Open Access Repository of Indian Theses

G, DHARUNYA

v 
 
 
 
ABSTRACT 
 
The thesis highlights the implications of nano-biotechnological 
approaches for fabrication of soft and hard tissue biomaterials. Ideal 
biomaterials should possess anti-microbial property, proteolytic stability and 
vascularizability for its acceptability in the host. The thesis describes the 
fabrication of multifunctional biomaterials using collagen/gelatin as natural 
biopolymer reinforced with nutraceuticals and biodegradable metals. The 
cumulative therapeutic efficacy of nutraceuticals and metal nanoparticles 
added in therapeutic and biomechanical properties to the scaffolds that 
resulted in better tissue regeneration. The thesis is distributed into 8 chapters. 
Chapter 1 elaborates Tissue engineering and its therapeutic significance. The 
methodologies and instruments used in this study are described in Chapter 2. 
Chapter 3 outlines the fabrication of collagen aerogels reinforced with 
wheatgrass nutraceutical for soft tissue regeneration. Additionally, the effect 
of biodegradable manganese on wheatgrass collagen aerogels towards wound 
healing is also described in this chapter. 
The role of curcumin as a cross-linker of collagen fibrils is detailed 
in Chapter 4 and the results suggested that curcumin in the aerogels spatio-
temporally inhibited MMP activity and induced angiogenesis with controlled 
anti-proteolytic activity. Fabrication of nanofibrous scaffolds for tissue 
regenerative applications in a core-shell manner for controlled release of 
curcumin from gelatin core are presented in Chapter 5. Chapter 6 and 7 
describes the application of glycoproteins form plant mucilage’s for the 
development of biomaterials in soft and hard tissue engineering. The 
experimental results obtained from the thesis provide cues for the 
vi 
 
 
development of nutraceutical/biodegradable metal based therapeutic 
biomaterials for soft and hard tissue engineering applicationsthat can 
overcome many setbacks associated with commercially available 
biomaterials. 
 
  


English

BIODEGRADABLE METAL NANOMATERIAL BASED BIO-INSPIRED MATERIALS FOR TISSUE ENGINEERING APPLICATIONS

Tumula, Nagaraju

Functional characterization of miRNA(s) that regulate(s) apoptosis

Apoptosis is an efficient and highly regulated type of programmed cell death by which unwanted and deleterious cells are removed from the body. It is highly active and tightly controlled phenomenon that plays a fundamental role in the maintenance of tissue homeostasis and development. Dysfunction of the apoptotic program is often implicated in variety of diseases. Evasion of apoptosis underlies tumorigenesis whereas progressive cell loss due to apoptosis is a pathological hallmark of various degenerative diseases including heart disease, atherosclerosis and neurodegenerative disorders.

microRNAs are small, 20-25 nt long, genome encoded non-coding RNA molecules which regulate gene expression post-transcriptionally. They were initially discarded as cellular detritus, but a decade ago, they have gained a overwhelming attention due to their huge number and each one of them targeting a vast number of functionally related genes. Reports in literature suggest that same microRNA can target multiple genes and thus influence many physiological processes together. Their profound impact on several patho-physiological processes including development, differentiation, proliferation, apoptosis and metabolism make them focus of interest for therapeutic intervention.

In the present thesis, we show that the over expression of hsa-miR-128 in human embryonic kidney (HEK293T) cells induces mitochondrial-mediated apoptosis as proved by the annexin assay, cell cycle changes, caspase activation, mitochondrial membrane disruption, release of cytochrome-c from mitochondria to cytoplasm. Furthermore, the hsa-miR-128 modulated the expression of a number of genes involved in apoptosis including Bax (Bcl-2 Associated X protein). Bioinformatically, Bax is predicted to be the target of hsa-miR-128. In the current study, we validated Bax as the target of hsa-miR-128. It negatively regulated the expression of Bax 3’UTR construct as shown by luciferase assay. Besides this, hsa-miR-128 also upregulated the expression of p53 and Bak.

To delineate the molecular mechanism(s) responsible for hsa-miR-128 induced apoptosis, gene expression profiling was carried out after hsa-miR-128 over expression in HEK293T cells. The microarray analysis yielded 182 differentially expressed genes and analysis of the gene expression data with Ingenuity Pathway Analysis (IPA) software revealed cell death among the top three molecular and cellular functions, which strengthened our previous study. The top three associated networks shown by IPA included cancer, cellular growth and proliferation, gene expression; hematological system development and function, hematological disease; cell cycle, cancer, DNA replication, recombination and repair. We further showed that hsa-miR-128 inhibited SIRT1 expression directly through a miR-128-binding site within the 3’UTR of SIRT1. miR-128 not only upregulated acetylated p53, its transcriptional activity and its targets (PUMA, Noxa) but also increased acetylation of FOXO3A and promoted FOXO3A translocation to the nucleus. Our study further demonstrated that miR-128 augmented the anti-tumor effect of the drugs that target the p53 pathway. We also observed that hsa-miR-128 induced apoptosis in wild type (WT) p53 (MCF-7) as well as in mutant p53-expressing (MDA-MB-231) cells in a p53- dependent and- independent manner via induction of PUMA. Pretreatment with PUMA and Bak siRNAs abolished miR-128 induced apoptosis in HCT116 p53+/+ and HCT116 p53-/- cells.

We here present the first evidence of hsa-miR-128 to be a new component joining the p53 network. This study emphasizes that miR-128 is a novel mitochondria targeted miRNA which can be further evaluated as a chemotherapeutic agent for human cancers since it induces apoptosis irrespective of p53 status. Introduction of hsa-miR-128 into in-vivo disease models would help evaluate the prospect of its use as a therapeutic in diseases known to occur because of deregulation of apoptosis. Further analysis also showed that PPAR?, LXR (liver X receptor) /RXR (retinoid  receptor) mediated cholesterol and fatty acid biosynthesis pathways are the most significantly enriched pathway. Our findings provided important evidence for the potential role of hsa-miR-128 in metabolism besides apoptosis, which needs further investigation.




METAL-FREE OXIDATIVE STRATEGIES FOR THE CONSTRUCTION OF HETEROCYCLIC COMPOUNDS: SYNTHESIS OF 1,2,4-THIADIAZOLES, 2-AMINOBENZO[d]THIAZOLES AND 1,2,4-TRIAZOLO[1,5-a]PYRIDINES

