isolation and identification of microbes for … · absorption and conversion of ammonia, nitrates...

ABSORPTION AND CONVERSION OF AMMONIA, NITRATES AND NITROGEN USING AQUAPONICS SYSTEM BY THE APPLICATION OF

MICRO ORGANISMS

GUIDE :-

ANANDA H V

ASSISTANT PROFESSOR

DEPT OF BIOTECHNOLOGY

PRESENTED BY :-

ARUN D K

7TH SEM

DEPT OF BIOTECHNOLOGY

SAPTHAGIRI COLLEGE OF ENGINEERING, BANGALORE-KARNATAKA

THE SOLUTION…....??

AQUAPONICS SYSTEM FOR THE STUDY OF AMMONIA CONVERSION

1. Designing an Aquaponics System by using Gold Fish .

2. Finding out the Concentration of Ammonia using Ammonia

Test Strips.

3. Absorption and Conversion Studies by the Plant Source of

Converted materials in the system.

4. Estimation of Dissolved oxygen Content.

5. Plant Growth Estimation

OBJECTIVES :-

Fig 1 :- Non interacting System for

study of Ammonia Conversion

Fig 2 :- Draining System fo Recirculation

Of water

WORKING :-

RESULTS

1. Study of Ammonia Conversion

2. Study of Dissolved Oxygen

3. Plant Growth Estimation

1. Decrease in the Ammonia content allows proper growth of

Aquatic Animals.

2. Fish waste is utilized as plant feed rather than being wasted.

3. Provides a truly organic form of nutrients for the plants.

4. 100 % Organic and Chemical free.

5. All weather solution.

Advantages :-

FUTURE PROSPECTS :-

ACKNOWLEDGEMENT

This Presentation was made possible with Funding from

Karnataka State council for Science and Technology.

Dr. Aswatha Kumar M , Principal , Sapthagiri college of

Engineering

Dr. Ananda S , Head Of Dept , Dept of Bio Technology

Mr. Ananda H V , Assistant Professor , Dept of Bio Technology

Mrs. Saranya D , Assistant Professor , Dept of Bio Technology

Mrs. Blessy baby Mathew , Assistant professor , Dept of Bio

Technology

Sapthagiri College of Engineering ,Bangalore

7. REFERENCES:

JAMES E. RAKOCY, MICHAEL P. MASSER AND THOMAS M. LOSORDO.

“RECIRCULATING AQUACULTURE TANK PRODUCTION SYSTEMS: AQUAPONICS—

INTEGRATING FISH AND PLANT CULTURE”. 2006

PETER M. VITOUSEK, JOHN D. ABER, ROBERT W. HOWARTH,GENE E.

LIKENS, PAMELA A. MATSON, DAVID W. SCHINDLER, WILLIAM H.

SCHLESINGER, AND DAVID G. TILMAN. “HUMAN ALTERATION OF

THE GLOBAL NITROGEN CYCLE: SOURCES AND CONSEQUENCES”.

1997

HTTPS://EN.WIKIPEDIA.ORG/WIKI/NITROGEN_CYCLE#MARINE_NITROGEN_CYCLE

HARRY AKO AND ADAM BAKER. “SMALL-SCALE LETTUCE PRODUCTION WITH

HYDROPONICS OR AQUAPONICS”. 2007

https://en.wikipedia.org/wiki/Nitrogen_cycle



















THANK

YOU ……

Absorption and conversion of ammonia and nitrites by using Nitrobacter and Nitrosomonas in

aquaponics system for the development of pathos , (40S_BE_0183)

Dept of Biotechnology, Sapthagiri College of Engineering, Bangalore Page 1

ABSTRACT

Aquaponics is a food production system that combines intensive aquaculture (raising

aquatic animals in tanks) with hydroponics (cultivating plants in a nutrient solution). In

Aquaponics system, Fish feed passes through fish and provides nutrients for plant growth. It is

recirculation aquaculture systems that incorporate the production of plants without soil. Plants

grow rapidly with dissolved nutrients that are excreted directly by fish or generated from the

microbial breakdown of fish wastes.

