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LU Umwelttechnik

Sustainability aspects of macronutrients

production and application

Tanja Wallner

1 Abstract

In this manuscript several mini projects about macronutrients are discussed

according to their production, field of application and according to their environmental

impact. The mini projects range from food industry (Xanthan), to clothes industry

(Cellulose) to biotechnology (Lipases) to bioenergy (Biodiesel). It is also discussed

which aspects of these projects cover the green chemistry principles.

The main findings include biochemical waste treatment and arguments for and

against animal testing, advantages of enzyme usage, cascade utilization, distribution

characteristics, closed reaction conditions, viscose process efficiency, fuel stability

and diesel emission reduction.

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2 Problematic

Sugars fats and proteins are renewable resources with many possibilities for

application.

Renewable resources are to be favored over fossil-based products, but the

production step doesn’t have to be neglected. The step from raw material to

commodity is essential to validate sustainability of the whole system.

Therefor I chose the following parts for my sustainability report about macronutrients:

• Characterization of an industrial scale bacterial produced polysaccharide –

Xanthan

Food

• Spinning viscose fibers from cellulose xanthate; dying of fibers by adsorption

and characterization of obtained fiber

Clothes

• Stability toward oxidation of Biofuels: Preparation and oxidation stability test of

Fatty Acid Methyl Esters (FAME) via Transesterification of Vegetable Oils with

Methanol

Fuel – Energy

• Purification and characterization of membrane proteins

Enzymes

2.1 Questions

2.1.1 Enzymes

Purification and characterization of membrane proteins

Membrane proteins are key player in the human metabolism. Insights into their

structure gives us better knowledge about their function and can be of great help to

treat overweight, diabetes and cancer. In addition to that lipases are also used as

environmentally friendly detergents. They are a very good example to understand

enzymes better. Enzymes make up a vast number of catalysts in various ecological

favorable reactions as for example waste water treatment. In my final concept I will

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try to give a brief introduction info protein engineering, structural characterization of

proteins and try to describe the gained knowledge in respect to science and human

health. I also try to critically evaluate the characterization methods in respect to

sustainability and green chemistry requirements. Maybe I also focus on animal

testing in biology.

2.1.2 Viscose

Spinning viscose fibers from cellulose xanthate; dying of fibers by adsorption and

characterization of obtained fiber

Afterwards we investigate the application of polysaccharides in the clothing industry.

Maybe we could compare commonly used fibers (either renewable (e.g. cotton) and

fossil based (e.g. elastane, polyamide etc.) with fibers from cellulose xanthate in

respect to sustainability

2.1.3 Fuels

Stability toward oxidation of Biofuels: Preparation and oxidation stability test of Fatty

Acid Methyl Esters (FAME) via Transesterification of Vegetable Oils with Methanol

In Addition to that we will synthesize biofuels out of triglycerides and test their

oxidation stability. As the synthesis is carried out on conditions very similar to

industrial production. Hopefully we will establish a greater understanding about

production processes and the problems of fossil fuel replacement. Maybe we could

also compare the stability of commonly used fuels. The substitution and the effects

on the engine and on the environment.

2.1.4 Xanthan

Characterization of an industrial scale bacterial produced polysaccharide – Xanthan

First, we will establish a reasonable data sheet for the poly saccharide Xanthan to

further use it as a bulk chemical and for food industry. Here it is important to get an

impression what green chemistry really is, how the measurements work and what

characteristics are needed to “convert” a raw material into an industrially usable

substance.

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3 State of the Art

3.1 Enzymes: Protein Lipases

Enzymes are sustainable widely used reagents in a vast number of processes. They

speed up reactions by lowering the activation energy to increase the efficiency of the

whole process. The aim of structural biology is to identify and classify unknown

enzyme structures. Although the amino acid sequence of the protein is known, there

is still no reliable method to predict or calculate the 3D folding. The so-called tertiary

structure (3D constellation of the amino acids sequence) can give important insights

into the function of the protein and the human metabolism, as well as strategies in

medical treatment. It enables the possibility to classify proteins and to gain an

improved understanding of biochemistry.