Gupta, Suvidha

vi 
ABSTRACT 
The demand for energy has been considerably increasing since the last few 
decades. This trend is expected to continue in the coming years. According to 
International Energy Outlook, the worldwide consumption of energy is projected to 
increase by 28% in the years between 2015−2040, while the consumption of petroleum-
based fossil fuels will increase from 190 to ~230 quadrillion British thermal units. 
Furthermore, the consumption of fossil fuels releases approximately 29 gigatons of CO2 
every year into the atmosphere, which in turn contributes to global warming. 
Considering the increased demand of petroleum-based fossil fuels, their effect on 
environment and their scarcity in the near future, alternative energy sources look more 
attractive and practically feasible than ever. Microalgae have emerged as an alternative 
renewable energy source because of their inherent property to accumulate high amount 
of lipid (up to 50 % of its dry cell weight (DCW)) which is then used for biodiesel 
production. Moreover, microalgae-based biofuels (biodiesel) are non-toxic, 
biodegradable, emit low Sulphur content and release high oxygen (10‒45%) as 
compared to petroleum-based fuels.  
In spite of these advantages, microalgae-based lipid and biodiesel production 
has not yet been done on a commercial scale due to several gaps in technology such as 
low biomass productivity, high production cost, variable temperature, and light 
intensities especially in tropical countries, and the use of wastewater as growth media. 
For the current study, mixotrophic cultivation of microalgae was chosen a route for 
lipid production due to higher biomass and lipid productivities under this mode of 
cultivation as compared to autotrophic cultivation. The present study has assessed 3 
microalgae species namely Chlorella sorokiniana, Scenedesmus obliquus, and 
Scenedesmus abundans for mixotrophic cultivation using a different concentration of 
glucose (0.5−5 g L−1), acetate (0.5−5 g L−1) and mixed substrate (glucose + acetate: 2−6 
g L−1). The mixed substrate (glucose:acetate−1:1) was found effective in enhancing 
biomass productivity (up to 330 mg L−1 d−1) of microalgae species by 5.4 times and 
reduce respiration by up to 40% as compared to when an individual organic carbon 
source was used. The C. sorokiniana accumulated highest amount of lipid (Lipid 
vii 
productivity: 29.8–36.2 mg L−1 day−1) among the three strains. The strategy of the 
mixing of organic carbon substrate (glucose and acetate) was also found effective in 
increasing the tolerance of microalgae species up to 6 g L−1 from 3 g L−1 as in case of 
individual organic carbon source. The biodiesel properties derived from fatty acid 
methyl esters (FAME) for individual and mixed organic carbon substrates met standard 
properties of biodiesel with respect to cetane number (> 47), Iodine value (< 120 g I2/ 
100g), degree of unsaturation and cold filter plug point.  
The production of microalgae biomass all over the world was reported to be 
about 9000 tons per year, with the production cost being anywhere between $20−$200 
per kilogram. There are two ways to reduce biodiesel production cost. One way is to 
focus on the production of high-value products i.e., omega fatty acids that have various 
health benefits. The second way is to use cheap organic carbon sources like wastewater 
instead of conventional organic carbon sources like glucose and acetate. The former 
approach was achieved by exposing the microalgae culture to red light spectra in an 
airlift photobioreactor (PBR) that can alter the biomass productivity and lipid quality 
of microalgae. The red light was found effective in enhancing biomass, lipid, and omega 
fatty acids (linoleic and linolenic acid) productivity of S. abundans and C. sorokiniana 
by ~2 times in airlift PBR. Mixotrophic condition strongly influenced nitrate 
consumption, and its early consumption was the key factor in getting higher total lipid 
content. Among acetate and glucose, the latter was found to be the more effective 
organic carbon to enhance biomass, lipid, and omega fatty acids productivity of C. 
sorokiniana. While comparing the effect of light spectra, both white and red lights were 
found to produce biodiesel of appropriate quality in mixotrophic mode (CN: 51–55) for 
both microalgae species. 
The present study used food product processing industrial wastewater for the 
latter approach. The pre-feasibility study for the cultivation of Chlorella species at 
different dilutions (0, 1.6 and 5x dilution) of synthetic and actual food product 
processing industrial wastewater reduced the freshwater requirement by 20%–60% and 
successfully removed COD, TN, and TP from wastewater. An integrated approach had 
also been studied to produce microalgal biodiesel using both raw (RW) and anaerobic 
digested wastewater (ADW) of food product processing industry without addition of 
viii 
extra nutrients or carbon source for cultivation. The addition of RW as a carbon source 
in ADW (RW + ADW), enhanced biomass and lipid productivities to 1.5‒2.9 times 
than that obtained with RW or ADW alone for C. sorokiniana, S. obliquus and S. 
abundans. The effluent obtained after microalgae growth contains COD (200 mg L‒1), 
TN (23 mg L‒1), and TP (8.8 mg L‒1) below the discharge limit. The FAME obtained 
with all three microalgae species for mixed wastewater (RW + ADW) met standard 
properties of biodiesel. Hence, the integrated approach i.e., the addition of carbon-rich 
RW into carbon-deficient but nutrient-rich ADW was a suitable solution for both in situ 
wastewater treatment and biomass generation for biofuels.  
As the biodiesel production process can only be scaled up in outdoor conditions, 
achieving high biomass and lipid productivities at uncontrolled environmental 
conditions especially at high irradiance and high-temperature in Nagpur region is the 
biggest challenge. Therefore, the present study assessed cultivation of microalgae 
species in airlift PBR in summer as well as in winter season. The outdoor study in 
summer season showed photoinhibition effect at high temperatures (47 °C) and 
irradiance conditions. The microalgae S. abundans can be considered to be a 
thermotolerant species as it can tolerate temperatures up to 45 °C with optimum 
biomass productivity of 162.5 mg L−1 day−1. The fed-batch cultivation strategy 
accumulated higher lipid content as compared to respective batch cultivation. The fed-
batch strategy favorably influenced consumption of acetate in light periods. The 
respiratory loss of carbon was 60−70% lower in fed-batch cultivation as compared to 
batch cultivation. Thus, the outdoor microalgae cultivation in summer season would be 
cost-effective under fed-batch strategy. Similarly, in winter season three microalgae 
species namely C. sorokiniana, S. obliquus, and S. abundans were successfully 
cultivated in a mixed substrate (3 g L−1 glucose + 3 g L−1 acetate). The fatty acid derived 
biodiesel properties obtained in all runs in summer and winter season also complied 
with International biodiesel standards (ASTM and EN14214) (CN: 51−63).  
  


Indoor and Outdoor Cultivation of Microalgae for Biofuel Using Mixotrophic Mode: Feasibility and Assessment Study

Delsa Dean, Deepika

Abstract
 
Increasing global concerns of climate change and energy crisis have created the need to switch from fossil energy to renewable energy. Renewable energy is viewed as an efficient alternative to compensate the escalating future fuel demands and deemed to be the futuristic energy source which is going to change the visage of energy scenario in the near future. Bioenergy derived from microorganisms is of great interest in the present world’s energy scenario due to its renewability and sustainability. Microbial fuel cell (MFC) is one such alternative technology where the microbial membrane potentials will be utilized for harnessing power by introducing the artificial electron accepting conditions. MFC is a hybrid bio-electrochemical device which facilitates direct transformation of energy stored in chemical bonds of substrate to electrical energy in the presence of fermentative bacteria under mild reaction conditions viz., ambient temperature and pressures. Biocatalyst present in the anode chamber of MFC generates electrons and protons upon substrate degradation and the potential difference between the respiratory system and electron acceptor facilitates generation of electricity. The present thesis focuses the importance and relevance of microbial fuel cell (MFC) as bio-electrochemical treatment (BET) system for the remediation of real-field complex pharmaceutical wastewater with simultaneous power generation. Utilization of wastewater for harnessing bioenergy by MFC is gaining importance due to its sustainable nature. The main scope of the study is to design and develop a hybrid membrane-less MFC/BET system. The performance of MFC/BET was compared with conventional anaerobic treatment to understand the influence of bioelectrochemical processes with electrode membrane assembly in the treatment of complex wastewater. Placing electrode in anaerobic microenvironment showed significant change in the electrocatalytic behavior of biocatalyst which also had a positive influence on treatment of complex wastewater. Bio-electrochemical analysis and electro-kinetic parameters were performed to understand significant variations in the behavior of biocatalyst, based on its residing microenvironment. Hence, the study designed a hybrid membrane-less single chamber MFC/BET with open-air cathode in an attached mode operation. The designed membrane-less fuel cell system demonstrated effective wastewater treatment along with electricity production. The biofilm developed on the electrode and on the solid media showed added benefits on both treatment and electrogenic activity. The study concludes that membrane-less MFC/BET is eco-friendly and cost-effective technique and could replace conventional unit processes existing in ETPs .



FMR1 gene mediated genotype and phenotype variation in POF and developmental delay Patients

RAUT, SANTOSH VIKRAM

Abstract

Pharmacognostic and Molecular Studies of Some Medicinal Plants from Family Cucurbitaceae

