A Major project report

on

Synthesis of Lignin Nanoparticles from Rice Straw and Studying its

Antimicrobial Effect.

Submitted in partial fulfilment of the requirement for the award of degree

BACHELOR OF TECHNOLOGY IN BIOTECHNOLOGY

SESSION (2015-2019)

National Institute of Technology

Raipur, (C.G)

Submitted by- Supervised by-

Anurag Mishra Dr. Pratima Gupta

8th Semester Head & Associate professor
B. Tech (Biotechnology) Department of Biotechnology

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 CERTIFICATE

This is to certify that the project work entitled “Synthesis of Lignin Nanoparticles from Rice
Straw and Studying its Antimicrobial Effect” is a bonafied work done by Anurag Mishra.
This project is submitted to National Institute of Technology, Raipur, Chattisgarh as record of
8th semester major project as a part of the Bachelor of Technology(B.tech) Biotechnology
curriculum. The research work is done at the Department of Biotechnology, National Institute
of Technology Raipur under my supervision. The project report either in a whole or in part has
not been previously formed or submitted anywhere else for dissertation.

Dr. Pratima Gupta,

Head & Associate Professor

Department of Biotechnology

NIT, Raipur (CG)

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 ACKNOWLEDGEMENT

I would like to thank our Project Guide Dr. Pratima Gupta, Department Of Biotechnology,
NIT Raipur for her consent support, help, motivation and guidance. Her constant
encouragement, constructive criticism and valuable guidance have been the cause for
successful completion of the project.
I am very much thankful to Mr. Anuj Rohatgi for his help and ideas given to me towards my
major project. I would also thanks to Dr. Vivek Singh Sir, for providing all the required material
and apparatus to me.
I gratefully acknowledge Dr. Pratima Gupta, Head & Associate Professor of the Department,
Biotechnology, NIT Raipur, for allowing me to work on this field and project in particular.
Finally, I wish to convey my warmest and deepest gratitude to my family members, to whom
I owe all my achievements, and to my friends for their endless support and encouragement.

Anurag Mishra

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 DECLARATION

The Project work entitled “Synthesis of Lignin Nanoparticles from Rice Straw and
Studying its Antimicrobial Effect” submitted, is a record of an original work done by us
under the guidance of Dr. Pratima Gupta, Department of Biotechnology, NIT Raipur. This
work is not copied either from other students work or from other source, except where due
reference or acknowledgement is made explicitly in the text, or has any part been written by
another person.

Anurag Mishra

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 Table of Contents

1. Introduction
2. Material and Methods
3. Results and Discussion
4. References

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Abstract

The present study is designed to enhanced production of amino acids by yeast strains isolated
from different sources including grapes , apples, peaches , fresh milk, honey, yoghurt and soil.
Several yeast isolates from these natural sources were isolated and many some of these isolates
were found examined for their potential to be producers of amino acids. These isolates were
grown in different pre-production media to enhance amino acidinitiate the production process.
The optimum temperature and pH for the growth of these isolates wereas found to be in
between 300C- 37°C. These isolates shows optimum growth in between and pH 6 to 8
respectively pH. . In the production media aA mixture of amino acids are were produced which
were estimated by ninhydrin assay mediated spectrophotometric analysis and found to be,,,
from …isolates respectively. Thin layer chromatography was done to know
quantitativelyseparate the amino acids from the mixture in order to detect/identify the presence
of specific amino acidsabout the obtained mixture of amino acids.The result showeds that it
contains mixture of glutamic acid, tyrosine and ,aspartic acid. To conclude the indigenously
isolated yeast isolates can be employed for as a alternative source overproduction for
production of the industrially important amino acids. Further studies are required to enhance
the amino acid production as well as to extract and purify the amino acids before they can be
thought to be exploited for commercial purpose.NBT1,NBT8,NBT13 are produced the mixture
of glutamic acid,aspartic acid,tyrosine.

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 INTRODUCTION

Biomass

Biomass is plant or animal material used for energy production, heat production, or in various
industrial processes as raw material for a range of products. It can be purposely grown energy
crops (e.g. miscanthus, switchgrass), wood or forest residues, waste from food crops (wheat
straw, rice straw, bagasse), horticulture (yard waste), food processing (corn cobs), animal
farming (manure, rich in nitrogen and phosphorus), or human waste from sewage plants.