3.1.1 Animal testing

For many biochemical studies animal testing is still a common method.

Recent estimates of worldwide annual laboratory animal use are imprecise and

unsubstantiated, ranging from 28–100 million.i In the following section positive and

negative aspects of animal testing are summarized:

3.1.1.1 Pro Animal Testing

• Animal testing has contributed to many life-saving cures and treatments.

• There is no adequate alternative to testing on a living, whole-body system.

• Animals are appropriate research subjects because they are similar to human

beings in many ways.

• Animals must be used in cases when ethical considerations prevent the use of

human subjects.

• Animals themselves benefit from the results of animal testing. (animal

medicine)

• Animal research is highly regulated, with laws in place to protect animals from

mistreatment.

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• Animals often make better research subjects than human beings because of

their shorter life cycles.

• Animal researchers treat animals humanely, both for the animals' sake and to

ensure reliable test results.

3.1.1.2 Con Animal Testing

• “Animal testing is cruel and inhumane.”

• Alternative testing methods now exist that can replace the need for animals.

• Animals are very different from human beings and therefore make poor test

subjects.

• Animal tests may mislead researchers into ignoring potential cures and

treatments.

• Drugs that pass animal tests are not necessarily safe.

• 95% of animals used in experiments are not protected by the Animal Welfare

Act.

• Animal tests do not reliably predict results in human beings. 94% of drugs that

pass animal tests fail in human clinical trials.

• Humane Society International compared a variety of animal tests with their in

vitro counterparts and found animal tests were more expensive in every

scenario studied.

3.1.1.3 Further Explanations:

“Animal testing is cruel and inhumane.” According to Humane Society International,

animals used in experiments are commonly subjected to force feeding, forced

inhalation, food and water deprivation, prolonged periods of physical restraint, the

infliction of burns and other wounds to study the healing process, the infliction of pain

to study its effects and remedies, and "killing by carbon dioxide asphyxiation, neck-

breaking, decapitation, or other means."

Alternative testing methods now exist that can replace the need for animals.

In vitro testing, such as studying cell cultures in a petri dish, can produce more

relevant results than animal testing because human cells can be used. Microdosing,

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the administering of doses too small to cause adverse reactions, can be used in

human volunteers, whose blood is then analyzed. Artificial human skin, such as the

commercially available products EpiDerm and ThinCert, is made from sheets of

human skin cells grown in test tubes or plastic wells and can produce more useful

results than testing chemicals on animal skin. Microfluidic chips ("organs on a chip"),

which are lined with human cells and recreate the functions of human organs, are in

advanced stages of development. Computer models, such as virtual reconstructions

of human molecular structures, can predict the toxicity of substances without invasive

experiments on animals.

Animals are very different from human beings and therefore make poor test subjects.

Animal tests may mislead researchers into ignoring potential cures and treatments.

Some chemicals that are ineffective on, or harmful to, animals prove valuable when

used by humans. Aspirin, for example, is dangerous for some animal species.ii

3.1.2 Laboratory waste management and Process design

As in many laboratories also in biochemistry laboratories there is a lot of waste

produced. It is necessary to sterilize biological active compounds. In addition to that

radioactive waste from biological tracers must be treated. But most of the time it’s

due to purity reasons that laboratory equipment is used one way. Further

convenience is also a great point. Therefor a thoughtful waste management is

needed. The most important point is to at first raise awareness about laboratory

waste causing a severe impact on the environment. In Addition to that an efficient

working strategy must be developed and passed to successors. As green chemistry

states, “waste prevention” in the first place is crucial and techniques which require

less hazardous substances are preferable.iiiIf the waste is inevitable a good

separation is crucial. Further information iv

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3.1.3 Environmentally friendly and benefiting enzymes

Enzymes are used in industries including agro-food, oil, animal feed, detergent, pulp

and paper, textile, leather, petroleum, and specialty chemical and biochemical

industry. They help to maintain an unpolluted environment through their use in waste

management.v

Examples:

