequences (CDSs) and 66 
RNA genes with mean G + C content of 57.7 %. RAST server based 
annotation of the whole genome, showed the presence of 437 subsystems. 
H. pylori have several genes for biosynthesis of cytosolic urease for its 
survival in the acidic environment of stomach lumen. Genome of strain M86 
and strain 229E  contains urease gene cluster out of which, UreA, beta 
subunit, UreB gamma subunit, UreC, alpha subunit, are part of core Urease 
enzyme, While four accessory proteins: UreD, UreE, UreF and UreG play 
important role in Ni2+ uptake and insertion into active site of apo-enzyme. A 
complete operon encoding the VirB gene involved in conjugative transfer is 
present in strain M86 and strain 229E. Genes encoding osmotic stress, 
oxidative stress HPIIb, cold shock GrpE, heat shock DnaK, periplasmic stress 
DegQ and protection from reactive oxygen species, sod are found. Genes 
predicated to encode flagellar biosynthesis protein FlhA and FlhB has been 
identified in genome of strain M86 and strain 229E which are likely elementary 
to adaptation of new lifestyle. Enterobactin synthesis clusters of entA, entB1, 
entB2, entC, entD, entE, entF, entG, entH genes were also observed in the 
genome sequence of strain M86 suggesting its ability of iron acquisition by 
siderophore production. Presence of membrane transport machinery with 
dominance of Dipeptide-binding ABC transporter, periplasmic substrate-
binding component was detected in the genome of strain M86.It has been 
found that all the clinical isolates and the type strains of Ochrobactrum were 
highly resistant to all forms of ß-lactams except imipenem. This resistance 
profile is consistent with the expression of the AmpC beta-lactamase 
VIII 
 
characterized in O. anthropi. The genome of strain M86 and strain 229E  
shows the presence of AmpC beta-lactamase gene which supports its 
resistance to ß-lactams antibiotic observed earlier. Prophages and putative 
phage like elements in the genome were identified using prophage-predicting 
PHAST Web server. Regions identified algorithmically as “intact” by PHAST, 
as well as regions sharing a high degree of sequence similarity and 
conserved synteny with predicted “intact” prophages, were identified as 
prophages A study involving genome sequencing and comparison of the 
strains isolated from non-ulcer dyspeptic individuals helps to understand the 
genomic features of its survival in the acidic condition of the stomach. 
The genomic properties of Ochrobactrum are poorly characterized and, as a 
consequence, their role in human health and disease remains unclear. 
Elucidation of the physiological properties and identification of genes 
putatively involved in the various metabolic pathways may lead to a better 
understanding of the survival of Ochrobactrum in acidic condition. Further 
studies involving large scale genome sequencing and comparison of the 
Ochrobactrum strains isolated from several other non-ulcer dyspeptic 
individuals will help us apprehend the genomic features of its survival in the 
acidic condition of the stomach. 
 
 
 
 
 
 
 
 


Studies in syntheses of Isocyanates and of Polymers using Isocyanates

SOMARAJU, SUSEELA

Abstract
Identification and characterization of anti-pathogenic agents from marine bacteria