Burning plant-derived biomass releases CO2, but it has still been classified as a renewable
energy source in the EU and UN legal frameworks because photosynthesis cycles the CO2
back into new crops. In some cases, this recycling of CO2 from plants to atmosphere and

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back into plants can even be CO2 negative, as a relatively large portion of the CO2 is moved
to the soil during each cycle.

Cofiring with biomass has increased in coal power plants, because it makes it possible to
release less CO2 without the cost associated with building new infrastructure. Co-firing is not
without issues however, often an upgrade of the biomass is beneficiary.

(Source: Academic Library)

Wood Biomass

Wood Biomass when used as a fuel has many environmental advantages over the use of
traditional fossil fuels. The main advantage is that wood is a renewable resource like solar,
water or wind power thereby offering a sustainable and dependable energy resource, unless
improperly managed. Other advantages of wood biomass energy is the fact that the amount of
carbon dioxide gas emitted during the combustion process is a lot less than when burning
conventional fossil fuel and as such has the ability to provide a much cleaner burning process.

For many decades now, sustainably controlled and produced wood biomass has become a
significant source of renewable energy while at the same time through forestry management
techniques maintaining the natural environment. Biomass is a collective term for organic matter
used as fuel and so covers a wide range of organically sourced materials which can be used for
energy production and other purposes. Amongst these organic substances is Wood
Biomass derived from forests, woodlands and other arboriculture activities from saw mills and
used as a source of wood fuel.

Wood biomass when used as a fuel can be safely stored and preserved in the form of logs or
chippings without any potential energy loss. The amount of energy that can be produced from
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wood biomass depends on the heat content of the material when it is dry. The storing of wood
prior to its burning reduces its moisture content thereby increasing its heating value and
efficiency.

The higher temperatures gained from burning drier wood also results in more heat per unit
weight of raw wood fuel, again increasing efficiency. Also, wood fuel contains minimal
amounts of sulphur and heavy metals and therefore is not a threat to acid rain pollution.

(Pic Source: Research Gate)

Cellulose, Hemicellulose and Lignin

Cellulose, hemicellulose and lignin constitute about 85–90% of lignocellulosic biomass;

organic extractives and inorganic minerals constitute the rest. Cellulose is the major structural
polymer of a plant cell wall and usually exists as long thread like fibres called microfibrils.

Cellulose

Cellulose is an organic compound with the formula (C6H10O5)n, a polysaccharide consisting of

a linear chain of several hundred to many thousands of β(1→4) linked D-
glucose units. Cellulose is an important structural component of the primary cell wall of green
plants, many forms of algae and the oomycetes. Some species of bacteria secrete it to

9
form biofilms. Cellulose is the most abundant organic polymer on Earth. The cellulose content
of cotton fiber is 90%, that of wood is 40–50%, and that of dried hemp is approximately 57%.

Cellulose is mainly used to produce paperboard and paper. Smaller quantities are converted
into a wide variety of derivative products such as cellophane and rayon. Conversion of
cellulose from energy crops into biofuels such as cellulosic ethanol is under development as
a renewable fuel source. Cellulose for industrial use is mainly obtained from wood
pulp and cotton.

Some animals, particularly ruminants and termites, can digest cellulose with the help
of symbiotic micro-organisms that live in their guts, such as Trichonympha. In human
nutrition, cellulose is a non-digestible constituent of insoluble dietary fiber, acting as
a hydrophilic bulking agent for feces and potentially aiding in defecation.

Hemicellulose

A hemicellulose (also known as polyose) is one of a number of heteropolymer (matrix

polysaccharides), such as arabinoxylans, present along with cellulose in almost all terrestrial
plant cell walls. While cellulose is crystalline, strong, and resistant to hydrolysis,
hemicelluloses have random, amorphous structure with little strength. They are easily
hydrolysed by dilute acid or base as well as a myriad of hemicellulase enzymes.