• Waste water treatment, Steroid binding in waste water treatment

• Microbiological biodegradation of petroleum contaminated soil

• Fermentation processes for bioethanol, Cellulases and ligninases for biofuel

• Microbiological enzyme production nucleases, DNA ligase and polymerases

• Xylanases for paper industry

• Proteases and lipases and amylases as detergents, desizining of textile fibers

, The application of cellulases for denim finishing and laccases for

decolorization of textile effluents and textile bleaching

• Acylase racemate cleavage

• Protease cheese production and patain to tenderize meat, Amylases,

betaglucanases, acetolactases for brewing industry, amylase starch

production vi

3.2 Viscose: Textiles

3.2.1 Comparison natural synthetic fibers

Fibers are divided into three main categories:

• Natural – like flax, wool, silk and cotton

• Manufactured – made from cellulose or protein

• Synthetic – made from synthetic chemicals.vii

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The difference between “manufactured” and “synthetic” fibers is that the

manufactured fibers are derived from naturally-occurring cellulose or protein, while

synthetic fibers are not. viii

In 2015 62% of the global fiber consumption was covered by fossil based synthetic

fibers, 25% by cotton, 6% by bio based synthetic fibers 1% by wool and 5% other

natural fibers.

figure 1 global fiber consumption

3.2.1.1 Advantages of synthetic bio-based fibers

Viscose is a man maid manufactured but not synthetic fiber made of chemically

designed cellulose units and therefore called regenerated cellulose fiber. The

advantage of fiber engineering is the possibility to adjust the fiber to our wishes.

Parameters like, thickness, tenacity, elasticity, resistance can be engineered within

the viscose process.

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figure 3 chemical reaction viscose process

3.2.2 Viscose process

In the viscose process, cellulose is treated

with caustic soda (aka: sodium hydroxide) and

carbon disulfide, converting it into a gold,

highly viscous liquid about the color and

consistency of honey. This substance gives its

name to the manufacturing process, called the

viscose process.

The viscous fluid is allowed to age, breaking

down the cellulose structures further to

produce an even slurry, and is then filtered to

remove impurities. Then the mixture is forced

through fine holes, called a spinerette, directly

into a chemical bath where it hardens into fine

strands. When washed and bleached these

strands become rayon yarn.ixx

The Chemical Reaction is shown below xixii

figure 2: viscose process scheme

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3.2.3 Byproducts their environmental impact and toxicity

This process needs sulfuric acid (H2SO4) and Carbon disulfide (CS2) and Hydrogen

sulfide (H2S) is produced as by product. Sulfuric acid, which is used in the spinning

step, is toxic for fish with a LD50 of 1.67 mg and human skin contact leads to burns

and irritation of the respiration when inhalating aerosols.xiiixiv Carbon disulfide is toxic

to organs, influences the reproductivity, irritates the skin and causes a severe damage

to the eyes when exposed.xvHydrogen sulfide, has a LD100 of 500-1000 parts per

million (ppm). Exposure to lower concentrations leads to such as 10-500 ppm, can

cause various respiratory symptoms that range from rhinitis to acute respiratory failure.

H2S may also affect multiple organs, causing temporary or permanent derangements

in the nervous, cardiovascular, renal, hepatic, and hematological systems. xvi

3.2.4 Alternatives: Lyocellverfahren

Pulp are dissolved in N-methylmorpholine N-oxide (NMO). The filtered cellulose

solution is then pumped through spinnerets, devices. The outcoming fibers are drawn

in air to align the cellulose molecules. The fibers are then immersed into a water

bath, where desolvation of the cellulose sets the fiber strands; the bath contains

some dilute amine oxide in a steady state concentration. Then the fibers are washed

with de-mineralised water. The Lyocell fiber next passes to a drying area, where the

water is evaporated from it. NMO exposure leads to irritation of the skin respiratory

system and eyes. Acute toxicity of amino oxide in fish, as indicated by 96h LC50

tests, is in the range of 1,000–3,000 ug L−1 for carbon chain lengths less than C14.