The World Health Organization (WHO) estimated that 80% of the population of developing countries relies on traditional medicines, mostly plant drugs, for their primary health care. Also, modern pharmacopoeia contains at least 25% drugs derived from plants and many others which are synthetic analogues built on prototype compounds isolated from plants (Anonymous, 1996). In India, about 25,000 plant-based formulations are used in traditional and folk medicine. In recent years herbal products has led to a quantum jump in volume of plant materials traded within and across the countries (Mukherjee & Wahile, 2006; Mukherjee, 2002). While the demand for medicinal plants is growing, some of them are being threatened in their natural habitat due to over exploitation. This may increase chances of adulteration and substitution in genuine material. 
In view of the multidisciplinary scientific development and industrialization, quality assurance of herbals has become mandatory. For achieving this, standardization plays a crucial role. It is not just an analytical operation for identification and assay of active principles; rather, it comprises complete information which guarantees consistent composition of all herbals. It involves different conventional and modern pharmacognostic methods published by WHO in year 2006. Recently, DNA markers have been applied for the authentication of medicinal and processed products of plant origins (Techen et al., 2014). DNA fingerprinting by molecular markers analysis are valid methods in distinguishing genuine and commercial plant material. It also helps to trace intra generic relationships (Sucher & Carles, 2008). This further compliments quality control, exact identification and authentication of crude drug (Zhao et al., 2006). Hence, search for new resources; their standardization using traditional as well as modern tools and their bio-efficacy study are becoming essential now days.
Family Cucurbitaceae is one of the largest, economically and medicinally important plant group comprising 800 species in 130 genera worldwide. It is observed genera like Bryonia, Bryonopsis, Citrullus, Corallocarpus and Momordica bear only bitter fruits, whereas Coccinia, Cucumis, Lagenaria, Luffa and Trichosanthes bear both bitter and non-bitter fruits. In Indian Traditional System of Medicine (ITSM) most of the bitter plant species have been recommended for curing wide array of disorders. Medico-practitioners also utilize these bitter plant species for formulation preparation. Voluminous work has been carried out on Momordica, Citrullus, Cucumis and Coccinia with respect to their, botany, phyto-chemistry, pharmacology and molecular studies (Dhiman et al., 2012). However, comparative studies of bitter and non-bitter fruit bearing closely allied species/ varieties/ forms from genus Lagenaria, Luffa and Trichosanthes have not been performed so far. Over the past decade several reports on adverse effects, suspected toxicity have been documented due to consumption of products containing species from these taxa (Puri et al., 2011; Sharma et al., 2006).Currently there are no full proof standards available for authentication of these species and their herbal products. 
In view of this, present study work was planned for evaluation of pharmacognostic and molecular studies on selected closely related species/ varieties/ forms of Lagenaria siceraria (Molina) Standl., Luffa acutangula (L.) Roxb., Trichosanthes dioica Roxb., and Trichosanthes cucumerina L., along with their wild relatives from genus Lagenaria, Luffa and Trichosanthes from family Cucurbitaceae. The comparative pharmacognostic studies have been developed as per WHO guidelines particularly for macro-characterization, micro-characterization and phyto-chemical contents. High Performance Thin Layer Chromatography (HPTLC) quantification of Cucurbitacin B and Inter-Simple Sequence Repeat (ISSR) marker based molecular profiles have been developed for these selected species along with their wild relatives. The selected species were also studied for the antioxidant capacities. These comparative baseline studies will definitely help for proper use of species. Results obtained have been compiled in the thesis form. The aspects studied and results obtained have been summarized as below. 
Chapter I: Introduction - This chapter gives an overview of Traditional Medicinal System (TMS) and rising global interest. Further, it provides brief scenario of Indian health care system with special emphasis on the role of herbal drug standardizations using traditional as well as advanced molecular techniques. The importance of present work with respect to the need of pharmacognostic and molecular evaluation of selected species from family cucurbitaceae has been pointed out.
Chapter II: Review Of Literature - This chapter is divided in to three sections where relevant literature about genera Lagenaria, Luffa and Trichosanthes is reviewed respectively. With in each genus, detailed information of selected species with respect to their origin and taxonomy, utility in TSM, pharmacognosy, phyto-chemical, pharmacological data and molecular studies are compiled. Problems related to adulteration and substitution of the selected species are reviewed and presented.   
Chapter III: Materials And Methods - The chapter describes detailed methodology and protocols used in the present study. 
	Collection, identification and voucher preparations of genuine plant materials were done as per standard protocol. The crude drug samples sold as ‘Kadu bhopla’, ‘Kadu turai’ and ‘Kadu padwal’ were collected mainly from Maharashtra and some parts of Gujrat.   
	The detailed pharmacognostic investigations were carried out on vegetative parts and fruits as per standard methods.
	The protocols for DNA extraction and Polymerase Chain Reaction (PCR) by using Inter simple sequences repeat (ISSR) markers were standardized. DNA amplification profiles were analyzed using NTSYSpc version 2.1.
	Antioxidant activity was evaluated by means of different spectrophotometic assays.
Chapter IV: Pharmacognostic and Molecular Studies on Lagenaria Siceraria (Molina) Standl. - The comparative pharmacognostic evaluation of non bitter and bitter forms of L. siceraria did not show major differences. However, chemical profiles of methanol extracts showed additional bands in bitter form. HPTLC method was developed for the estimation of Cucurbitacin B (Bitter component). The result indicated that non bitter form is completely devoid of bitter principle. The antioxidant potential of bitter form was slightly higher than the non bitter one. In molecular profiling using the ISSR markers no variation was observed between non bitter and bitter forms of L. siceraria. However, ISSR markers were useful to detect adulteration or substitution by other species.
Chapter V: Pharmacognostic and Molecular Studies on Luffa Acutangula (L.) Roxb.
- The comparative pharmacognostic evaluation of Luffa acutangula var. acutangula and Luffa acutangula var. amara (Roxb.) Clarke showed very few differentiating characters. The size of flower, fruit and leaf epidermal characters in var. amara were distinct. In HPTLC analysis, var. acutangula was devoid of cucurbitacin B, whereas it was detected in vegetative parts and fruits of var. amara. The antioxidant potential of var. amara is significantly higher than var. acutangula. In molecular profiling using ISSR markers var. acutangula and var. amara could be distinguished clearly based on the polymorphic profiles with four ISSR markers. In analysis of market samples, based on ISSR marker profiles adulteration by other Luffa species and Momordica charantia L. could be identified.   
Chapter VI - Pharmacognostic and Molecular Studies on Trichosanthes Dioica Roxb. and Trichosanthes Cucumerina L. – In comparative pharmacognostic studies, T. dioica and varieties of T. cucumerina i.e. var. cucumerina and var. anguina could be differentiated. However; there were no major differences between non bitter and bitter varieties of T. cucumerina. In HPTLC analysis presence of Cucurbitacin B in vegetative parts of T. dioica and vegetative parts as well as fruits of T. cucumerina var. cucumerina was detected. However fruits of T. dioica and vegetative parts as well as fruits of T. cucumerina var. anguina were devoid of cucurbitacin. Antioxidant potential of T. cucumerina var. anguina was the highest followed by T. cucumerina var. cucumerina and T. dioica respectively. In molecular profiling using ISSR markers all the three species/varieties could be distinguished clearly based on amplification profiles of seven primers. In analysis of market samples based on ISSR amplification profiles adulteration by other Trichosanthes species and Momordica charantia L. could be identified.       
Chapter VII: Summary, Conclusions and Future Directions 
The result of work are summarized as follows 
Pharmacognostic standardization:
	In macroscopic and microscopic studies the diagnostic differences were found in T. dioica and T. cucumerina (two varieties) which can be used for identification of these species. 
	However, in comparative macroscopic and microscopic studies, no variation was observed in vegetative and floral characters except shape of ovary and fruits in wild relative of L. siceraria, L. acutangula (two varieties) and T. cucumerina (two varieties).
	In all the selected species, significant variations were observed in physicochemical values viz. percentage of ash, protein and amino acid contents in non bitter and bitter forms/varieties. 
	Tannins, phenols, flavonoids, glycosides and alkaloids were present in all species. While steroides and terpenes were found only in bitter form/varieties. 
	Tannin and polyphenol content were found to be higher in fruits of bitter forms/varieties species under study. 
HPTLC profiling and estimation of biomarker: 
	TLC fingerprints of petroleum ether and methanol extracts of selected species have been developed. The numbers of spots and their respective RF values were documented. 
	To our knowledge this is a first complete report on chromatographic studies and development of HPTLC method for estimation of Cucurbitacin B from vegetative parts and fruits of selected plant species. Cucurbitacin B is estimated only in bitter forms/varieties of L. siceraria, L. acutangula, T. cucumerina var. cucumerina and vegetative part of T. dioica. Thus, it can be used to differentiate closely related form/varieties. 
	Further, the developed HPLTC method was successfully applied to detect presence of Cucurbitacin B in the market samples procured under name of Kadu bhopla, Kadu dhodka, Patola. This method can be useful to detect adulteration and substitutions in market samples of selected species. 
Molecular studies: 
	ISSR marker based DNA profiles were used for the first time in identification of closely related non bitter and bitter forms/varieties of L. siceraria, L. acutangula, T. dioica and T. cucumerina. Specific primers based DNA profiles were found useful to differentiate close related species and varieties. 
	In L. acutangula (both varieties), primers UBC 808 and 809 produced polymorphic/specific bands and in T. cucumerina (both varieties), primers UBC 812, 822, 887, 888, and 889 amplified species specific bands. These profiles are useful in correct identification of these varieties. 
	However, in case of L. siceraria bitter and non bitter form, selected ISSR markers failed to produces any variation in collected set of DNA. 
	Further, species specific primers successfully applied for investigation of market samples. This is first report of application of ISSR primers based molecular techniques for detection of adulterant with closely related species and establishment of genetic relationships among the species with in genus Luffa and Trichosanthes.
	The different antioxidant assays demonstrated bitter forms/varieties of selected species have higher antioxidant capacity which may be due to increased amount of tannin, polyphenol and bioactive compounds like Cucurbitacins.  
Conclusions: 
Authentication of closely related medicinal plants is an especially critical when there is direct application of plant material in medicinal treatment or as a botanical supplement. The safety, efficacy and quality information obtained by applying advanced chromatographic and molecular techniques will serve as a rapid and confirmatory tool in medicinal plant research, thereby benefiting the entire pharmaceutical industry. The present work is comprehensive report on voucher specimen, macro and microscopic data, chemical profiling and DNA fingerprinting information of important medicinal plant, L. siceraria, L. acutangula, T. dioica and T. cucumerina and/with their wild relative. This data/information would be clearly beneficial for the authentication procedure, and for providing safe product to consumers.     