The Setup of Aquaponics system is based on non-interacting tanks concept by using gold fish.

Identification of micro-organism associated with the nitrogen cycle- Nitrobacter and

Nitrosomonas are selected. The Ammonia and nitrates concentration are measured by using

ammoniatest strip in ppm. Decreasing in the ppm level shows the biological conversion of

ammonia and nitrates by using Nitrobacter and, Nitrosomonas. The pothos (money plant) is

selected based on plants growing in water concept. The Dissolved oxygen is tested based on

wrinkle’s method. Absorption & conversion studies by the microorganisms studied. The room

temperature has maintained (25-270C), Plant growth is estimated based on the development of

the plant in the duration of one month. Proper circulation of water is maintained in the

aquaponics system regular period of time intervals.

Keywords: Aquaponics System, Nitrite, Nitrate, Ammonia, Nitrobacter, Nitrosomonas, Pothos




OBJECTIVES

Set up of Aquaponics system by using Gold Fish

Finding out concentration of Ammonia using Ammonia test strips

Identification of micro-organism associated with the nitrogen cycle- Nitrobacter and

Nitrosomonas

Media preparation, Sub culturing and proper inoculation of organisms to the system

Selection of plants based on water growing plants

Proper temperature and pH maintenance

Absorption & conversion studies by the plant source of converted materials in the system

Dissolved oxygen in samples are estimated by using Wrinkles method

Plant growth estimation

Recirculation of water to the system regularly




INTRODUCTION

Aquaponics is a food production system that combines intensive aquaculture (raising

aquatic animals in tanks) with hydroponics (cultivating plants in a nutrient solution). In

Aquaponic system, Fish feed passes through fish and provides nutrients for plant growth.

Aquaponic systems are recirculation aquaculture systems that incorporate the production of

plants without soil. Plants grow rapidly with dissolved nutrients that are excreted directly by fish

or generated from the microbial breakdown of fish wastes.

The nutrient rich effluents from the aquaculture component are circulated through the

hydroponic component where a proportion of these nutrients are taken up by the plants before

the water is returned to the fish tanks [1]. For a proper nitrogen cycle to take place there should

be adequate resources such as nitrogen source, microorganisms, and plants [2]. So as to contain

all of this in a single process we are using aquaponics system. The Nitrite, Nitrate and Ammonia

content in the effluent of Fish Tank is used for the growth of plant, Many Vegetables can be

grown in water culture using nutrients either provided by aquaculture effluents.

In closed recirculating systems with very little daily water exchange (less than 2 percent),

dissolved nutrients accumulate in concentrations similar to those in hydroponic nutrient

solutions. Dissolved nitrogen, in particular, can occur at very high levels in recirculation

systems.

Fish excrete waste nitrogen, in the form of ammonia, directly into the water through their

gills. Bacteria convert ammonia to nitrite and then to nitrate, Ammonia and nitrite are toxic to

fish, but nitrate is relatively harmless and is the preferred form of nitrogen for growing higher

plants such as fruiting vegetables.

Aquaponic systems offer several benefits. Dissolved waste nutrients are recovered by the

plants, reducing discharge to the environment and extending water use (i.e., by removing

dissolved nutrients through plant uptake, the water exchange rate can be reduced). Minimizing

water exchange reduces the costs of operating Aquaponic systems in arid climates and heated

greenhouses where water or heated water is a significant expense. Having a secondary plant crop

that receives most of its required nutrients at no cost improves a system’s profit potential. The




daily application of fish feed provides a steady supply of nutrients to plants and thereby

eliminates the need to discharge and replace depleted nutrient solutions or adjust nutrient

solutions as in hydroponics. The plants remove nutrients from the culture water and eliminate the

need for separate and expensive bio filters.

