Microbial CellulosePrinciples of Biochemistry

Subject Code-MB101

Presented byDeepika Rana Monika Yadav Roll no-1601 Roll no.-1605M.Sc. Microbiology M.Sc. Microbiology 1st Semester 1st SemesterMD University, Rohtak MD University, Rohtak

•Cellulose is a major constituent of plant cell walls, providing strength and rigidity and preventing the swelling of the cell and rupture of the plasma membrane that might result when osmotic conditions favour water entry into the cell. •Each year, worldwide, plants synthesize more than 1011 metric tons of cellulose, making this simple polymer one of the most abundant compounds in the biosphere. •The structure of cellulose is simple: linear polymers of thousands of (β1→4) linked D-glucose units, assembled into bundles of about 36 chains, which aggregate side by side to form a micro-fibril.

USES OF CELLULOSE1. Paper2. Guncotton3. Cellophane4. Movie film5. Frames6. Toys7. Cellulosic ethanol

SOURCES OF CELLULOSE1. Wood (40-50%)2. Cotton (90%)3. Dried hemp (45%)4. Microbes (Varies)

•Microbial cellulose, sometimes called bacterial cellulose, is a form of cellulose that is produced by bacteria.

•Bacterial cellulose is an organic compound with the formula (C₆H₁₀O₅)n produced from certain types of bacteria.

•The glucan chains are held together by inter- and intra- hydrogen bonding.

•Inherent Purity: free of hemicellulose, lignin, pectin, wax

•Moldable in cultivation. Carbon Sources used:Glucose, fructose, sucrose, molasses, glycerol Corn steep liquor, potato effluent, grape pomace, whey lactose Tea, cola nut, Saccharified food waste •Natural network structure, High Crystallinity: ~85%, High DP

•High Carbon-to-Cellulose Conversion Efficiency •Typical cell converts 108 glucose molecules to cellulose per hour

Scanning electron microscopy images of BCmembrane from static culture of A. xylinum (a) andbacterial cell with attached cellulose ribbons (b).www.sciencedaily.com

Overview of Bacterial Cellulose Production and ApplicationFaezah Esa*, Siti Masrinda Tasirin, Norliza Abd RahmanDepartment of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600Bangi, Selangor, Malaysia

A wet microbial cellulose pellicle being removed from a culture

en.wikipedia.org

Bacterial sources•Cellulose can be found in many microorganisms like fungi, bacteria, and algae. It is also found in small quantities in brown algae (Phaeophyta), most of the red algae (Rhodophyta) and most of the golden algae (Chrysophyta). 

•In some fungi(oomycetes), cellulose forms as an inner cell wall layer.

•Bacteria that produce cellulose include Gram-negative bacteria species such as Acetobacter, Azotobacter, Rhizobium,Pseudomonas, Salmonella, Alcaligenes, and Gram-positive bacteria species such as Sarcina ventriculi.

• The most effective producers of cellulose are Acetobacter xylinum, A. hansenii, and A. pasteurianus. Of these, A. xylinum is the model microorganism for basic and applied studies on cellulose due to its ability to produce relatively high levels of polymer from a wide range of carbon and nitrogen sources.

Chemical structure of cellulose

Scanning electron micrographShowing bacterial cellulosefibres

microbialcellulose.blogspot.com

Biosynthesis of Microbial Cellulose•The synthesis of bacterial cellulose is a multistep process that involve two main mechanisms: the synthesis of uridine diphosphoglucose (UDPGIc), followed by the polymerization of glucose into long and unbranched chains (the β-1→4 glucan chain).

• The production of UDPGIc starts with carbon compounds (such as hexoses, glycerol, dihydroxyacetone, pyruvate, and di-carboxylic acids) entering the Krebs cycle, gluconeogenesis, or the pentose phosphate cycle depending on what carbon source is available.

•It then goes through phosphorylation along with catalysis, followed by isomerization of the intermediate, and a process known as UDPGIc pyrophosphorylase to convert the compounds into UDPGIc, a precursor to the production of cellulose.

•The polymerization of glucose into the β-1→4 glucan chain has been hypothesized to either involve a lipid intermediate or not to involve a lipid intermediate. If the bacteria use lipid to initiate new chains, it cannot be sterols-bacteria don’t contain sterols.

Handbook of Polymer Nanocomposites. Processing, Performance and Applicationedited by Jitendra K. Pandey, Hitoshi Takagi

Biochemical Pathway for Cellulose Synthesis GK-Glucokinase PGM-PhosphoglucomutaseUGP-UDP glucose pyrophosphorylaseFBP-Fructose-1,6 bisphosphatase

CS-Cellulose Synthase PFK-PhosphofructokinaseFK-FructokinasePGI-Phosphoglucose isomerase

Glucose

Glucose-6-phosphate

Glucose-1-phosphate

UDP-Glucose

Cellulose

Glukokinase

Phosphglucomutase

UDP-glucose pyrophosphorylase

Cellulose Synthase

Biosynthesis of Cellulose In Acetobacter xylinum

•The complex enzymatic machinery that assembles cellulose chains spans the plasma membrane, with one part positioned to bind the substrate, UDP-glucose, in the cytosol and another part extending to the outside, responsible for elongating and crystallizing cellulose moleculesin the extracellular space.

