Cellulose biosynthesis. Synthesis of sucrose and polysaccharides Text of the scientific work on the topic "Biosynthesis of bacterial cellulose by the culture of Medusomyces gisevii"
The cell wall is formed as a result of the development of the median lamina. Immediately after the complete division of the plant cell nucleus in the telophase of mitosis, it forms across the dividing cell phragmoplast.It consists of many flattened membrane vesicles - phragmos,containing components of the cell wall. The cytoskeleton is involved in their construction. All polysaccharides of the cell wall, with the exception of cellulose, are synthesized in the Gol'dji apparatus. They are packed into vesicles that are transported to the growing
the median plate and merge with it. The median plate increases towards the plasmalemma and joins with it, dividing the two daughter cells. Finally, the newly formed cell wall connects to the pre-existing primary cell wall.
Almost all "non-cellulose" components of the cell wall - polysaccharides, structural proteins, a wide range of enzymes - are formed in the Golgi apparatus and in its vesicles are coordinatedly directed to the cell wall.
So far, no genes have been identified that encode polysaccharide synthases involved in the synthesis of the backbone of "noncellulose" polymers. Genes for several fucosyl and galactosyltransferases have been identified that attach individual sugars to the main chain.
The only polymers that are synthesized from the outside of the plasma membrane are cellulose and callose. The expediency of this becomes obvious if we take into account the large length of the formed cellulose microfibrils and the need for their filigree packing into the cell wall. Callose differs from cellulose in the presence of β 1 → 3-D-glucan chains, which can form helical duplexes and triplexes. Callose is formed in several types of cells at certain stages of cell wall formation, for example, in the germinating pollen tube or the median lamina of dividing cells. Callose can also be synthesized during stress reactions or in response to fungal infection.
Cellulose synthesis is catalyzed by multimeric enzyme complexes located at the ends of elongating cellulose microfibrils. These terminal complexes are clearly visible under an electron microscope.
Figure: 1.30. Scheme of the structure and work of cellulose synthase
In some seaweeds, the terminal complexes of cellulose synthesis are linear; in all angiosperms, they form rosette structures. Terminal complexes appear in the plasmalemma membrane at the moment of activation of cellulose synthesis.
The initial substrate for cellulose synthase is UDP-glucose. It is formed by the enzyme sucrose synthase directly from sucrose. A number of isoforms of this enzyme are found in the plasma membrane. They are associated with cellulose synthase and can supply UDP-glucose directly to its catalytic center (Fig. 1.30).
More recently, several plant genes have been identified that encode enzymes for cellulose synthesis, in particular the CesA genes, which are intensely expressed in cotton fibers during the active synthesis of cellulose of the secondary cell wall. The polypeptides encoded by these genes have eight transmembrane domains and a mass of about 110 kDa. The discovery of the CesA genes made it possible to identify a number of other genes encoding cell wall polysaccharide synthases.
In plants, in the process of photosynthesis, not only phosphoric esters of sugars or simple sugars are formed, but also more complex forms of carbohydrates - sucrose, starch, fiber. The decomposition of complex forms of carbohydrates to simple ones also proceeds very quickly. This is observed, for example, during seed germination, aging of vegetative organs, etc. The simple sugars or their phosphorus esters formed during decomposition flow into the reproductive organs, where complex carbohydrates are synthesized from them again. And, finally, in plants the processes of mutual transformations of carbohydrates proceed very easily.
The interconversion of monosaccharides takes place through phosphoric esters of sugars or their uridine diphosphate derivatives (UDP derivatives). UDP-derivatives of sugars are one or another sugar combined through two phosphoric acid residues with uridine, for example:
Fig. 1. Uridine diphosphate glucose
Synthesis of sucrose
Sucrose is the most important ligosaccharide produced in plants, in the form of which bound carbon and energy are transported throughout the plant. It consists of an α-D-glucose residue in its pyranose form, linked by a glycosidic bond to β-D-fructose in its furanose form. Since the anomeric carbon atom of both monosaccharides is involved in the formation of the glycosidic bond, their hemiacetal groups are blocked and none of the rings can open. Thus, sucrose is a non-reducing sugar (does not reduce Fehling's and Benedict's reagents), and except for its extreme sensitivity to acid hydrolysis, it is chemically inert. When heated with acids, or under the action of sucrase (invertase), sucrose is hydrolyzed, forming invert sugar - a mixture of glucose and fructose. Sucrose is highly water soluble and has a sweet taste.