Aquaponic systems require substantially less water quality monitoring than separate

hydroponic or recirculation aquaculture systems. Savings are also realized by sharing operational

and infrastructural costs such as pumps, reservoirs, heaters and alarm systems. In addition, the

intensive, integrated production of fish and plants requires less land than ponds and gardens.

Aquaponic systems do require a large capital investment, moderate energy inputs and skilled

management. Niche markets may be required for profitability.

Most of the fecal waste fish generate should be removed from the waste stream before it

enters the hydroponic tanks. Other sources of particulate waste are uneaten feed and organisms

(e.g., Bacteria, Fungi and Algae) that grow in the system. If this organic matter accumulates in

the system, it will depress Dissolved Oxygen (DO) levels as it decays and produce carbon

dioxide and ammonia. If deep deposits of sludge form, they will decompose anaerobically

(without oxygen) and produce methane and hydrogen sulfide, which are very toxic to fish. This

enhances microbial activity and increases the mineralization rate.

A major concern in aquaponics systems is the removal of ammonia, a metabolic waste

product excreted through the gills of fish. Ammonia will accumulate and reach toxic levels

unless it is removed by the process of nitrification in which ammonia is oxidized first to nitrite,

which is toxic, and then to nitrate, which is relatively non-toxic. Two groups of naturally

occurring bacteria (Nitrosomonas and Nitrobacter) mediate this two-step process. Nitrifying

bacteria grow as a film (referred to as bio film) on the surface of inert material or they adhere to

organic particles. Bio filters contain media with large surface areas for the growth of nitrifying

bacteria. Aquaponic systems have used bio filters with sand, gravel, shells or various plastic

media as substrate. Bio filters perform optimally at a temperature range of 77 to 86 °F, a pH

range of 7.0 to 9.0, saturated DO, low BOD (<20 mg/liter) and total alkalinity of 100 mg/liter or

as additional surface area is provided by plant roots and a considerable amount of ammonia is

taken up by plants.




Fig No 01: Aquaponic System

Nitrification efficiency is affected by pH. The optimum pH range for nitrification is 7.0 to

9.0, although most studies indicate that nitrification efficiency is greater at the higher end of this

range (high 8s). Most hydroponic plants grow best at a pH of 5.8 to 6.2. The acceptable range for

hydroponic systems is 5.5 to 6.5. The pH of a solution affects the solubility of nutrients,

especially trace metals. Essential nutrients such as iron, manganese, copper, zinc and boron are

less available to plants at a pH higher than 7.0, while the solubility of phosphorus, calcium,

magnesium and molybdenum sharply decreases at a pH lower than 6.0. Compromise between

nitrification and nutrient availability is reached in aquaponics systems by maintaining pH close

to 7.0. Nitrification is most efficient when

For maximum growth, plants in aquaponics systems require 16 essential nutrients. These

are listed below in the order of their concentrations in plant tissue, with carbon and oxygen being

the highest. The essential elements are arbitrarily divided into macronutrients, those required in

relatively large quantities, and micronutrients, those required in considerably smaller amounts.

Three of the macronutrients—Carbon (C), Oxygen (O) and Hydrogen (H)—are supplied by




water (H2O) and carbon dioxide gas (CO2). The remaining nutrients are absorbed from the

culture water. Other macronutrients include Nitrogen (N), Potassium (K), Calcium (Ca),

Magnesium (Mg), Phosphorus (P) and Sulfur (S). The seven micronutrients include Chlorine

(Cl), Iron (Fe), Manganese (Mn), Boron (B), Zinc (Zn), Copper (Cu) and Molybdenum (Mo).

These nutrients must be balanced for optimum plant growth. High levels of one nutrient can

influence the bioavailability of others.

Maintaining high DO levels in the culture water is extremely important for optimal plant

growth, especially in aquaponics systems with their high organic loads. If DO is deficient, root

respiration decreases. This reduces water absorption, decreases nutrient uptake, and causes the

loss of cell tissue from roots. The result is reduced plant growth. Low DO levels correspond with

high concentrations of carbon dioxide, a condition that promotes the development of plant root

pathogens. Root respiration, root growth and transpiration are greatest at saturated DO levels.