•Freeze-fracture electron microscopy shows these terminal complexes,also called rosettes, to be composed of six large particles arranged in a regular hexagon. Several proteins, including the catalytic subunit of cellulose synthase, make up the terminal complex.

•Much of the recent progress in understanding cellulose synthesis stems from genetic and molecular genetic studies of the plant Arabidopsis thaliana, which is especially amenable to genetic dissection and whose genome has been sequenced.

•Cellulose production depends heavily on several factors such as the growth medium, environmental conditions, and the formation of by products. •The fermentation medium contains carbon, nitrogen, and other macro and micro nutrients required for bacteria growth. •Bacteria are most efficient when supplied with an abundant carbon source and minimal nitrogen source. •Glucose and sucrose are the most commonly used carbon sources for cellulose production, while fructose, maltose,  xylose, starch, and glycerol have been tried. Sometimes, ethanol may be used to increase cellulose production. •The problem with using glucose is that gluconic acid is formed as a by product which increases the pH of the culture and in turn, decreases the production of cellulose.

•Addition of extra nitrogen generally decreases cellulose production while addition of precursor molecules such as amino acids and methionine improved yield. Pyridoxine, nicotinic acid, p-aminobenzoic acid and biotin are vitamins important for cellulose production whereas pantothenate and riboflavin have opposing effects.

•According to experimental studies, the optimal temperature for maximum production was between 28 and 30 °C. For most species, the optimal pH was between 4.0-6.0. Controlling pH is especially important in static cultures as the accumulation of gluconic, acetic, or lactic acid decreases the pH far lower than the optimal range. Dissolved oxygen content can be varied with stirrer speed as it is needed for static cultures where substrates need to be transported by diffusion.

Reactor based production•Static and agitated cultures are conventional ways to produce bacterial cellulose. •Both static and agitated cultures are not feasible for large-scale production as static cultures have a long culture period as well as intensive manpower and agitated cultures produce cellulose-negative mutants alongside its reactions due to rapid growth.•Thus, reactors are designed to lessen culture time and inhibit the conversion of bacterial cellulose-producing strains into cellulose-negative mutants. Common reactors used are the rotating disk reactor, the rotary biofilm contactor (RBC), a bioreactor equipped with a spin filter,

and a reactor with a silicone membrane.

BC pellicle formed in static culture.

BC pellets formed in agitated culture.

Bacterial CelluloseProf. Dr. Eng. Stanislaw Bielecki1, Dr. Eng. Alina Krystynowicz2, Prof. Dr. MariannaTurkiewicz3, Dr. Eng. Halina Kalinowska4

Rotating Disc Reactor

TYPES OF CELLULOSE

Genus Cellulose type

Acetobacter Extracellular pellicle,ribbons

Achromobacter Ribbons

Aerobacter Fibrils

Agrobacterium Short fibrils

Alcaligenes Fibrils

Pseudomonas Non-distinct

Rhozobium Short fibrils

Sarcina Amorphous

•Bacteria from the genera Aerobacter, Acetobacter, Achromobacter, Agrobacterium, Alacaligenes, Azotobacter, Pseudomonas, Rhizobium, and Sarcina synthesize cellulose.

•However, only the Gluconacetobacter produce enough cellulose to justify commercial interest. The most extensively studied species is Gluconacetobacter xylinus, formerly known as Acetobacter xylinum and since reclassified as Komagataeibacter xylinus.

•G. xylinus extrudes glycan chains from pores into the growth medium. These aggregate into microfibrils, which bundle to form microbial cellulose ribbons. Various kinds of sugars are used as substrate. Production occurs mostly at the interface of liquid and air.

Cellulose pellicle formed by Gluconacetobacter persimmonis GH-2.

www.omicsonline.org

Differences with plant celluloseSome advantages of microbial cellulose over plant cellulose include:•Finer and more intricate structure•No hemicellulose or lignin to be removed•Longer fiber length: much stronger and wider•Can be grown to virtually any shape and thickness•Can be produced on a variety of substrates•The formula of the media used and the strain of Acetobacter xylinum will determine the quality of the pellicle•More absorbent per unit volume

Fig. 1 Schematic model of BC microfibrils(right) drawn in comparison withthe `fringed micelles'; of PC fibrils(Iguchi et al.,2000)

Disadvantages for commercial useSome issues that have prevented large-scale commercialization so far include:•High price (about 50 x more than plant cellulose)Due to the inefficient production process, the current price of bacterial cellulose remains too high to make it commercially attractive and viable on a large scale.