Biomass Conversion:
Thermal conversions

Straw Bales

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 Wood Pulp Bagasse
(Pic Source: Wikipedia)
Thermal conversion processes use heat as the dominant mechanism to upgrade biomass into a
better and more practical fuel. The basic alternatives are Torre faction, pyrolysis,
and gasification, these are separated principally by the extent to which the chemical reactions
involved are allowed to proceed (mainly controlled by the availability of oxygen and
conversion temperature).

There are other less common, more experimental or proprietary thermal processes that may
offer benefits, such as hydrothermal upgrading. Some have been developed for use on high
moisture content biomass, including aqueous slurries, and allow them to be converted into
more convenient forms.

Chemical conversion

A range of chemical processes may be used to convert biomass into other forms, such as to
produce a fuel that is more practical to store, transport and use, or to exploit some property of
the process itself. Many of these processes are based in large part on similar coal-based
processes, such as the Fischer-Tropsch synthesis. Biomass can be converted into multiple
commodity chemicals.

Biochemical conversion

As biomass is a natural material, many highly efficient biochemical processes have

developed in nature to break down the molecules of which biomass is composed, and many
of these biochemical conversion processes can be harnessed. In most cases, microorganisms
are used to perform the conversion process: anaerobic digestion, fermentation,
and composting.

Glycoside hydrolases are the enzymes involved in the degradation of the major fraction of
biomass, such as polysaccharides present in starch and lignocellulose. Thermostable variants

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are gaining increasing roles as catalysts in biorefining applications, since recalcitrant biomass
often needs thermal treatment for more efficient degradation.

Electrochemical conversion

Biomass can be directly converted to electrical energy via electrochemical (electrocatalytic)

oxidation of the material. This can be performed directly in a direct carbon fuel cell, direct
liquid fuel cells such as direct ethanol fuel cell, a direct methanol fuel cell, a direct formic
acid fuel cell, a L-ascorbic Acid Fuel Cell (vitamin C fuel cell), and a microbial fuel cell. The
fuel can also be consumed indirectly via a fuel cell system containing a reformer which
converts the biomass into a mixture of CO and H2 before it is consumed in the fuel cell.

Lignin
Lignin is a versatile class of biomaterial, second most abundant biopolymer after the cellulose
and the first one aromatic compound. Monomeric units of the lignin (monolignols),
hydroxyphenyl(H), guaiacyl (G) and syringyl (S) moieties joined together to form complex
three-dimensional amorphous structure. Lignin joins cellulose and hemicellulose in the plant
cell and provides strength to the plant. Lignin has a number of functional groups, like phenolic
and aliphatic hydroxyl groups, methoxy groups, terminal aldehyde groups, etc. presence of
these functional group make lignin important feedstock for the organic compound. Besides
these functional groups, lignin is biodegradable, biocompatible, antioxidant with no
cytotoxicity and their soil nutrients improvement by microbial degradation makes it an ideal
precursor for developing environmentally friendly nanoscale materials. Lignin is available in
the form of industrial/agricultural biomass as waste and causes environmental pollution, lignin
is among the low-cost biomaterial whose cost is based mostly on transportation to a processing
facility. Green chemistry stimulates to utilize renewable resource feedstock for chemical
industries as well as for academia to synthesized different chemicals. Biomass and industrial
waste as feedstock for different synthesis promoted for the sack of sustainability of the
environment.

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 (Source: Wikipedia)

Lignin is a highly branched and amorphous biomacromolecule with variable composition

dependent on the plant source. Lignin is unique as a plant cell wall biopolymer due to its
heterogeneity, lack of defined primary structure and aromatic functionality. However, lignin
can be rationalized as the polymerized product of the three basic phenylpropane monomers as
repeating units known collectively as “monolignols”: p-coumaryl alcohol, coniferyl alcohol

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and sinapyl alcohol.

(Source: Wikipedia)

Structure and Composition

The composition of lignin varies from species to species. An example of composition from
an aspen sample is 63.4% carbon, 5.9% hydrogen, 0.7% ash (mineral components), and 30%
oxygen (by difference), corresponding approximately to the formula (C31H34O11)n.