Design synthesis and biological screening of nitrogen containing heterocycles using conventional and modern methods

GUPTA, NIDHI

 
Exploring the role of versican and its regulation by microRNA in Multiple Myeloma  
 
 
The abnormal growth of malignant plasma cells in Multiple Myeloma (MM) require bone 
marrow (BM) niche consisting of proteoglycans, cytokines, etc. Versican (VCAN), a chondroitin 
sulfate proteoglycan promote progression in solid tumors but there is dearth of literature in MM. 
Hence, we studied the involvement of VCAN in MM and its regulation by microRNAs as a 
therapeutic approach. 30 MM patients and 20 controls were recruited and BM Stromal Cells 
(BMSCs) were isolated by primary culture. Molecular levels of VCAN, miR-144, miR-199 & 
miR-203 were examined in study subjects and cell lines. The involvement of VCAN in myeloma 
pathogenesis was studied using BMSCs-conditioned medium (BMSCs-CM) and recombinant 
versican G3 domain. The regulation of versican was studied by microRNA mimics and inhibitors. 
Elevated expression of VCAN was observed in patients especially in bone marrow stroma while 
microRNA expression was significantly lower and showed negative correlation with VCAN. 
Moreover, BMSCs-CM showed presence of VCAN which upon supplementing to MM cells alter 
parameters in favour of myeloma progression, however, this effect was neutralized by VCAN 
antibody. The downstream signaling of VCAN was found to activate FAK and STAT3 which 
subsides by using VCAN antibody. The recombinant VCAN spanning G3 domain also leads to 
the alteration in cancer hallmarks in favor of myeloma pathogenesis with downstream activation 
of FAK and STAT3 signaling. The transfection of microRNA mimics (miR-144 and miR-199) 
in primary BMSCs leads to the significant knockdown of transcript and protein levels of VCAN. 
The inclusion of microRNA mimics transfected BMSCs-CM reversed the pro-myeloma effect of 
BMSCs-CM in the myeloma cells. The microRNA mediated knockdown of VCAN interferes 
with the paracrine signaling of VCAN and impede the activation of FAK and STAT3 signaling 
in myeloma cells. The regulation of VCAN by microRNAs was also studied by transfecting 
microRNAs inhibitors to myeloma cell lines. Inhibition of microRNAs by anti-miR oligos in 
myeloma cells resulted in significant over-expression of VCAN. The microRNA antagomirs 
mediated upregulation of VCAN observed to contribute in myeloma pathogenesis with 
downstream activation of FAK and STAT3 signaling. Thus, VCAN was observed as a paracrine 
mediator in the cross-talk of BMSCs and myeloma cells in BM microenvironment which could 
be regulated by miR-144 and miR-199. Therefore, these findings suggest exploring VCAN as 
novel therapeutic target and utilization of microRNAs as therapy to regulate VCAN for better 
management of MM.  
 


EXPLORING THE ROLE OF VERSICAN AND ITS REGULATION BY MICRORNA IN MULTIPLE MYELOMA

DUMALA, NARESH

SYNOPSIS 
Introduction  
The applications of nano particles (size < 100 nm) (NPs) across the various fields are expanding 
rapidly because of their unique physicochemical features compared to their bulk analogues. 
According to the market survey, the production of metal oxide NPs was estimated to rise from 2, 
70, 041 tons in 2012 to 16, 63, 168 tons by 2020. On the other hand it has been reported that the 
novel properties of these NPs could cause unpredictable outcomes due to their interactions with 
biological systems. This increasing presence of NPs in the ecosystem urges us to study their 
potential toxic impact on health and to understand as well as assess their adverse effects on 
environment. Among the known metal oxides, nickel oxide (NiO) NPs have attracted wide 
scientific interest due to their use in solar cells, lithium batteries, LEDs, resistive memory chips, 
and sensors. The applications of NiO-NPs as chemical catalysts, conduction paste, magnetic 
materials, and in ceramics are worth mentioning. They are also used in films (electrochromic), 
electronics, devices for storing energy, fuel cells, inks for printing, and for wastewater treatment. 
Hence, in the current investigation the toxicity of NiO-NPs was evaluated using in vitro, in vivo 
and eco (Allium cepa) toxicity systems. 
Statement of problem 
The rapid rise in manufacturing and use of NiO-NPs raises a concern of intentional and 
unintended delivery into the environment with possible adverse implications for human and 
environmental health. The lack of toxicological data on NiO-NPs makes it difficult to determine 
the risk associated with them. Thus, there is an urgent need to develop rapid, accurate and 
efficient testing strategies to assess toxic effects of these particles on humans. Characterization of 
materials is an essential part to understand their behaviour in biological systems prior to 
toxicological testing. Therefore in this study, characterization of NiO-NPs was performed to 
provide the basis for understanding the properties of NPs that determine their biological effects. 
We have investigated whether NiO-NPs exposure can cause environmental and human health 
risk, with a special focus on genotoxic, biochemical, histological and biodistribution patterns. In 
vitro (human peripheral blood lymphocytes), in vivo (acute and 28 day repeated oral exposure in 
albino Wistar rats) and plant bio systems (Allium cepa) were tested with NiO-NPs for toxicity. 
The data generated in this study may be useful in predicting the possible health hazards that may 
arise from the introduction of NiO-NPs into the environment and give an estimate of human 
health risk. 
Methodology used & sample results 
Characterization of NiO-NPs 
The NiO-NPs were characterized by Transmission electron microscopy (TEM), Dynamic Light 
Scattering (DLS), Laser Doppler Velocimetry (LDV) and Brunauer Emmett Teller (BET) 
analysis. The size obtained by TEM for NPs was 15.6 (± 7.6) nm and showed cubic crystalline 
lattice structure by X-ray Diffraction (XRD) analysis. The mean hydrodynamic diameter and 
z  in Milli Q water and RPMI 1640 media was measured by DLS and was found 
to be 189.9 (±17.1) and 285.9 (±19.6) nm, whereas the zeta potential of NiO-NPs was 36.3 and 
24.16 mV, respectively. Electrophoretic mobility of the NiO-NPs in Milli Q and RPMI 1640 
medium was 2.85 (±0.3) and 1.4 (±0.4) , respectively when studied by LDV. The 
specific surface area was found to be 115.9 (±5.3) m² g-1 by BET analysis. 
Toxicity assessment of NiO NPs on human peripheral blood lymphocytes 
The in vitro parameters investigated were cytotoxicity, genotoxicity and oxidative damage in 
human peripheral blood lymphocytes. The cytotoxicity studies were conducted with 0.1, 1, 5, 10, 
20, 50 and 100 µg ml-1 concentrations of NiO-NPs. The result of trypan blue dye exclusion and 
MTT assays revealed that NiO-NPs reduced cell viability in a dose dependent manner. The IC50 
for NiO-NPs was determined as 23.58 µg ml-1 after 24 h of exposure. Genotoxicity studies 
performed with 12.5, 25 and 50 µg ml-1 concentrations of NiO-NPs revealed a significant dose 
dependent increased in DNA damage and micronucleus formation. The oxidative stress analysis 
for ROS by DCFDA dye, and increased levels of MDA by lipid peroxidation assays were noted 
upon increasing the concentration of NiO-NPs. Morphological assessment of the lymphocytes 
upon exposure to NiO-NPs showed that the mechanism of toxicity was apoptosis. Thus, the 
preliminary mechanism of NiO-NPs for cytotoxicity on lymphocytes was assumed to be 
oxidative stress-mediated apoptosis and DNA damage. 
Toxicity assessment of NiO-NPs by using in vivo studies (acute and 28 day repeated oral dose) 
In vivo study of NiO-NPs is essential because animal systems are extremely complicated and the 
interaction of the NPs with biological systems could lead to novel biodistribution, clearance, 
immune response, and metabolism patterns. In vivo examination was carried out for 
genotoxicity, biochemical, oxidative stress, hematological, histopathology parameters and 
biodistribution patterns after acute and 28 day repeated oral dose exposures in Wistar rats. The 
genotoxicity evaluation was performed by using comet, MNT and CA assays. The antioxidant 
enzyme status and lipid peroxidation profile were determined to examine the ROS generation 
and oxidative stress levels.  
 The results of comet assay revealed significant (P < 0.001) DNA damage at 500 mg kg-1 
bw dose in the PBL, kidney and liver cells of female rats at the 24-h sampling time. The result of 
MNT and CAs assays was in agreement with the comet assay data after single oral dose. The % 
tail DNA values of PBL, liver and bone marrow cells of male and female rats showed dose 
dependent increment and significant at the higher dose (200 mg kg-1) treatment after 28 days 
repeated oral exposure. The frequency of MN-PCEs and CAs in bone marrow cells of rats 
revealed clastogenic events and supported well the comet assay results of repeated oral dose 
study. These results provided the preliminary evidence that the NiO-NPs are capable of inducing 
genotoxicity when administered through the oral route after acute and repeated exposure 
regimens. Histological analysis showed hepatic damage and alterations in the tissue architecture 
of kidneys in the female rats exposed to acute doses of NiO-NPs. On the other hand, histological 
studies performed in rats after 28 days repeated oral dose exposure depicted mild to severe 
alterations in the vital organs including brain. The observed histological alterations might be 
attributed to the distribution profile of these NiO-NPs, which indicated that a significant quantity 
of Ni content was distributed to all the visceral organs. NiO-NPs induced substantial biochemical 
perturbations and imbalance in the antioxidant enzyme profiles in a dose dependent manner in 
both acute and repeated oral dose toxicity. Activation of the hepatotoxicity marker enzymes, 
aminotransferases, was recorded in serum and liver, whereas inhibition was observed in kidney. 
The activity of antioxidant enzymes was also altered by NiO-NPs in a dose-dependent manner 
and found to be significant at the higher doses of exposure. Lipid peroxidation profile in rats 
treated with acute and repeated oral doses of NiO-NPs provided further evidence for the NiO-
NPs capability for inducing ROS and oxidative stress. Biodistribution analysis helps in 
understanding the absorption, distribution, metabolism and excretion rates (ADME kinetics) 
along with the organ specific accumulation after acute and 28 days of repeated oral treatment. 
Biodistribution of NiO-NPs revealed a maximum accumulation of Ni in the liver tissue at the 24-
h sampling time. Moreover, the results of 28 days study showed significant accumulation of Ni 
in all tissues and clearance via urine and feces (elimination kinetics) which was noted to be dose 
dependent. Overall, the results obtained from the in vivo investigations revealed NiO-NPs 
produced significant increase in DNA damage, histopathological changes, alterations in 
biochemical enzyme profiles and induced oxidative stress along with distribution of Ni content 
in vital organs. The low-dose (125 mg kg-1) treated rats in acute oral dose toxicity study were 
asymptomatic, which gives an important clue about the NOAEL. Furthermore, the results of the 
present study indicated that the NOAEL concentration for NiO-NPs is <50 mg kg-1, when 
exposed repeatedly via the oral route in rats.  
Toxicity assessment by using A. cepa (plant) bio-system as eco-toxicity indicator 
The optical and fluorescent microscopy results gave a comprehensive view on the impact of the 
NiO-NPs on A. cepa root tip cells. A variety of chromosomal aberrations (CAs), like laggard, 
clumped, disturbed, sticky anaphase, chromosomal break, bridge and c-mitosis were found upon 
interaction of root tip cells with various concentrations (5, 10, 20 and 40 µg ml-1) of NiO 
particles. Results indicated a dose dependent increase in CAs, decrease in mitotic index (MI) and 
the effect was more significant (P<0.05).  The results were directly proportional to the 
concentration of NiO NPs treatment to the A. cepa root bio-system. The genotoxic effect on root 
tip cells was evaluated using comet assay in isolated nuclei showed an increased % tail DNA 
with a notable decrease in % head DNA. After exposure of NiO-NPs, there was a significant 
increase in production of superoxide (O2
.-), hydroxyl (.OH) and hydrogen peroxide (H2O2) when 
examined in root elongation zone of A. cepa with histochemical staining using Nitro blue 
-Diaminobenzidine (DAB), whereas, no alteration was observed in 
the maturation zone. Oxidative stress analysis revealed increased levels of O2
.-, H2O2, 
.OH 
radicals and MDA upon increasing the concentration of NiO-NPs. The elemental analysis by 
using ICPAES showed a concentration and time dependent increase in the internalization of Ni 
into the root tip cells. 
Conclusions 
In this study, a range of in vitro, in vivo and plant model approaches have been applied to assess 
the possible toxic behaviour of NiO-NPs. The result of in vitro studies revealed NiO NPs 
reduced cell viability, produced oxidative stress in dose and time dependent manner. The present 
investigation revealed acute and 28 day repeated exposure to NiO-NPs beyond the threshold 
levels might lead to serious consequences including DNA damage and ROS production. The 
toxicity (genotoxicity, biochemical, histopathology and biodistribution) was validated by 
standard protocols. The blood chemistry and lipid profiles are evidence for the toxic effect of 
NiO-NPs. The induced DNA damage may be attributed to increased oxidative stress which 
correlated with histological alteration and presence of particles in the various tissues. The ROS 
induced oxidative stress may be the most frequently observed mechanism of DNA damage after 
NiO-NPs exposure. The NiO-NPs treated A. cepa root tip cells indicated a dose-dependent 
increase in DNA damage, cytological changes and decrease in MI compared to controls. The 
interaction of root cells with NPs resulted in the generation of ROS, which was found to be a 
strongly dependent on the dose. 
From this investigation it can be concluded that in comparison to controls, NiO-NPs 
induced significant changes in the target biochemical as well as genotoxic parameters in a dose 
and time dependent but not gender dependent manner. The smaller size, large surface area and 
may be the ions of NPs could have contributed to the obtained toxic effects. The data from the 
current investigation suggested a moderate toxicity risk with nano NiO. Therefore NiO particles 
can be considered as chemical substances that induce toxicity at high doses of exposure. 
However, more studies are needed to understand the exact mechanism of toxicity by extending 
these studies further using toxico-proteomics and metabolomics approaches. This knowledge 
may help to develop new approaches for the design of safe NP based consumer and medical 
products. 
Possible Mechanism of Toxicity of NiO-NPs 
 