Because of high-price substrates: sugarsLow volumetric yields

•Lack of large-scale production capacity. Traditional production methods cannot produce microbial cellulose in commercial quantities, so further advancements with reactor based production must be achieved to be able to market many microbial cellulose products.•Timely expansion and maintenance of the cell culture for production 

Functions•One continuing mystery surrounding microbial cellulose is its exact biological function.• A. xylinus, since been renamed as Gluconacetobacter xylinus and more recently as Komagataeibacter xylinus, is a successful and prevalent bacterium in nature, frequently finding a home in rotting fruits and sweetened liquids.• The most familiar form of microbial cellulose is that of a pellicle on the top of a static cultured growth media. It has, thus, been hypothesized that cellulose acts as a floatation device, bringing the bacteria to the oxygen-rich air-media interface. •This hypothesis has largely been discredited by experiments conducted on submerged oxygen-permeable silicone tubes that show that cellulose grows well submerged if enough oxygen is present. Others suspect that cellulose is used to immobilize the bacteria in an attempt to keep it near the food source, or as a form of protection against ultraviolet light.

ApplicationsBacterial cellulose has a wide variety of current and potential future applications. Food•The oldest known use of bacterial cellulose is as the raw material of nata de coco, a traditional chewy, translucent, jelly-like foodstuff produced by the fermentation of coconut water, which gels through the production of microbial cellulose by Acetobacter xylinum.  •It has also been used as a thickener to maintain the viscosity in food and as a stabilizing agent. Due to its texture and fiber content, it has been added to many food products as a dietary fiber. A specific example is Cellulon ®, which is a bulking agent used as a food ingredient to act as a thickener, texturizer, and/or calorie reducer.•Microbial cellulose has also been used as an additive in diet beverages in Japan since 1992, specifically kombucha, a healthy tea based drink .

Biofiber bio cellulose microbial cellulose disposable face facial sheet

It is being tested in the textile industry, with the possibility of manufacturing cellulose based clothing

www.snipview.com

www.alibaba.com

The worlds first bio-cellulose � �membrane transducer of some Sony headphones a number of years ago.Biocellulose is actually grown by special bacteria, and then treated to be suitable for manufacturing.  The end result is a material perfect for speakers that is about as stiff as aluminum, but quite a bit lighter, to keep distortion to a minimum.  Having bacteria grow your parts is novel, but probably not the quickest nor the most cost effective

BIOCELLULOSE MEMBRANE IN HEADPHONES

Paper from bacterial cellulose Due to microbial cellulose's higher purity and microfibril structure, it may prove to be an excellent candidate for an electronic paper substrate. Microbial cellulose can be fashioned into sheets approximately 100 micrometers thick, about the thickness of normal paper, by a wet synthesis process.. In papermaking, it is used as an ultra-strength paper and as a reticulated fine fibre network with coating, binding, thickening and suspending characteristics.

www.adream2012.eumicrobialcellulose.blogspot.com

Microbial cellulose is biocompatible and non-toxic, making it a good candidate material for medical applications. So far it has found a commercial role in some wound dressings. There is on-going research to evaluate a possible role for bacterial cellulose in the following applications:•Scaffolds for tissue engineering•Synthetic dura mater•Bladder neck suspension•Soft tissue replacement•Artificial blood vessels

MEDICAL USES•The microbial cellulose molds very well to the surface of the skin, providing a conformal covering even in usually difficult places to dress wounds, such as areas on the face.•Another microbial cellulose commercial treatment product is XCell produced by the Xylos Corporation, which is mainly used to treat wounds from venous ulcers. •In addition to increasing the drying time and water holding abilities, liquid medicines were able to be absorbed by the microbial cellulose coated gauze, allowing them to work at the injury site

www.intechopen.com

•Axcelon leverages bacterial cellulose •expertise with Nanoderm launch

•It has been tested and successfully used as a wound dressing, especially in burn cases. •Microbial cellulose products, such as Biofill ®, Dermafill®, have been developed. 

biotuesdays.com

www.dermafill.com

REFERENCES

Microbial Cellulose Utilization: Fundamentals and BiotechnologyLee R. Lynd, Paul J. Weimer, Willem H. van Zyl and Isak S. Pretoriushttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC120791/

Microbial cellulose - Wikipedia, the free encyclopediahttps://en.wikipedia.org/wiki/Microbial_cellulose

Bacterial cellulose - Wikipedia, the free encyclopediahttps://en.wikipedia.org/wiki/Bacterial_cellulose

Production and application of microbial cellulosewww.sciencedirect.com/science/article/pii/S0141391097001973

Overview of Bacterial Cellulose Production and Applicationwww.sciencedirect.com/science/article/pii/S2210784314000187

LEHNINGER A.L., Nelson D.L., Principles of Biochemistry, M.M. Cox. Worth Publishing.

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