As a biopolymer, lignin is unusual because of its heterogeneity and lack of a defined primary
structure. Its most commonly noted function is the support through strengthening of wood
(mainly composed of xylem cells and lignified sclerenchyma fibers) in vascular plants.

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The lignols that crosslink is of three main types, all derived from phenylpropane: 4-hydroxy-
3-methoxyphenylpropane,3,5-dimethoxy-4-hydroxyphenylpropane, and
4ydroxyphenylpropane. The former tends to be more prevalent in conifers and the latter in
hardwoods.

Lignin is a cross-linked polymer with molecular masses in excess of 10,000 u. It is

relatively hydrophobic and rich in aromatic subunits. The degree of polymerization is difficult
to measure, since the material is heterogeneous. Different types of lignin have been described
depending on the means of isolation.

Three monolignol monomers are precursors, all of which are methoxylated to various
degrees: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These lignols are
incorporated into lignin in the form of the phenylpropanoids p-hydroxyphenyl (H), guaiacyl
(G), and syringyl (S), respectively. Gymnosperms have a lignin that consists almost entirely of
G with small quantities of H. That of dicotyledonous angiosperms is more often than not a
mixture of G and S (with very little H), and monocotyledonous lignin is a mixture of all
three. Many grasses have mostly G, while some palms have mainly S. All lignin contains small
amounts of incomplete or modified monolignols, and other monomers are prominent in non-
woody plants.

Lignin Nano Particles

Preparation of lignin nanoparticles and their different high Tec applications is the best way to
utilizes the huge amount of biowaste from agriculture and industries. Recently interests are
grown in the synthesis of LNP and have synthesized by different methods such as CO2
saturation, solvent exchange of pure lignin, acetylated lignin and dialysis, flash precipitation
methods by sonication, water-in-oil microemulsion methods as well as precipitation methods
with pH lowering by acid. LNP were also acted as micro-emulsion to synthesize metal/metal-
oxide nanoparticles. Lignin precursor and the nanoparticle synthesis technique affect the
physical properties of these particles, wide variation of the surface properties of the LNP makes
them suitable for different environmental-friendly applications. Nanoparticle synthesis from
lignin is environmental being and in accordance with the green chemistry principal, as it utilizes
lignin available in the form of industrial/ agricultural waste.

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Applications of LNP
Nanoparticles dispersed epoxy coatings are being potentially used for corrosion protection of
underlying substrate. Different types of metal/metal oxide nanoparticles, conducting polymers,
clay nanoparticles, graphemes. were used to disperse in epoxy to enhance its anticorrosive
behaviour and overcome its limitation. There is scant literature available on the lignin-based
coatings and inhibitors for corrosion protection, and no literature was found on LNP dispersed
epoxy coatings for corrosion protection. These LNP can be used as anticorrosive nanofillers
for epoxy coatings to protect CS substrate.

Objective:

On the basis of facts and evidences the present work / study was designed to study the
biocidic property of Lignin nanoparticles which were extracted from Rice Straw,

Effect of antimicrobic property of Lignin Nanoparticles produced through Ultra Sonication

on E.coli was studied and presented.

Methods and Materials:

1.Media composition for Nutrition Broth 100ml: (Nutrition media)

Peptone 0.5g

Beef Extract 0.3g

Sodium Chloride 0.5g

Distilled Water

Final pH (at 36°C) 6.5±0.2

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2. E.coli 200µl

3. Rice Straw (Biomass)

4.Nutrient Agar for Agar well diffusion (30ml):

Peptone 0.15g

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 Beef Extract 0.09g

Sodium Chloride 0.15g

Agar 0.6g

Distilled Water

Methods:
Lignin Extraction:

Biomass that we have taken is Rice Straw, it was pre-treated and shredded in small
pieces for better extraction. The Biomass was taken and 0.5% H2SO4 was added to it. The
Solid loading was 10% This mixture was then covered properly and was Autoclaved for 60
minutes. After autoclaving it should be allowed to cool and the extract was collected in a test
tube. The remaining biomass was washed with distilled water and was put in large petri dish
in hot air oven for its other uses.