Publications from the thesis work  
1. 
  
2. 
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3. 
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4. 
  
 
 


IN VITRO, IN VIVO AND ECO-TOXICOLOGICAL ASSESSMENT OF

NICKEL OXIDE NANOPARTICLES

CARPENTER, JITENDRA

 
 
v 
 
Abstract 
In this doctoral thesis, cavitation techniques (ultrasound and hydrodynamic) have been 
utilized with an intensifying approach for producing oil in water (O/W) emulsions with 
higher attributes such as minimum droplet size, enhanced stability and higher encapsulation 
efficiency. This research work aims to advance the understanding of ultrasonic and 
hydrodynamic cavitation based emulsification process. The motivation of this work was to 
understand the fundamentals related to the kinetic stability of oil in water emulsions of 
cavitationally prepared emulsions which are yet to be fully elucidated. This research work 
deals with the detailed study on the formulation, stability and encapsulation characteristics of 
mono and multilayer nanoemulsions prepared using ultrasonic and hydrodynamic cavitation. 
It also considers the impact of ultrasonic and hydrodynamic cavitation parameters on the 
physicochemical properties of emulsions as well as the significance of the effect of these 
cavitation process parameters on the emulsion properties such as droplet size, encapsulation 
stability and chemical stability. 
This thesis contains an introduction outlining the background information on the emulsion 
science and fundamentals of cavitation techniques for the generation of O/W emulsions. The 
overall study in this research work consists of four major parts which are described as 
follows: 
In the first part of thesis, the mustard oil in water nanoemulsions stabilized by Span 80 and 
Tween 80 were prepared using ultrasonication with detailed optimization of the various 
process conditions. Effects of various operating parameters such as HLB (Hydrophilic 
Lipophilic Balance) value, surfactant volume fraction (φS), oil volume fraction (φO) and 
power amplitude were investigated and optimized on the basis of minimum droplet size and 
stability of nanoemulsions. It was observed that minimum droplet size of about 87.38 nm 
was obtained within 30 min of ultrasonication at an optimum HLB value of 10, φS of 0.08 
(8%, v/v), φO of 0.1 (10%, v/v) and ultrasonic power amplitude of 40%. The detailed 
statistical analysis was also conducted using response surface methodology and Analysis of 
variance technique to describe the significance of the process parameters on the emulsion 
droplet size. It has been observed that the oil fraction was found to be the most significant 
factor affecting the droplet size of emulsion. The significance level was found to be in the 
 