The thesis entitled ‘Identification and characterization of anti-pathogenic agents from marine bacteria’ documents the hidden potentials of marine bacteria like the antibiofilm, anti-quorum sensing and anti-virulence properties. Since resistance to antibiotics is the striking factor that predisposes therapeutic breakdown and subsequent increase in morbidity and mortality, novel targets like biofilms, cell to cell communication (quorum sensing, QS) and production of virulence factors are of prime interest and are targeted for the development of novel drugs.  These phenotypes were shown crucial for the virulence and pathogenesis of the bacterial or fungal pathogens, besides it is widely believed that targeting these phenotypes will not let the pathogen to develop resistance. Epibiotic bacteria were explicitly studied knowing their defensive role in protecting the host from invading pathogens.  Bacteria associated with marine ecosystem are reported to play such a defensive role since the ecosystem is highly populated and a constant rivalry between the organisms takes place for nutrients, space, etc.  The potential of such bacteria to inhibit other bacteria were exploited to inhibit human bacterial and fungal pathogens.
	Marine samples like sediment, mangrove rhizosphere soil, sea water, sea grass and sea weed were collected from the Karankadu mangrove forest region in Palk Bay and 149 morphologically distinct bacteria were isolated from them.  Nineteen morphologically different bacteria isolated from coral mucus of Acropora digitifera in an earlier study were taken from the laboratory’s culture collection repository, Biobank.  All the marine bacteria were primarily screened for their ability to inhibit QS in an indicator bacterium, Chromobacterium violaceum ATCC12472.  Nine coral associated bacteria and 37 other bacteria from Karankadu region were found to possess anti-QS activity and are further studied against Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Serratia marcescens, Candida albicans and C. tropicalis which play a crucial role in nosocomial and device associated infections in human.
	A coral associated actinomycete CAA-3, identified as Streptomyces akiyoshiensis, displayed potential antibiofilm activity against S. aureus, its clinical isolates and its drug resistant counterpart, Methicillin Resistant S. aureus (MRSA).  Another actinomycete (G9d) isolated from the seaweed Gracilaria gracilis, identified as Kocuria marina significantly inhibited the production of extracellular polymeric substance (EPS) production and biofilm formation in S. epidermidis. Two other coral associated bacteria called Delftia tsuruhatensis (CAB23) and Stenotrophomonas maltophilia (CAB41) were found to inhibit the biofilms
P. aeruginosa and S. marcescens.  Apart from inhibiting the biofilms, the extracts of CAB23 and CAB41 effectively inhibited the production of QS regulated virulence factors like LasA protease, LasB elastase, protease, pyocyanin and pyoverdin in 
P. aeruginosa and prodigiosin, total protease, lipase and swarming motility in S. marcescens.  K. marina was also found to inhibit the biofilms of C. albicans and C. tropicalis.  The extract of K. marina inhibited the production of secreted aspartyl proteinase (SAP), a major virulence trait in C. tropicalis that plays a crucial role in host tissue damage and immune evasion.  Purification and structural characterization of the active principles revealed the presence of Perfluoroeicosane in a fraction active against S. epidermidis and Gancidin W in a fraction active against C. tropicalis.
	On the whole, the study explains the potential of marine bacteria with special reference to actinomycetes as a potential source for anti-pathogenic agents which could replace the antibiotics in treatment of infectious diseases.  Further, this study unveils various leads for future research which will deliver fruitful products for use in the field of medicine.




A STUDY OF MAGNETIC AND ELECTRICAL PROPERTIES OF SOME SPINELS

PATIL, SAMPATRAO

Abstract
Modulating Conformation and Organization of Protein Assemblies Through Interfacial Rheology
This thesis describes a perspective on the role of interfacial rheology on the conformations of different classes of proteins at air/buffer, water/solvent and solid/solution interfaces and possible folding/misfolding events that lead to both organized and disorganized assemblies of proteins near the interface. Further, the role of interfacial viscosity in the adsorption and adhesion of the proteins to solid substrates has been analyzed. The influence of pH, electrolytes and viscogens on the rheological properties for different classes of proteins and poly aminoacid has been analyzed.




SPECTROSCOPIC AND KINETIC STUDIES ON MOLECULAR STRUCTURE AND REACTIVITY

POL, P.G.

  
 
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Synopsis 
Tsunami hazards were greatly underestimated along the coasts of countries 
bordering the northeastern Indian Ocean until the occurrence of the 26 December 
2004, Mw
The 2004 experience was unprecedented in South Asia. Unaffected by tsunami 
hazards in the past, tsunami geology is a nascent field for most South Asian 
researchers. Very little background field data is available on the deformational 
features of great earthquakes along this plate boundary and the depositional 
characteristics of extreme coastal surges, such as tsunamis and storms. Where do we 
begin our search for evidence of past tsunamigenic earthquakes? How best can we use 
 9.2 earthquake and its ensuing tsunami. Sourced off the coast of northern 
Sumatra, on the plate boundary between the Indo-Australian and Eurasian plates, the 
rupture of the 2004 earthquake propagated ~1300 km northward. The magnitude of 
this earthquake and the reach of its tsunami exceeded all known precedents, based on 
instrumental and historic records. The coseismic deformational and post-tsunami 
depositional features facilitated opportunities to conduct tsunami geology studies 
along the coasts of countries bordering the Indian Ocean. Several questions are being 
posed, the answers of which have implications for tsunami hazard assessment. How 
did this plate boundary behave prior to and after the great earthquake? Was the 2004 
earthquake the first of its kind on the Sumatra-Andaman plate boundary? If it had a 
predecessor, when did it occur and was it a true predecessor in terms of its rupture 
dimensions and tsunamigenic potential? What types of depositional evidence are 
preserved and how can we use them to develop the history of past tsunamigenic 
earthquakes? Researchers are exploring the affected regions and using the imprints 
left by the 2004 event, to address these questions. 
 
There are two components to this study: one, a seismotectonic analysis of the 
region from the perspective of plate driving forces and their relative roles in the 
interseismic and post-seismic phases. This study uses global data catalogs like the 
NEIC PDE (National Earthquake Information Centre Preliminary Determination of 
Epicenters) and the Global Centroid Moment Tensor (CMT) solutions for earthquake 
source parameters to understand the along-strike variations in seismicity patterns 
before and after the 2004 earthquake.  
 