LC50 values for chain lengths greater than C14 range from 600 to 1400 ug L−1.xvii

The amine oxide used to dissolve the cellulose and set the fiber after spinning is

recycled. 98% of the amine oxide is typically recovered. Since there is little waste

product, this process is relatively eco-friendly.xviii

The main advantage is the closed cycle process.

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3.3 Fuels: FAME

3.3.1 Fatty acid methyl esters (FAME)

FAME are a type of fatty acid ester that are derived by transesterification of fats with

methanol. The molecules in biodiesel are primarily FAMEs, usually obtained from

vegetable oils by transesterification. They are used to produce detergents and

biodiesel.

figure 4 transesterification reaction resulting in FAME xix

3.3.2 FAME Production

Transesterification of a vegetable oil is carried out with methanol under catalysis with

potassium hydroxide. For homogenization of the reaction mixture a high-speed stirrer

Ultra-Turrax is used. The reaction is carried out in 2-steps (more steps in industrial

scale), after which the formed glycerol is separated by centrifugation. The obtained

methyl esters are washed with water and dried.

3.3.3 Problems of fossil fuel replacement

Fossil based Diesel is a mixture of hydrocarbons without oxygen in the structure. In

contrast to that bio-based Diesel is a mixture of fatty acid methyl esters with oxygen

in the ester bond. Because of that fact Bio Diesel is less stabile to oxidation than

fossil-based Diesel. Therefore, Oxidation Stability has to be tested to assure a good

driving performance and to be prone of engine damages. The low aromatic and sulfur

content in biodiesel reduces SO2 emissions and particle emissions. In addition to that

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lower hydrocarbon, CO and fine dust emissions are emitted because of the higher

oxygen content.

In contrast to that NOx emissions are higher are observed. These components also

depend on (injection time, ignition delay and adiabatic flame temperature. xx

3.3.4 Emissions/exhaust fumes

The main pollutants are listed below:

• Carbon dioxide (CO2)

• Carbon monoxide (CO)

• Nitrogen oxides (NOx)

• Ammonia (NH3)

• Polycyclic aromatic hydrocarbons

• Volatile organic compounds

• Sulfur dioxide (SO2)

• Particle emissions

• Soot

• Fine dust xxi

3.3.4.1 Catalytic converters

These are the most common catalysts for exhaust fume waste treatment

• Tree-way catalytic converter

• The NOx-Reduction catalyst

• Selective catalytic reduction via urea

• Particle filter

The Tree-way catalytic converter converts carbon monoxide, nitrogen oxide and

hydrocarbons into carbon dioxide, nitrogen and water.

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The reaction steps are:

Reduction of nitrogen oxides to nitrogen

Oxidation of carbon monoxide to carbon dioxide

Oxidation on unburnt hydrocarbons to carbon dioxide and water

figure 5 reaction in the converter xxii

3.4 Xanthan: Food Applications

3.4.1 Xanthan polysaccharide

Xanthan Gum is a 1,4-linked beta-D-glucose polysaccharide with five sugars

containing different residues. It forms helical fibers. The indicated (distribution)

molecular weight of Xanthan is around 2000 kDa.

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figure 6 Xanthan Gum structure

In Aqueous solution it forms gums and gels and is used as flocculant, binder. Film

former, lubricant and friction- reducer. It is industrially produced via fermentation with

the bacterium Xanthomonas campestris. Due to aggregation most of the material

characteristics an only be given in form of distributions. In general, it is highly viscose

also in low concentrations. It shows high viscosity at low shear rates and a high

elastic modulus. It is stable to ionic variations, heat, change in pH, shear stress,

enzymes, and chemicals like salts, acids and bases. It is used in food industry for

texture and freezing stability in baked goods, beverages, confectionery, dairy

products, desserts and dressings. In addition to that it is used in personal care

applications as in dispersed oil phases, shampoos, make-up, lotions and creams,

and liquid soaps. Further it is used in pharmaceutical applications and oral care

applications like toothpastes. In industrial applications it is used in acid-based

cleaners, animal feed, surface coatings, carpet printing and fire-fighting foam xxiii

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3.4.2 Datasheet (relevant numbers)

A useful datasheet has to include all relevant characteristics of the molecule.