 
vi 
 
order as φO > φS > %Amp > Time > HLB. The stability of the nanoemulsion was measured 
through visual observation and it was found that the unstable emulsions got separated within 
24 hours whereas, stable emulsions never showed any separation until 90 days. In addition to 
that, the kinetic stability of the prepared nanoemulsions was also assessed under centrifuge 
and thermal stress conditions. The emulsion stability was found to be unaffected by these 
forces as the droplet size remained unchanged. The ultrasound prepared emulsion was found 
to be stable even after 3 months of storage at ambient conditions without any visual evidence 
of creaming and phase separation and also remained kinetically stable. FTIR analysis of the 
emulsions at different sonication conditions was carried out to examine the possible impact 
of ultrasonically induced chemical effects on the oil structure during emulsification and it 
was found that the oil molecular structure was unaffected by ultrasonication process. This 
work illustrates the formation and stability of mustard oil in water nanoemulsion using 
ultrasound cavitation which may be useful in food and cosmetic based applications. 
In the second part, nanoemulsions were prepared with the same system but with a different 
cavitation technique i.e. hydrodynamic cavitation (HC) and compared with ultrasonication 
process. In this work, detailed geometrical analysis of hydrodynamic cavitating devices was 
carried out to investigate the effect of geometry (orifice and venturi of different shapes) and 
geometrical parameters on the formation and stability of mustard oil in water nanoemulsion. 
The optimization of geometrical parameters was carried out based on the lowest droplet size 
obtained and droplet size reduced per unit pass. It was observed that the single hole orifice 
plate of circular shape having lower perimeter and higher flow area than other orifice 
devices, was found to be an efficient device which produced emulsion of lowest droplet size 
i.e. 87 nm at an optimum CV of 0.19 (10 bar optimum operating pressure) in 90 min of 
processing time. A mathematical correlation was also developed using dimensionless 
analysis approach for understanding the effect of various operating and geometrical 
parameters of HC. The mathematical expression to calculate the droplet size was found to be 
the function of dimensionless numbers such as Reynold number, Weber number and 
cavitation number. Moreover, the kinetic stability of the nanoemulsion was also assessed 
under centrifugal and thermal stress conditions and it was found to be unaffected by these 
forces as no change in the droplet size observed during the assessment. The prepared 
nanoemulsions were found to be stable up to 3 months indicating the potential of HC over 
 
 
vii 
 
high energy techniques for producing highly stable nanoemulsion on a large scale. 
Furthermore HC was found to be 11 times more energy efficient than acoustic 
cavitation/ultrasonication in the preparation of nanoemulsion. 
In the third part, a different kind of emulsion i.e. multilayer O/W emulsion was prepared 
using layer by layer approach which is a potential system that can be utilized particularly in 
food and pharmaceuticals for the encapsulation of various nutraceuticals. The ultrasonication 
method was used for the preparation of multilayer O/W emulsion stabilized with whey 
protein isolate (WPI) and sodium alginate (SA). Ultrasonication was employed in both batch 
and recirculating flow configurations. The effect of process parameters such as pH, SA 
concentration, and sonication time on the droplet size, zeta potential, morphology, physical 
and oxidative stability of the multilayer emulsion was studied using batch process. It was 
observed that the emulsion prepared at pH 5, 0.2 wt% SA and 60 sec sonication had good 
bilayer interaction between WPI and SA molecules. Moreover, it was found that sonication, 
if given for a controlled time period can improve the physical and oxidative stability of the 
emulsion. Further, the ultrasonication based recirculating flow configuration (RFC) was 
utilized for the preparation of multilayer emulsion at the optimum operational conditions 
obtained in the batch studies. Emulsions prepared using RFC were found to have better 
physical and oxidative stability than using batch ultrasonication at the optimum flow rate of 
0.5 L/min with 6 recirculation passes. RFC was found to be 2.5 times more energy efficient 
than batch ultrasonication process for the synthesis of multilayer emulsions.   
In the fourth part of this thesis, the multilayer emulsion prepared using ultrasonication 
process was utilized as a carrier system for the encapsulation of a bioactive compound i.e. 
curcumin. Curcumin is a natural polyphenol compound which is obtained from the turmeric 
plant, having numerous health promising benefits. In order to deliver the curcumin into the 
human body, it is necessary to develop an efficient carrier system for its encapsulation such 
that the physico-chemical properties of curcumin can be preserved during the storage. In this 
study, the encapsulation stability, antioxidant activity and release properties of curcumin 
encapsulated in the primary emulsion (PE, stabilized by whey protein isolate) and secondary 
emulsion (SE, stabilized by double layer of whey protein isolate/sodium alginate) prepared 
using ultrasonication was analyzed. It was observed that the formation of a double layer 
 
 
viii 
 
coating of secondary biopolymer over the primary coated droplet enhanced the encapsulation 
efficiency and antioxidant activity of the curcumin during the storage of 3 weeks. Moreover, 
the multilayer emulsions were freeze dried in order to see the effect of dehydration of 
emulsion on the stability of multilayer coated droplets. FTIR analysis indicated the presence 
of all the constituents including curcumin after the freeze drying of the emulsions. SEM 
images showed that the microstructure of emulsion droplets was found to be uniformly 
distributed in case of SE. The antioxidant activity of curcumin encapsulated in SE was found 
to be higher during storage whereas it was significantly reduced in other encapsulated 
systems like PE, olive oil and ethanol. In vitro release of curcumin from the multilayer 
emulsion was carried out under the simulated intestinal conditions of pancreatin enzyme and 
bile salt. The maximum release of 73% and 64% was obtained in SE and PE, respectively 
within 2 h of digestion. The different kinetic models such as zero order, first order and 
Higuchi model were fitted to understand the release kinetic of curcumin. The best fitting was 
obtained with zeroth order kinetic model based on the regression analysis of the data. 
Overall this study provided useful information on the formation of multilayer emulsion as a 
carrier system for better protection and controlled release of curcumin for the food and 
pharmaceutical applications. 
Overall this research work highlights the efficient utilization of both the cavitation 
techniques i.e. ultrasound and hydrodynamic cavitation for the preparation of different oil in 
water emulsions which has potential application in food, pharmaceutical and cosmetic 
industries. Efforts have been made to improve the efficiency of the ultrasonication process 
not only to get the high stability emulsion but also to improve the throughput capacity of the 
emulsification process. On the other side, the HC proved its potential for the synthesis of 
emulsion on a large scale with desired stability and thus can be a good substitute to the other 
high energy extensive techniques used in the industries. The potential of cavitationally 
prepared multilayered emulsion for the encapsulation of bioactive compound was thoroughly 
investigated and it was concluded that the cavitation process is a viable and feasible 
technique for the production of emulsions as an encapsulating system for the various 
bioactive compounds that are useful in food, cosmetic and pharmaceutical industries. 
 


STUDY ON CHARACTERIZATION, STABILITY AND ENCAPSULATION ACTIVITY OF MONO AND MULTILAYER EMULSION (OIL IN WATER) PREPARED USING CAVITATION

Amradi, Naresh Kumar

SYNOPSIS 
 
1. Preamble  
Sustainable waste management practices play a critical role in minimizing environmental 
pollution and also promote the transformation towards a green society (Lee et al., 2014). 
Conventional waste management practices eliminate the potential advantage of waste as 
feedstock and also emit greenhouse gases. So in order to switch towards the environmental 
sustainability and biobased economy efficient waste management practices need to be developed 
embedded with resource recovery (Bonk et al., 2015; Venkata Mohan et al., 2016). Extensive 
scientific studies were being evaluated with the waste a resource to produce the value-added 
products apart from its remediation. Various solid, liquid and gaseous waste (CO2) originating 
from municipal bodies, domestic (kitchen/food waste) and industrial could be used as a feedstock 
for biobased products synthesis (Puyol et al., 2017). Therefore, all major sectors, including 
environment expertise, industrialists and economist are pushing to recover and regain valuable 
products from waste resources (Marshall et al., 2013). It is important to explore individual and 
combined technologies to convert specific wastes to a wide spectrum of value-added products 
(Karmee, 2016; Venkata Mohan et al., 2016). 
Apart from the solid waste, liquid waste is also one of the major waste which generated day to 
day human activities. Biological treatment of this wastewater generates huge quantities of waste 
activated sludge (WAS) and according to the estimates WAS production rate is 0.1-30.8 kg per 
person equivalent per year (Liu et al., 2017). Huge quantities of WAS is significantly increasing 
the disposal cost in the biological waste treatment process. In this context, proper WAS disposal 
practices are essential to avoid its further environmental contamination. Conventionally, WAS is 
being disposed off through landfills, incineration, and biomethanization. Landfilling requires big 
open lands and emits greenhouse gases and incineration necessitates high thermal energy inputs. 
In addition to the negative impacts on the environment, the potential use of WAS as a resource is 
also lost in these conventional practices (Salsabil et al., 2010). Alternatively, WAS could be used 
as a resource for sustainable biological processes and these are known as eco-friendly and 
economically reliable processes (Tao et al., 2016, Liu et al., 2017).  
 