  
 
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the 2004 tsunami and its deposits as a proxy? What problems are encountered in the 
interpretations? This thesis addresses these questions in part and presents observations 
from the Andaman Islands (the ~400 km, northern segment of the Sumatra-Andaman 
subduction zone) and the southeast coast of India, towards developing a reliable 
database of tsunami geology for 2004-type events. 
 
The premise is that regions affected by the 2004 earthquake are more likely to 
conserve signatures from older events. Based on the stratigraphic context of the proxy 
and quality of age estimates, this work presents evidence for past earthquake related  
deformation and tsunami deposition. In this work we use deformational and 
depositional features from the Andaman Islands, falling within the 2004 rupture zone 
and from one location on the Tamil Nadu coast of India (Kaveripattinam). From a 
perceptive understanding of the features related to tectonic deformation of the 
Sumatra-Andaman subduction zone, we have selected the Andaman segment that 
demonstrates explicit evidence for deformation and tsunami deposition through 
geomorphological and stratigraphic features, which are key to our exploration. A gist 
of each chapter is given below. 
 
The introduction (chapter 1) presents the background, motivation and scope 
of this work and the organization of this thesis, also summarizing the contents of each 
chapter. Chapter 2 provides a review of literature on subduction zone earthquakes 
and updates on tsunami geology, to place this study in the global context. The next 
two chapters discuss the seismotectonics of the Sumatra-Andaman plate boundary, the 
important earthquakes and their source processes. In chapter 3 we discuss the 
Andaman segment (from 10–15° N), characterized by relatively lower level 
seismicity, but distinctive, as it falls within the northern limit of the 2004 rupture. The 
deformational and depositional features here are better exposed due to availability of 
land straddling the hinge line separating the areas of 2004 uplift and subsidence. Here, 
the pre-2004 earthquakes used to occur along a gently dipping subducting slab, up to 
a depth of about 40 km. Post-2004, the earthquakes moved up-dip, extending also to 
the outer-rise and outer-ridge regions, expressing post-earthquake relaxation [Andrade 
and Rajendran, 2011]. The southern Nicobar segment (5–10° N) differs from the 
Andaman segment in its style of deformation and seismic productivity. The 
decreasing obliquity of convergence, the likely influence of a subducting ocean ridge 
  
 
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on the subducting plate and the character of the subducting oceanic plate make this 
segment distinctly different. In chapter 4 we present an analysis of its seismotectonic 
environment based on the well-constrained focal mechanisms of historic and recent 
earthquakes. We report that left-lateral strike-slip faulting on near N-S oriented faults 
control the deformation and the style of faulting is consistent to ~80 km within the 
subducting slab [Rajendran, K. et al., 2011]. The 11 April 2012 sequence of 
earthquakes on the subducting oceanic plate, between the Sumatra Trench and the 
Ninety East Ridge are the more recent among the oceanic intraplate earthquakes that 
demonstrate the reactivation of N-S oriented fossil fractures.  
 
The limited availability of land and the 2004 coseismic deformation 
dominated by subsidence, followed by prolonged waterlogging makes exploration 
difficult in the Nicobar segment. Thus, we focus on the Andaman Islands for 
deformational and depositional evidence, using observations that can be corroborated 
through multiple proxies and depositional environments that are not prone to other 
coastal surges, such as cyclones and storms. The criteria for selection of sites, 
evaluation of deposits and determination of limiting ages are discussed in chapters 5 
through 9. In chapter 5 we discuss different types of coastal environments and their 
response to high-energy sea surges. We also give a brief review of the comparative 
analyses of storm and tsunami deposits, a highly debated issue and then discuss 
important characteristics of these two deposits, using examples from the 2004 tsunami 
and the 2011 Thane cyclone that affected parts of the Tamil Nadu coast.  
 
An important component of tsunami geology is the ability to identify and 
select datable material from tsunami deposits and chose an appropriate method for 
dating (chapter 6). The types of material used vary from peat layers, peat-rich soil, 
gastropod shells, wood, charcoal, organic remains such as bones, coral fragments, 
pottery sherds and buried soil. Techniques such as AMS Carbon-14 and 
Thermoluminescence are commonly used with appropriate calibrations and 
corrections. In addition to the dates generated in this study (based on wood and shell 
dates) we use some previous dates from the entire stretch of the rupture within the 
Indian Territory and assign a relative grading to these ages, based on the quality 
criterion evolved in this study. We believe that this is the first attempt to segregate age 
  
 
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data obtained from coastal deposits, and assign them a specific quality grading based 
on their environment of deposition and the type of material dated. 
 