Climatic factors also are important for hydroponic plant production. Production is

generally best in regions with maximum intensity and daily duration of light. Growth slows

substantially in temperate greenhouses during winter because solar radiation is low.

Supplemental illumination can improve winter production, but is not generally cost effective

unless an inexpensive energy source is available. Water temperature is far more important than

air temperature for hydroponic plant production. The best water temperature for most hydroponic

crops is about 75 °F.

Dissolved nutrients are measured collectively as Total Dissolved Solids (TDS), expressed

as ppm, or as the capacity of the nutrient solution to conduct an Electrical Current (EC),

expressed as millimhos/cm (mmho/cm). In a hydroponic solution, the recommended range for

TDS is 1,000 to 1,500 ppm (1.5 to 3.5 mmho/cm). In an aquaponics system, considerably lower

levels of TDS(200 to 400 ppm) or EC (0.3 to 0.6 mmho/cm) will produce good results because

nutrients are generated continuously.

Concern with aquaponics systems is nutrient accumulation. High feeding rates, low water

exchange and insufficient plant growing areas can lead to the rapid buildup of dissolved nutrients

to potentially phototoxic levels. Phyto toxicity occurs at TDS concentrations above 2,000 ppm or




EC above 3.5 mmho/cm. Because aquaponics systems have variable environmental conditions

such as daily feed input, solids retention, mineralization, water exchange, nutrient input from

source water or supplementation, and variable nutrient uptake by different plant species,

Aquaponics systems are quite simple to operate when fish are stocked at a rate that

provides a good feeding rate ratio for plant production. Aquaponic systems are easier to operate

than hydroponic systems or recirculating fish production systems because they require less

monitoring and usually have a wider safety margin for ensuring good water quality. Operating

small aquaponics systems can be an excellent hobby.

Systems can be as small as an aquarium with a tray of plants covering the top. Large

commercial operations comprised of many production units and occupying several acres are

certainly possible if markets can absorb the output. The educational potential of aquaponics

systems is already being realized in hundreds of schools where students learn a wide range of

subjects by constructing and operating aquaponics systems. Regardless of scale or purpose, the

culture of fish and plants through aquaponics is a gratifying endeavor that yields useful products

food.




3. MATERIALS

3.1 Set up of Aquaponics system by using Gold Fish

The aquaponics system is constructed by using fish tank, having four Gold Fish (shown

in the Figure No 02, tank for the growth of plants and the additional tank for the recirculation of

water based on the non-interacting tank system shown in the Figure No 03. Proper food is given

to the fishes regularly twice in a day.

Fig No 02: Gold fish

Fig No 03: Non interacting system used for the Aquaponics set up

3.2 Finding out concentration of Ammonia using Ammonia test strips

The ammonia concentration is estimated by using Ammonia Test Strips as shown in the

Figure No 04

Fig No 04: Ammonia Test Strips




3.3 Identification of micro-organism associated with the nitrogen cycle- Nitrobacter and

Nitrosomonas

As per the review of literature, Nitrosomonas and Nitrobacter are selected for the

conversion of ammonia and nitrites in the fish tank.

3.4 Media preparation, Sub culturing and proper inoculation of organisms to the system

The media as prepared by using the fallowing nutrients as shown in the Table No 01 & 02

and maintained aseptic conditions. The proper procedure were followed for the sub culturing and

inoculation of organisms

Table No 1: Media Composition for the Nitrobacter

Solution 1:-

Sl No Nutrients Composition

1 MgSO4 0.2g

2 K2HPO4 1g

3 FeSO4 50 mg

4 CaCl2 20 mg

5 MnCl2 2 mg

6 Na2MoO4 1 mg

7 Distilled water 1 L

Adjust pH to 8.5 with NaOH Sterilization at 121°C for 15 min.