Renewable sugars and nutrient-rich hydrolysate extracted from the organic solid waste (OSW) 
could be considered as an alternative and potential resource biobased industries (Venkata Mohan 
et al., 2015). Acidogenic fermentation is emerging technology, which is compatible with various 
composite waste (high strength) biodegradation with concurrent production of biohydrogen (H2) 
and volatile fatty acids (VFA) or short-chain carboxylic acids (SCA) (Wang et al., 2014; Venkata 
Mohan et al., 2016; Dahiya et al., 2015). VFA‘s are the soluble intermediate metabolic products 
of acidogenic fermentation and which are majorly composed of acetic acid (AA: C2), propionic 
acid (PA: C3) and butyric acid (BA: C4) and valeric acid (IVA: C5). Further, SCA‘s are 
considered as platform chemicals and act as building blocks for various high-value product 
synthesis including alcohols, ketones, esters, medium-chain fatty acids (caproic/capric acid), etc. 
(Lee et al., 2014). Medium-chain carboxylic acids (MCA) are the superior to VFA‘s as they have 
a longer hydrophobic carbon chain and low oxygen/carbon ratio, which increases the energy 
density and makes its separation simple (Steinbusch et al., 2011). MCA‘s are identified as C6-C12 
acids namely, caproic acid (C6),  Oenanthic acid (C7),  Caprylic acid (C8),  Pelargonic acid (C9), 
and Capric acid (C10).  Among these acids, the biological production of caproic acid from VFA 
and alcohols utilizing chain elongation pathway is gaining perceptible interest. Besides this, VFA 
can be used as precursors for the production of biopolymers in an integrated process (Arslan et 
al., 2016). Biopolymers are the biodegradable polymers produced by microbiomes when 
subjected to stress conditions such as availability of excess carbon and limitation of nitrates and 
phosphates required for growth (Venkata Mohan and Reddy., 2013). Since their first report by 
Lemogine in 1926 (Anderson and Dawes., 1990), PHA‘s have attracted a great deal of attention 
due to their biodegradability, chemical-diversity, biocompatibility, and manufacture from 
renewable carbon resources. In order to enhance PHA production from wastes/wastewaters, 
many strategies have been proposed and of them, the integration of acidogenic fermentation with 
the aerobic process has emerged out as one of the viable strategy (Venkata Mohan et al., 2010). 
Acidogenic fermentation (AF) of wastes generates effluents that are rich in VFA need treatment 
prior to disposal (Dahiya et al., 2015). These VFAs can be effectively used as a source of carbon 
by aerobic bacteria for their growth and also their survival during carbon depleted conditions by 
storing these VFA as PHA (Reddy et al., 2012; Wu et al., 2016). Through this multiproduct 
approach, the intrinsic load of the waste can be reduced thereby reducing environmental 
pollution while obtaining products of high commercial interest (Reddy et al., 2013).  
Waste valorization is the process of transforming organic-rich substrate into valuable products 
like, renewable chemicals, biofuels, and materials. The globe is tuning the magnitude of the 
waste management problems and has been working towards the development of advanced waste 
remediation technologies with resource recovery as the main goal (Amulya et al., 2015; Venkata 
Mohan et al., 2016). On the other hand, there is a platform exists for the devotement of 
sustainable technologies that could follow the ‗trash to cash‘ concept by producing industrially 
important chemicals, materials, and fuels which will reduce the dependency on fossil-based fuels 
and products but also help in addressing the environmental sustainable remediation issues (Bing 
et al., 2016; Chen et al., 2016). Concept of biorefinery analogous to today‘s petroleum refineries 
is also showing interest. Biorefinery systems work by utilizing bio-based feedstock to produce a 
range of bio-products like fuel, platform chemical, animal feeds, etc.  in a closed-loop approach 
(Pradel et al., 2016; Pleissner et al., 2016; Venkata Mohan et al., 2016). Many scientists working 
with waste believe that it contains potentially huge amount of energy to replace a major fraction 
of the global energy crisis if it can be economically converted to sustainable energy forms 
(Kannengiesser et al., 2016).  
 
2. Rationale of the Study  
Organic municipal solid waste per capita generation in major Indian cities ranges from 0.2 Kg to 
0.6 Kg, out of which the about 90-95% is being dumped as waste (Dube et al., 2014;Venkata 
Mohan et al., 2018). The calorific value of Indian solid waste is between 600 and 800 Kcal/Kg 
and the density of which is in between 330 and 560 Kg/m
3
. Organic solid waste (OSW) is 
conventionally disposing of through landfilling, incineration and biomethanization. Landfilling 
requires large open lands and emits the greenhouse gases into the environment. Further, the 
process of incineration requires high thermal energy input and releases the secondary pollutants 
into the environment. In addition, these conventional disposal practices are eliminating the 
potential of OSW and are no longer sustainable due to its high economic inputs and negative 
impacts on the environment. Alternatively, sustainable biological conversion is an eco-friendly 
process and economically reliable (Lee et al., 2014).  The heterogenic composition of OSW and 
embedded polysaccharide content in it does not suit its direct supplementation as a biological 
substrate.  Studies were reported with various individual pretreatment methods to harness the 
maximum sugars from OSW like physical (thermal conversion, sonication), chemical (acid, 
base), enzymatic (amylase, cellulases, mixture of enzymes) (Ho et al., 2013, Hernández et al., 
2015, Hafid et al., 2015, Shokrkar et al., 2018). Each individual method has its own advantages 
and disadvantages, wherein the physical method is easy to operate but the resulting sugar yields 
are lower. On the other side, chemical method is suitable for effective degradation of 
polysaccharides, but the acid or base concentrations need to be optimized to avoid the formation 
of the inhibitor and moreover, the resulting sugar yields are lower. Advantageously, enzymatic 
hydrolysis is more specific towards the carbohydrate depolymerization with no/low inhibitors 
formation, but the major concerns of this method is the requirement for longer reaction time 
apart from the high cost of enzymes (Lee et al., 2013). Therefore, there is a need to develop an 
effective pretreatment method for maximum sugar extraction from OSW and its entirety 
utilization as a renewable resource. Since the selection of an appropriate pretreatment method 
largely depends on the composition of waste. Additionally, optimization of biological process is 
pre-requisite to maximize the process efficiency and to obtain higher yields. In this context, 
experimental were evaluated to harness the maximum sugars from OSW followed by its 
effective utilization in the subsequent biological processes for the production of various biobased 
products such as biohydrogen, short-chain carboxylic acids, medium-chain carboxylic acids, 
biopolymers and bioethanol in the integrated multiproduct approach.  
 