In case of a polymer the chemical composure (REACH registered Monomer),

dimensions, geometry and field of application and are needed. In Addition to that also

interactive properties and dynamic properties (mobility, stability, sheer stress

behavior) must be considered.

As we speak of a colloidal system most of the parameters are given as distributions.

For example:

• Mass distribution

• A distribution for the number of molecules

• Volume distribution

• Degree of polymerization distribution

• Viscosity distribution

It is possible to arithmetically calculate the mean values of these distributions, but it

has to be considered that even though they are comparable to mean values of other

substances, they include muss less information than the whole distribution.

Additionally, the relevance of mean values derived from distribution decreases with

increasing width of distributions and rather are zero-approach estimations than

significant information. Range of distribution, percentages of upper and lower limiting

components, dominant mass or molar fractions and distribution of distinct and

separated populations of components or continuous broad distribution provide much

more information about investigated distributions than mean values.xxiv

3.4.3 Green chemistry

Green Chemistry is an attempt to design chemical processes and commercial

products without toxics and waste. It’s a sustainable approach to include energy

conservation, waste reductant and life cycle considerations.

The main principles of green chemistry are:

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1. Prevent waste

2. Design safer chemicals and products

3. Design less hazardous chemicals syntheses

4. Use renewable feedstock chemicals

5. Use catalysts not stoichiometric reagents

6. Avoid chemical derivates

7. Maximize atom economy

8. Use safer solvents and reaction conditions

9. Increase energy efficiency

10. Design chemicals and products to degrade after use

11. Analyze in real time to prevent pollution

12. Minimize the potential of accidents

In addition to that also main principles of green engineering are formulated:

1. Inherent Rather Than Circumstantial

2. Prevention

3. Atom Economy

4. Less Hazardous Chemical Syntheses

5. Designing Safer Chemicals

6. Safer Solvents and Auxiliaries

7. Design for Energy Efficiency

Use of Renewable Feedstocks

8. Reduce Derivatives

9. Catalytic reagents (as selective as possible) are superior to stoichiometric

reagents.

10. Design for Degradation

11. Real-time analysis for Pollution Prevention

12. Inherently Safer Chemistry for Accident Preventionxxv

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3.4.4 Cascade usage

Further it is fundamental to consider a certain kind of process design hierarchy to

make a process as efficient as possible. This hierarchy called cascade utilization.

This approach prefers the application of renewable resources at first on the food

marked, followed by the chemical market and as the last step biomass for energy

production.

4 Materials Methods Results and perspektives

4.1 Enzymes

To solve a protein structure an assessable source of the protein of your interest is

needed. There are two main approaches to isolate proteins either from an already

available organic sample or in the laboratory via microbiological expression.

figure 7 Steps to get a crystal structure

As a next step, the bacterial colony has to be harvested. The purification step starts

afterwards. Different proteins require different purifications techniques. These

techniques (MBP Column's, Ni-NTA Affinity Resin, Strep Tactin to name just a few)

can be implemented with the help of new technologies like cloning and side directed

mutagenesis. If no bacterial expression is used, the harvesting can be neglected but

there are less possibilities to implement the above-mentioned methods. In the case of

the Lipo-Protein-Lipase (LPL) purification Heparin Colum's are chosen, because a

big domain of LPL shows a very high affinity to them. Afterwards, the separation

Get source / microbiological

expression

Isolate and adjust Protein

Crystalize or solve Protein

Measure structural date

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according to the volume of the Lipase in solution is done by Size Exclusion

Chromatography.

a) b) c)

figure 8: purification chromatograms: Chromatography and Electrophoresis Gels

a) First manual heparin purification. This step: washes Casein and Lactoglobulin out of the solution

b) Second heparin purification step

c) Result of the size exclusion purification:

now the Antithrombin 3 cannot be observed anymore

After the purity is checked by spectrometric measurements and Electrophoresis Gels,

a technique to get structural data has to be chosen. For small molecules NMR is

suitable. In case of larger molecules like proteins, solid state NMR is possible. X-Ray

measurements are chosen is this case because they are the most established

technique at that moment. It should be mentioned that this method is relatively

reliable, but it needs a protein crystal to diffract on.