Solution 2:-


1 NaNO2 6g

2 Distilled water 100mL

To 100 mL of solution I, and 5 mL of solution II is added




Table No 02: Media composition for the Nitrosomonas

Solution 1:-


1 K2HPO4 1g

2 MgSO4 0.2g

3 CaCl2 20 mg

4 FeSO4 50 mg

5 MnCl2 2 mg

6 Na2MoO4 1 mg

7 Distilled water 1 L

Add 0.5 % CaCO3 to the medium after adjusting the pH

Solution 2 :-

Sl No Nutirents Composition

1 NH4Cl 3g

2 Distilled water 100 mL

To 100.0 mL of solution I, and 5.0 mL solution II is added

Fig No 05: Media, Autoclave, Incubater, Laminar air flow




3.5 Selection of plants based on water growing plants

Based on the review, Pothos is selected for the present work. It is shown in the figure

Fig No 06: Pothos Plant (Money Plant)

3.6 Absorption & conversion studies by the plant source of converted materials

For the absorption and conversion study, the ammonia and nitrates level is estimated by using

the Ammonia and Nitrite strips and the level of ammonia and nitrates and its conversion is

analyzed and tabulated in results

Fig No 07 :- Nitrite and Nitrate strips

3.7 Proper temperature and pH maintenance

The proper temperature is measured by using thermometer and maintained and pH is

measured by using pH meter and maintained by adding proper amount of acid or base.




Fig No 08 :- pH Meter

3.8 Dissolved oxygen in samples are estimated by using Wrinkles method

Dissolved oxygen is estimated by using wrinkles method by fallowing the below procedure

Collect 10 mL of sample. Add 0.10 mL of conc. H2SO4 and 4 drops of KMNO4

solutions.

After 5 mins, add 0.1 mL of potassium oxalate solution ,and 0.5 mL of alkaline KI

Titrate the above with sodium thio sulphate solution. The iodine liberated from KI is

directly proportional to the Dissolved Oxygen in the sample.

3.9 Plant growth estimation

Plant growth is estimated by measuring the plant length for every 3 days once. The plant

length is measured by using scale and the development is tabulated.

3.10 Recirculation of water to the system regularly

Recirculation of water is done by using the motor, which is connected to the drain tank.

Water is collected to the tank from the plant tank.




4.0 METHODOLOGY

Setting up of an Aquaponics system by using gold fish

Estimation of Ammonia and nitrates concentration in the water

Media preparation, Inoculation and sub culturing of bacteria

Introducing bacteria into the Aquaponics system

Estimation of ammonia for every 24 hours by using Ammonia Test Strip

Maintain pH by using pH meter for every 24 hour duration

Maintain the temperature (25-270C)

Estimation of dissolved oxygen for every 24 hours

Every 24 hours 2% fresh water addition

Plant growth estimation for every 72 hours

Recirculation of water regularly




5.0 RESULTS

5.1 Set up of Aquaponics system by using Gold Fish

Aquaponics system is developed based on non-interacting system by using fish tank

having the gold fish, motor, plant growing tank having the plant Pothos (Money Plant) and

collection tank or the drain tank. The setup of the aquaponics system based on the materials

having the principle of Non interacting system is showed in the figure No 03

5.2 Identification of micro-organism associated with the nitrogen cycle- Nitrobacter and

Nitrosomonas

As per the review of literature, Nitrosomonas and Nitrobacter are selected for the

conversion of ammonia and nitrites in the fish tank.

5.3 Media preparation, Sub culturing and proper inoculation of organisms to the system

Nutrient agar media was prepared, inoculation of Nitrobacter and Nitrosomonas, sub cultured

periodically, introducing the microorganism into the fish tank, regularly proper feeding of the

food

5.4 Finding out concentration of Ammonia using Ammonia test strips

Ammonia estimation by using ammonia strip regularly for period of 24 hours once. Less

ppm of ammonia indicates the conversion of ammonia and nitrites by using microorganisms

5.5 Proper temperature and pH maintenance

For the proper growth of plant, optimum Temperature and pH should be maintained. pH

is an important factor for the growth . So any changes in the pH can be adjusted by adding

suitable acids or bases. The pH will be measured using pH meter. Measured pH will be

monitored and tabulated in the results. Based on the results, Proper pH is maintained to help the

growth of plant.