Chapter 7 presents results of our investigations in the Andaman Islands, 
which cover ~30% of the rupture area. A coseismically subsided mangrove from 
Rangachanga (Port Blair, east coast of South Andaman) led us to a former subsidence 
during AD 770–1040, which we believe is the most convincing evidence for a 
previous tectonic event. Data based on inland deposits of coral and organic debris 
yielded a younger age in the range of AD 1480–1660. Both these dates fall in the age 
brackets reported from other regions of this plate boundary (mainly Sumatra) as well 
as distant shores of Sri Lanka, Thailand and mainland India. To understand the nature 
of distant deposits, we present observations from Kaveripattinam, an ancient port city 
on the east coast of India, where a high-energy sea surge deposit, found 1 km inland is 
attributed to a paleotsunami. The inland location of this archeological site at an 
elevation of 2 m and characteristics of the deposit that help discriminate it from 
typical storm deposition provide clinching evidence in favor of a 1000-year old 
regional tsunami (chapter 8).   
 
In chapter 9 we discuss the results of our study. We evaluate the nature of 
deformation/deposition and the calibrated age data in the context of their 
environments. Ages based on the organic material associated with coral debris (at Hut 
Bay and Interview Island) and the remains of mangrove roots, 1 m below the present 
ground level (at Port Blair) are considered as reliable estimates, due to their sheltered 
inland location and the in situ root horizon used for dating. Age data from 
Kaveripattinam is also considered reliable, based on its inland location beyond the 
reach of storm surges, sediment characteristics typical of tsunami deposition and ages 
based on multiple methods and samples. The age data based on the sites presented in 
this thesis are more conclusive about the 800 to 1100 AD and 1250 to 1450 AD 
tsunamis, and the former is represented from regions closer to the 2004 source as well 
as distant shores reached by its tsunami. 
 
Chapter 10 presents our conclusions and the scope for future studies. We 
present this as the first study of its kind in the northeastern Bay of Bengal, wherein 
the coseismic vertical coastal deformation features along an interplate subduction 
  
 
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boundary and a variety of tsunami deposits are used to categorize depositional 
environments and ages of paleoearthquakes and tsunamis.  To our knowledge, this is 
the first study of its kind where the effects of a recent tsunami have been used to 
evaluate paleodeposits based on their respective environments of occurrence. Our 
results have implications for tsunami geology studies in coastal regions prone to 
tsunami hazard. 
  


SYNTHESIS AND PHYSICAL PROPERTIES OF SOME NEW PHOSPHORS

RAO, BALACHANDRA

Abstract
Site suitability analysis for solid waste disposal in Bangalore using GIS
Urbanization has brought forth several maladies and suffering to human kind, besides bringing economic and cultural development in its folds.  Due to increasing industrialization and population growth large quantities of waste are being generated in different forms such as – solid, liquid, sludge and gasses.  Each city produces tons of solid waste daily from house holds, hospitals, industrials offices, market centers etc.  The enormous generation, improper storage and unscientific disposal of solid waste can affect air and water quality, land use and public health.  One of the serious and growth potential problems in most large urban areas is the shortage of land for waste disposal.  Although there are some efforts to reduce and recover the waste, disposal in landfill is still the most common method for waste destination.  An inappropriate landfill site may have negative environmental, economic and ecological impacts.  Therefore it is imperative that accurate, economically practical and environmentally sound waste disposal sites are located, so that their impact on the environment and public is minimized. It would require volumes of multidisciplinary data to be collected, stored and analyzed.  One approach particularly well suited to the management of such database is the Geographic Information System (GIS).

Bangalore has an estimated metropolitan population of 75 lakhs making it India’s third most populous city and fifth largest metropolitan area.  Total municipal solid waste generated per day is around 3000 tons and out of this only 1000 tons is being scientifically managed.  Keeping this in view, the present proposal aims to identify suitable sites for disposal of municipal solid waste generated in Bangalore city using GIS and remote sensing.  GIS can be used to analyze the existing spatial relationships between various geologic, topographic, hydrologic and physiographic characteristics of the area which will help in identifying suitable landfill sites.  Various input spatial map layers including settlements, roads, topography, geology, land use, geomorphology, soil, aquifers and surface water are prepared using the survey of India toposheets, satellite data, ground data and collateral data with the help of Arc GIS software.

The present study, with the help of GIS and remote sensing provides the selection of environmentally friendly disposal sites in a quick and cost-effective manner, thus supplying reasonable, convenient and administratively transparent solutions to the waste disposal problem. It not only gives the information about the existing waste disposal sites but also the possible suitable sites that could be utilized for waste disposal. The data generated from the study can be accessed by number of users to facilitate decision making. The data generated would serve as a baseline for many Government organizations like Municipal Corporation of Bangalore, Karnataka State Pollution Control Board (KSPCB), for various developmental activities




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