3. Scope of the Research 
The present study comprehensively evaluates the renewable resource potential of organic solid 
waste for the production of various biobased products such as reducing sugars, short-chain 
carboxylic acids, biohydrogen, medium-chain carboxylic acids, biopolymers, bioethanol and 
struvite in a closed-loop approach. With the existing scope in the literature, the experiments were 
designed and evaluated with the following objectives.  
o Effective strategy for organic solid waste (OSW) pretreatment for maximizing sugar 
solubilization using various individual and combined methods like physical, chemical, 
physicochemical, enzymatic and bio-electrohydrolysis. 
o Evaluation of waste-derived renewable sugars as main feedstock in the fermentation 
process towards the production of short-chain carboxylic acids (C2-C5), biohydrogen 
(Bio-H2) and bioethanol  
o Integrated utilization of SCA as a carbon source for the production of biopolymers (PHA: 
polyhydroxyalkanoates) and medium-chain carboxylic acids (MCA: C6-C10) in the 
secondary biological process.  
o Evaluation of biorefinery process with multiproduct valorization design in a circular loop 
Renewable sugars obtained from the organic solid waste was used as a substrate for the 
production of short-chain carboxylic acids (C2-C5), biohydrogen (Bio-H2), medium-chain 
carboxylic acids (C6-C10), biopolymers (PHA), bioethanol and struvite in the biorefinery 
framework. Organic fractions present in waste could replace the fossil based raw materials for 
the synthesis of biobased products in the biorefinery approach. These renewable products have a 
good commercial interest and also the production of which using waste reduces the carbon load 
on the environment. Although it would be presumptuous to believe that waste will be able to 
completely meet the world‘s energy needs, it is still achievable if a coalesced approach to recycle 
and reuse waste is developed, wherein the waste coming from one industry or sector becomes the 
raw material for another. However, this leads to the development of the biobased circular 
economy with less environmental impact and an utmost generation of bioenergy and other bio-
based products. In line with the above mentioned facts, to address the entirety use of organic 
solid waste the present study strategically designed and evaluated in the multiproduct approach 
with the integrated cascading approach.   
4. Summary of Results 
The pervasiveness of organic solid waste (OSW) and the energy content embodied in it makes 
waste one of the most potential and affordable feedstocks for the production of multiple biobased 
products. This will address the dual challenges, resource scarcity and increasing energy demand. 
Usually, the composition of waste is dissimilar, based on its material distribution and origin the 
pretreatment method will differ. The obtained result from the study illustrates the influence of 
various pretreatment methods based on the origin of OSW. Hybrid (PSC) pretreatment strategy 
with dilute acids (H2SO4/HCl) resulted in relatively higher RS solubilization from vegetable 
waste and deoiled algal biomass sugar solubilization. On the other side, waste activated sludge 
(WAS) pretreatment showed the good organic and nutrient solubilization with alkaline-catalyzed 
conditions pretreatment than corresponding acid-catalyzed method. Specifically, WAS alkaline 
catalyzed conditions facilitated the leaching of the cell wall and thus resulted effective 
disintegration of WAS with increased soluble COD concentration. However, the combination of 
physical and chemical methods (physicochemical) with acid/alkaline-catalyzed conditions 
induced good sugar solubilization than the consequent individual methods. Additionally, WAS 
pretreatment with novel alkaline bioelectrohydrolysis system (BES) also resulted in good SCOD 
solubilization and resulted the sludge flocs disintegration. On the contrary, low SCOD 
solubilization resulted in non-electrochemical control system (AnT). FE-SEM morphological 
examination was supported well with the WAS refractory structure degradation and the EDX 
analysis showed the changes in elemental composition with the course of pretreatment.  
Acidogenic fermentation of waste derived renewable sugars as a substrate for the production of 
biohydrogen and short-chain carboxylic acids (VFA: C2-C5) resulted the good yields. The 
experiments were studied at various redox conditions such as pH-4, 6, 7 and 10. Higher 
biohydrogen and VFA production resulted at pH-6 condition (H2; 1.22 l and VFA: 0.62 g VFA/g 
RS) than corresponding other experimental variations. Operational pH showed a marked 
influence on the production and compositional distribution of biogas and VFA. Higher AA 
concentration in systems operated with pH-6, 10 and 7 contributed to higher DOA, while in the 
case of pH-4 system BA contributed a major fraction of DOA. Fermentation time also played a 
key role in the governance of VFA profiles. Specifically, in the case of pH-7 condition, longer 
retention time consumes the accumulated VFA towards methane production. On the other side, 
lower retention time (48 h) wrapped further VFA consumption towards the methane in all the pH 
conditions. First-order kinetic models were studied to fermentative biobased products synthesis 
to understand the stable process performance under practical conditions.  
Besides, the resulted VFA was also reutilized as a substrate in the integrated biological processes 
for medium-chain carboxylic acids and biopolymers production due to its simple organic nature. 
Medium-chain carboxylic acids (MCA) are the superior to VFAs as they have a longer 
hydrophobic carbon chain and low oxygen/carbon ratio, which increases the energy density and 
makes its separation simple. MCA are majorly composed of C6-C12 namely, Caproic acid (C6),  
Oenanthic acid (C7),  Caprylic acid (C8),  Pelargonic acid (C9), and Capric acid (C10).  Among 
these acids, the biological production of caproic acid from short-chain acids/ VFAs and alcohols 
utilizing chain elongation pathway is studied using VFA as an electron acceptor and ethanol as 
an electron donor. The obtained results showed the good yields (4.73 g/l) than the corresponding 
hydrogen as an electron donor. Additionally, the utilization of VFA as a substrate for biopolymer 
production (PHA: polyhydroxyalkanoates) also resulted in good yields than renewable sugars. 
Enriched mixed microbial cultures were used for PHA production, wherein PHA producing 
bacteria was examined using a fluorescent microscopy, flow cytometry and confocal microscope 
with Nile blue staining. Thereafter, the characterization of extracted PHA was studied using 
NMR, FT-IR, XPS, XRD and thermal analysis. These obtained results showed the structural 
correlation with polyhydroxybutyrate (PHB). Thereafter, PHB films were prepared using acetic 
acid as a casting agent and which resulted in good high-quality films than conventional 
chloroform films. Acetic acid is relatively safe and cost-effective solvent which can bring down 
the cost of film preparation and wider the application of PHB films in the medical applications. 
Additionally, to increase the strength of the extracted PHB, nanocellulose composites were 
prepared by using different concentrations of PHB. The obtained PHB nanocellulose composite 
film showed a higher strength than individual PHB.  
The energy crisis, escalating crude oil prices and environmental aberrations upsurge the need for 
potential renewable fuels like bioethanol. The 3G ethanol produced from deoiled algal biomass 
(DAB) residue is gaining interest owing to the sustainable supply of the feed and the possibility 
of waste valorization. Subsequent utilization of DAB derived sugars showed the significant 
variations in the bioethanol production, wherein in higher bioethanol production was found at pH 
5.5 followed by pH 5.0 and pH 6.0. In addition, struvite was also produced from organic rich 
hydrolysate resulted from the WAS. Struvite is known as a phosphate fertilizer with a crystalline 
solid structure composed of magnesium, ammonium and phosphorus (Mg.NH4PO4.6H2O). The 
precipitated struvite yield was 0.18 g/L which resulted at most suitable pH 9.0 to 9.5. The FTIR 
structural analysis of struvite revealed a broad asymmetric band in between 3600 and 2200 cm
-1
, 
which can be attributed to O-H and N-H stretching vibrations.  
 
 
 


Microbial Transformation of Solid Waste for Renewable Chemicals, Energy and Polymers in the Biorefinery Framework

KUMAR, RAJESH

Page | i  
 
ABSTRACT 
 As the fossil fuels are depleted rapidly, and the byproducts of fossil fuels are 
responsible for polluted environment, it becomes a necessity to develop an eco-friendly 
and renewable energy source. To overcome the problem of air pollution by the fossil fuel 
and due to energy requirement, our energy dependence on renewable energy sources 
should be high. Hydrogen has been widely deliberated as an attractive energy carrier 
with high efficiency for developing an environmental friendly and cost-effective 
sustainable energy system. However, besides the hydrogen energy, the main issue with 
other energy sources is energy density and area dependency.  
  Fuel cell is that unit, which is used to convert hydrogen in the other form of 
energy. The advantage with the H2 fuel cell has water, the only byproduct obtained after 
the energy generation. But the primary requirement for lifetime and efficiency of the fuel 
cell is the purified H2 from the impurities or separated H2 from other gases. So the 
separation/purification of the H2 is the solution for a new renewable energy source, 
prevent the environment from global warming as well as useful for controlling the air 
pollution.  
 The primary aim of our research work is to find out the eco-friendly technology 
through which we can separate/purify hydrogen. There are many types of method or 
process, which are used to separate/purify the hydrogen gas from the mixture of other 
gases. Moreover, membrane-based technology for the H2 separation/purification has 
attracted considerable response due to inherent benefits over other separation methods. 
The membrane-based separation process is not only cost-effective but also 
environmentally friendly as well.  
 Membrane-based gas separation, suffer from a problem in the form of trade-off 
relationship between selectivity and permeability. So, the control over selectivity and 
permeability of the gases is the main goal for the membrane-based gas separation. For 
the gas separation with the membrane is depends on two parameters; diffusion 
coefficient (D) and solubility coefficient (S). For gas separation application, we have 
used different types of porous polymeric membranes and functionalized these 
membranes to attach the gas sensitive nanoparticles. For the H2 selective membranes, 
we have synthesised and deposited palladium nanoparticles in the functionalized porous 
membranes. 
Page | ii  
 
 Functionalization is an effective method for different functionalities for the 
membrane and subsequently desired nanomaterials can be incorporated into the polymer 
matrix. Initially, Polycarbonate (PC) with functionalized and non-functionalized 
MWCNTs composite membranes have been prepared. The alignment of the MWCNTs 
is controlled by high magnetic field. Pd nanoparticles were synthesised chemically and 
deposited on PC-MWCNTs composite membranes. In the obtained of permeability and 
selectivity data, significant improvement has been found with the use of functionalized 
MWCNTs and Pd nanoparticles.  
 If membranes will be functionalized by a specific functional group then it can 
improve the selectivity of hydrogen. We have functionalized porous PET membranes 
having pore diameter     ~ 0.2 µm with carboxyl group. The effect of functionalization 
time on permeability and selectivity of hydrogen over other gases in PET membranes 
have been observed. Functionalized PET membranes were also used for the H2 
separation application with 6 hours attachment of the Pd nanoparticles. Functionalization 
the specific group and attachment of Pd nanoparticles enhanced the H2 permeability as 
well as selectivity.   
 Further, to functionalized porous PC membranes, we have used a reliable and 
low-cost UV irradiation method with the fixed time difference. To enhance hydrogen 
selectivity, palladium nanoparticles have deposited into the pores as well as on the 
surface membranes. We have also use binder polyvinylpyrrolidone (PVP), for the 
palladium nanoparticles to improve the hydrogen selectivity over other gases. Due to the 
functionalization and maximum attachment of palladium nanoparticles, these 
membranes have high permeability as well as selectivity of hydrogen. Significant results, 
easy fabrication process and low cost of these membranes gives an opportunity to use 
for commercial use. 
  


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