The crystallization was the main challenge on this project. There are attempts to

crystallize LPL since 1970. To crystallize a relatively amorphous protein, a very high

purity is needed. The yield of an 8L Milk purification is approximately 1mg of Protein.

The pure protein was mixed with a vast amount of (approx. 2000) different

standardized crystal screen conditions. Only 0,5% of these conditions showed a

crystal-like behavior. Weekly microscopical examination for the duration of 6 months

showed 5 promising protein crystals were picked and measured on the X-Ray anode.

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Most of them where salt crystals caused by the crystal screen conditions itself. Only

one crystal could be a LPL crystal but it didn‘t diffract.

figure 9: (top down left right) Promising crystal conditions with their position and exact chemical condition

Crystal Screen 29.4. (P- Buffer) A6-6, 0.2 M Magnesium chloride hexahydrate, 0.1 M TRIS hydrochloride pH 8.5,

30% w/v Polyethylene glycol 4,000

Index 29.4 (P- Buffer), D1-37, 25% w/v Polyethylene glycol 1,500

JCSG 12.5, A1-1, 0.2 M Lithium sulfate 0.1 M Sodium acetate 4.5 50 % w/v PEG 400

Diffracting crystal, bMGL-C14 complex initial

After these projects, other experiments had been planned for ssNMR measurements,

Binding Studies and other reagents to stabilize LPL (e.g. GPIHBP1 Peptide Fragment

experiments and other Assays)

4.2 Viscose

In order to design a durable viscose fiber with new features the raw material and

biopolymer cellulose has to be broken down via the viscose process. In this lab we

got a sample of Cellulosexantate from Lenzingxxvi. We made solutions with a different

amount of cellulose nano-crystals and compared the resulting fiber thickness. In

addition to that we also measured the reactant (Cellulosexantate) and the product

(viscose) via IR spectroscopy

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Produced viscose fiber

After the viscose process two monomer glucose subunits contain a maximum of one

xanthate residue. xxvii

figure 10 Cellulose xanthate after the viscose process

figure 11: IR spectra viscose fiber

We tried to wash off all Xanthate residues. Infrared measurements showed that this

washing step hast to be improved to get rid of every single sulfur, indicated by the peak

at 1640 1/cm for double bonds (in our case C=S double bonds).

4.3 Fuels

FAME

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The FAME was produced through esterification of vegetable oils and analytically

characterized as follows:

Ester content:

• FT-Infrared Spectroscopy (FT IR)

• Thin Layer Chromatography (TLC)

• Gas chromatography with flame ionization detection (GC FID)

• Gelpermeation Chromatography (GPC/SEC)

Oxidation stability:

• Petrooxy,

• Ranzimat

Viscosity and density

• Viskosimeter

GC FID Measurements are standardized EN 14214 norms. The ester content has to

be above 96,5 %.

Table 1: results biofuels

Öl Pflanze IV

(Iod-

Zahl)

[g

I2/100g]

Oxidationsstabilität Estergehalt [%]

PetroOxy

® [min]

Ranzima

t [h]

FTIR TLC

(circa

-

Wert)

GC-

FID

GPC

A Olive 75,1 26,68 14,61 87,1

8

90 96,8 85,1

4

B Raps 105,5 26,03 3,20 88,6

9

85 88,4 82,5

5

C Sonnenblum

e

133,7 13,91 2,52 92,1

4

85 99,0 90,9

4

D1 Distel - - - 88,9

8

- - 84,8

5

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D2 Distel 91,78 25,87 1,32 90,9

7

90 95,9 86,5

2

E Maiskeim 119,3 23,83 4,27 91,9

1

85-90 88,1 83,0

7

Table 2results viscosity density

Öl Pflanze

Dichte [kg/m3]