As per the reviews, the optimum temperature is 25-270C. So throughout the work All the

objectives are maintained with Room temperature around 24-28oC. In future objectives, the




optimum Temperature for the growth of pothos by using aquaponics system will be estimated

and determined.

5.6 Absorption & conversion studies by the plant source of converted materials

Adsorption studies were done measuring and analysis the ammonium content in the fish

tank and the ammonium content in the tank where the plant is kept growing. The difference in

the ammonium level shows that the biological conversion of ammonia and nitrates in to nitrites

by using microorganisms Nitrobacter and Nitrosomonas. The ammonia level differences in the

fish tank and the plant tank shows the adsorption and conversion by the microorganisms. These

results were tabulated in the Table No 03 and the variation in the ammonia, nitrates and nitrite

level is graphically presented in the Fig No 07

Days Ammonia Nitrite Nitrate

1 0.8 0 0

2 2 0.1 0

3 3.1 0.5 0

4 3.9 0.9 0

5 5.2 1.5 0.2

6 6.8 2 0.4

7 6.5 3.4 0.5

8 6.2 4.9 0.6

9 6 5.5 1.1

10 5.7 7.5 1.3

11 5.2 7.4 1.8

12 5.1 7.1 2.1

13 4.8 6.8 2.4

14 4.2 6.5 3.1

15 3.8 5.5 3.6

16 3.5 4.8 4

17 2.9 4.3 4.6

18 2.5 3.7 5.2

19 2.3 3.1 6.5

20 2.1 2.5 7.9

21 2.3 2 8

22 2.1 1.4 8.2

23 1.9 1.2 8.5

24 1.8 1.3 8.8

25 1.9 1.2 8.6

Table No 03: Ammonia, Nitrites and Nitrates concentration




The above charts show the conversion of Ammonia into Nitrites and into Nitrates, which

indicates the flow of nitrogen from the excreta of the Fish into the plant in the form of Nitrates

Fig No 09: Conversion of Ammonia, Nitrite and Nitrate

5.7 Dissolved oxygen in samples are estimated by using Wrinkles method

Dissolved oxygen is estimated for the proper survival of fishes for every 24 hours, and

results are tabulated in the Table No 04, if the Dissolved Oxygen (DO) level is very low means

it’s very difficult to survive the plants. So every day DO will check and maintained the proper

levels by adding additional water and by removing the fish waste (excretes) and the uneaten feed

which will be deposited in the floor of the tank. As per the review, 7-9ppm will be suitable for

the proper fish growth

No of

days

Dissolved

Oxygen in ppm

No of

days

Dissolved

Oxygen in ppm

1 8.8 13 7.5

2 8.6 14 7.3

3 8.2 15 7.2

4 7.9 16 7.8

5 7.7 17 7.6

6 7.5 18 7.4

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25 30

PP

M b

u u

sin

g te

st s

trip

s

No of days

CONVERSION LEVELS

Ammonia

Nitrite

Nitrate




7 7.6 19 7.2

8 7.7 20 7.6

9 7.8 21 7.5

10 8 22 7.4

11 7.9 23 8

12 7.7 24 7.7

Table No 04: Dissolved oxygen level in ppm

Fig No 10: Dissolved Oxygen level in fish tank

5.8 Plant growth estimation

Plant growth is estimated by measuring the plant length for every 3-4 days, the selected

plant pothos growth is measured by using the plant body length. The length will be measured by

using the scale. The plant growth is measured with regular intervals of 3 days for a month. The

results are tabulated in Table No 05 and the growth of the plant is shown in Fig No 09

No of

Days

Pothos Plant

Growth(cms)

3 0.6

6 1

9 1.7

12 2.7

15 3.9

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

D O

IN

PP

M

DISSOLVED OXYGEN IN ppm

Column2




18 5.5

21 7.1

24 8.5

Table No 05: Plant Growth

Fig No 11: Plant Growth Estimation

5.9 Recirculation of water to the system regularly

Recirculation of water is done by using the motor which is connected to the drain tank.