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And allow the yeast isolates for the production of Amino acids.Lignin Nanoparticle
preparation

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The extracted liquid containing Lignin was taken Aqueous suspensions of lignin (0.7%) were
used. The lignin suspensions were sonicated for 60 min. Finally, a homogeneous stable
Nano dispersion was obtained. The samples resulted in sonication of lignin were dried in mild
conditions to be used in further analysis. Ultrasonication was performed by using an ultrasonic
horn at 20 KHz frequency, 600W power.
The liquid subjected to sonicator was taken after 60 minutes for proper sonication and
nanoparticle formation.

Ultra sonicator

Agar well Diffusion preparation for LNP antimicrobial activity check

E. Coli Broth was prepared by adding E. Coli strain to the Nutrient Broth. And was set in
incubator Shaker at 37ºC for 3 Days at 70 rpm for proper growth of the microbe in the media.
Nutrition agar was poured in Petri dish and was allowed to solidify. Three wells were made in
the gel for pouring LNP of different concentration. E. coli Broth was taken and was spread on
the surface of the gel for proper growth.

Addition of LNP of various concentration to agar well for its antibacterial activity

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Lignin extract was taken and diluted to 70%. 5ml of extract was added to 10ml of distilled
water and 15 ml solution was prepared which was put in Hot Air Oven for 3 Days at 100ºC for
precipitation, in order to obtain solid Lignin Nano particles. After 3 days, the solid residue left
was crushed carefully and was transferred to an Eppendorf tube.

The 3 wells on the petri dish were marked 1,2,3 for different lignin concentrations i.e.
10mg,20mg,30mg respectively. Lignin Nano particles were poured carefully in their
respective wells and were put inside the incubator for 72 hours at 37ºC.

Results and discussion:

1. Figure 1 shows the obtained solid lignin nanoparticles. Figure 2 shows the antimicrobic
activity of LNP in agar well diffusion method. (No activity by lignin is seen in all 3 wells).

Figure 1

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 Figure 2

Results and Discussion:

Discussion:

Gupta et al. 2018 reported

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The present study reports production of ……… (amino acids name) from (isolates name)
using (media). The obtained quantity are significantly good . hence these isolates can be
considered as potential candidates for commercial amino acid production after further level of
studies

Figure 3: thin layer chromatography resultTLC plate showing standard and test samples

Conclusion :

My aim is toAim of this study was to determine whether the yeast

isolates(NBT1,NBT8,NBT13, g3,g4) are producing amino acids. !After performing qualitative
( spectrophotometric ) and quantitative ( thin layer chromatography ) study, I came toit was
confirmed that my yeast isolates are NBT1,NBT8,NBT13 producing a mixture of amino
acids…………….. The highest quantity of amino acids produced were …. , …. And ….
Through …, … and … isolate in …., …. And … media respectively. NBT1 produces glutamic
acid while NBT81 producing tyrosine.NBT13 produces glutamic acid as well as probable
producer of aspartic acid. Isolatee G3 and G4 did not show any significant amino acid
production neither any spots of amino acids were obtained in TLC analysis. These amino acids
are can be further extracted and purified for the future commercial use.To conclude the
indigenously isolated yeast isolates can be employed as for good alternative sources of the
industrially important amino acids in future after further level of studies.

References:

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1. O. U. Rahman, S.Shi, J. ding, D. Wang, S. Ahmad and H. Yu, New J. Chem., 2018, DOI:
10.1039/C7NJ04103A.
2.Obtaining lignin nanoparticles by sonication Iulian Andrei Gilca , Valentin I. Popa , Claudia
Crestini
3. A simple process for lignin nanoparticle Preparation. Miikka Lievonen,a Juan José Valle-
Delgado,*a Maija-Liisa Mattinen,Eva-Lena Hult, Kalle Lintinen,c Mauri A. Kostiainen,c Arja
Paananen, Géza R. Szilvay,b Harri Setäläb and Monika Österberg
4. Antibiotic Properties of Lignin Components J. Z~MEK, B. Ko~iKOVX, J. AVGUSTiN and
D. JONIAK.
5. Infrared and Antimicrobial Studies on Different Lignins NADAA, M. A.1, EL-DIWANYA,
I.2, E LSEAFEIA, M.3

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