Viskosität

[mm/s2]

Öl

Methylest

er Öl

Methylest

er

A Olive 898,9 868,4

39,40

7 6,829

4.4 Xanthan

Not finished yet

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5 Summary

Enzymes are sustainable widely used reagents in a vast number of processes. They

speed up reactions by lowering the activation energy to increase the efficiency of the

whole process. Structural Biology gives insights into the mechanism of this molecular

machines and helps to gain a better understanding and more efficient biological

processes. As in every laboratory also in biochemistry labs many different kind of

wastes are produced and depending on the scientific topic also animal testing can be

include.

Xanthan is produced microbiological with help of enzymes. This polysaccharide is

used according to cascade process principle. (Food before Chemicals - Chemicals

before Bioenergy). In addition to that renewable resources and non-hazardous

substances are used during the production but it is very difficult to get feasible

characteristics of the product.

As a green chemistry principle (Minimize the potential of accidents) cellulose

production is performed in a closed circle to prevent emissions but there are still

improvements to make in the field of waste treatment.

As the last step of cascade utilization energy can be processed from biomass. In this

case fuels with a low iodine value are more stable than fuels with a high iodine value.

In addition to that we could use waste from agricultural overproduction, biorefineries

or butchery waste. Further it should be mentioned that biodiesel causes less

emissions than fossil-based Diesel. It would be possible to add more Biodiesel

(nowadays 7%) to the daily used engine fuel.

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i http://www.animalexperiments.info/studies/numbers/worldwide.html

ii https://animal-testing.procon.org/

iii https://www.acs.org/content/acs/en/greenchemistry/what-is-green-

chemistry/principles/12-principles-of-green-chemistry.html

iv https://www.ncbi.nlm.nih.gov/books/NBK55885/

v https://www.ncbi.nlm.nih.gov/pubmed/15493529

vi https://de.wikipedia.org/wiki/Enzym#Enzyme_in_der_Technik

vii http://www.coatsindustrial.com/en/information-hub/apparel-expertise/know-about-

textile-fibres#fibre%20classification 27.05.2018

viii https://oecotextiles.wordpress.com/category/fibers/viscose/

ix https://oecotextiles.wordpress.com/tag/lyocell/

x https://oecotextiles.wordpress.com/category/fibers/viscose/

xi http://costfp1205.com/wp-content/uploads/2017/04/13.-

Roder_final_COST_Stockholm_March_2017.pdf

xii https://de.wikipedia.org/w/index.php?title=Viskosefaser&oldid=177831417.

xiii https://echa.europa.eu/de/registration-dossier/-/registered-dossier/8699/6/2/2.

xiv https://en.wikipedia.org/wiki/Sulfuric_acid#Industrial_hazards

xv www.seilnacht.com/Chemie/ch_cs2.html

xvi https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2850187/

xvii https://en.wikipedia.org/wiki/Amine_oxide#Environmental_safety

xviii https://de.wikipedia.org/wiki/Lyocell#Herstellung

xix https://en.wikipedia.org/wiki/Fatty_acid_methyl_ester

xx https://de.wikipedia.org/wiki/Biodiesel#Abgasemissionen

xxihttps://www.ivt.tugraz.at/images/stories/Files/Skripten/Folien_Sams_Schadstoffbild

ung_Teil_1_2016.pdf

xxii https://secure.lambdapower.co.uk/TechNotes/Tech-10.asp

xxiii https://www.cpkelco.com/products/xanthan-gum/ visited 2018.05.24

xxiv http://ahgroup.at/user_files/SE-&-VO/lecture07/polymerCharacteristics.php

xxv https://www.acs.org/content/acs/en/greenchemistry/what-is-green-

chemistry/principles/12-principles-of-green-chemistry.html

xxvi https://www.lenzing.com/produkte/ 22.05.2018

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xxvii http://kirste.userpage.fuberlin.de/chemistry/kunststoffe/viskose.htm. (Accessed:

2nd June 2018)