The purpose behind the tank is to save the water. Based on the review the 2% of water is added

regularly to make it fresh for the fish

Fig No 12 :- Draining tank for Recirculation of Water

0

2

4

6

8

10

3 6 9 12 15 18 21 24

pla

nt

gro

wth

in

cm

s

No of days for the plant growth

POTHOS

PLANT

GROWTH




6.0 FUTURE PROSPECTS

Other effective parameters for the plant growth have to be study.

Fish feeding effect on generation of ammonia have to be study

Dissolved oxygen effect on fish growth

Possibility study of other plants

Optimum conditions calculation for the growth of pothos

TDS calculation in the water samples

Nutrients effects study on plants

Removal of excretes by using biofilms

BOD study




7. REFERENCES:

1. James E. Rakocy, Michael P. Masser and Thomas M. Losordo. ―Recirculating

Aquaculture Tank Production Systems: Aquaponics—Integrating Fish and Plant

Culture‖. 2006

2. Peter m. vitousek, John d. aber, Robert w. howarth,Gene e. likens, Pamela a. matson,

David w. schindler, William h. schlesinger, and david g. Tilman. ―Human alteration of

the Global nitrogen cycle: Sources and consequences‖. 1997

3. Wikipedia- Nitrogen cycle

4. Harry Ako and Adam Baker. ―Small-Scale Lettuce Production with Hydroponics or

Aquaponics‖. 2007

5. K. Arivalagan, S. Ravichandran, K. Rangasamy And E.Karthikeyan , ―Nanomaterials and

its Potential Applications‖ , International Journal of ChemTech Research, Vol. 3, No.2,

pp 534-538.

6. E. V. Korotkaya, ― BIOSENSORS: DESIGN, CLASSIFICATION‖, AND

APPLICATIONS IN THE FOOD INDUSTRY, ISSN 2308-4057. Foods and Raw

Materials Vol. 2, No. 2, 2014.

7. Jesper deClaville Christiansena, Catalina-Gabriela Potarnichea, ―NANOMATERIALS IN

BIOMEDICAL APPLICATIONS‖, Department of Mechanical and Manufacturing

Engineering Aalborg University Aalborg, Denmark

8. Ahmed Touhami, ―Biosensors and Nanobiosensors: Design and Applications‖, Physics &

Astronomy Department, University of Texas at Brownsville One west university

boulevard, Brownsville, Texas, 78520, USA

9. Y.-H. Yun, E. Eteshola, A. Bhattacharya, Z. Dong, J.-S. Shim, L. Conforti, D. Kim, M. J.

Schulz, C. H. Ahn, and N. Watts, ―Tiny medicine: nanomaterial-based

biosensors‖,Sensors 2009,9, 9275-9299.

10. S. Itoh, M. Kikuchi, Y. Koyama, K. Takakuda, K. Shinomiya, and J. Tanaka,

―Development of an artificial vertebral body using a novel biomaterial,

hydroxyapatite/collagen composite,‖ Biomaterials, 2002, 23, 3919-3926.

11. T. Ikoma, T. Muneta and J. Tanaka, ―Fabrication and animal experiment of

nanocomposites of hydroxyapatite collagen and polysaccharides,‖ Key Eng. Mater.,

2000, 192, 487–490.

12. M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya and J. Tanaka, ―Selforganization

mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro

and its biological reaction in vivo,‖ Biomaterials, 2001, 22, 1705–1711.

13. S. Itoh, M. Kikuchi, Y. Koyama, K. Takakuda, K. Shinomiya and J. Tanaka,

―Development of a hydroxyapatite/collagen nanocomposite as a medical device,‖ Cell

Transplant., 2004, 13, 451–461.

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