Sucrose is used as a food product, as well as in the production of surfactants (sucrose esters with higher acids). The main source of sucrose production is sugar beet, which contains up to 23% sucrose, and sugar cane, the stems of which contain 10-18% sucrose. It has now been established that sucrose is synthesized not only in chloroplasts, but also in the cytoplasm of photosynthesizing cells from UDP-glucose and fructose-6-phosphate, arising from
dihydroxyacetone phosphate. This substance is formed during photosynthesis in chloroplasts and then enters the cytoplasm. In non-photosynthetic tissues (for example, in the endosperm of germinating castor beans), the formation of sucrose from UDP-glucose and fructose-6-phosphate also occurs in the cytoplasm of cells.
Sucrose (cane, beet sugar) is the most widespread disaccharide in nature. In plants, it is formed from glucose and fructose. The first stage is glucose phosphorylation:
Glucose + ATP → glucose-6-phosphate + ADP,
then glucose-6-phosphate is isolated to glucose-1-phosphate. Glucose-1-phosphate combines with UTP, resulting in the cleavage of pyrophosphate acid and the formation of a compound of glucose with uridine diphosphoric acid (UDP) - uridine diphosphate glucose.
At the same time, fructose phosphorylation occurs under the action of the fructokinase enzyme with the participation of ATP:
fructose + ATP → fructose-6-phosphate + ADP.
After this, UDP-glucose interacts with fructose-6-phosphate with the participation of the enzyme sucrose-phosphate-UDP-glucosyltransferase. Finally, the resulting sucrose-6-phosphate is hydrolyzed by the phosphatase enzyme to form free sucrose. Thus, for the biosynthesis of one sucrose molecule, 3 high-energy phosphate bonds are required, this reaction is irreversible. In non-photosynthetic tissues of some plants, for example, in sugar beet roots, potato tubers and others, sucrose can be formed from free fructose:
UDP-glucose + fructose ↔ sucrose + UDP.
The reaction is catalyzed by the enzyme sucrose-UDP-glucosyltransferase and, depending on the conditions, can be directed both towards synthesis and towards the decomposition of sucrose.
Starch synthesis
Starch is a reserve polysaccharide of plants, and it can be stored in a plant for a long time, or consumed quickly enough. For a long time, it is stored in many seeds, tubers and rhizomes and is used only when these organs germinate. For a short time, starch is formed in chloroplasts during the period of rapid photosynthesis, and in the subsequent dark period it is consumed and flows out of the leaves in the form of sucrose. Starch is always formed and stored in the form of starch grains found in plastids - chloroplasts or amyloplasts. Starch grains are highly organized structures, the shape and size of which are very diverse, but often characteristic of a given plant species. The shape of the grains can be spherical, ovoid, lenticular, or irregular; the size can range from 1 to 100 microns. The largest starch grains are found in potatoes, and the smallest in rice and buckwheat. Starch grains contain up to 20% water (of which 10% is chemically bound to starch) and a number of concentric layers of starch. Starch grains are formed by layering newly formed layers on top of pre-existing ones.
The content of minerals in starch is very low - 0.2–0.7%, they are mainly represented by phosphoric acid. Some high-molecular fatty acids (palmitic, stearic, etc.) are found in starch, the content of which reaches 0.6%. Starch is a mixture of two polysaccharides - amylose and amylopectin. Amylose molecules are long, unbranched chains containing from 100 to several thousand glucopyranose residues linked by glycosidic bonds. X-ray diffraction studies have shown that amylose molecules have a helical structure with a diameter of 1.3 nm with six consecutive glucose residues per spiral turn.
Amylose molecules are soluble in hot water, but the resulting solution is unstable and then spontaneous amylose precipitation occurs, known as retrogradation. This is due to the tendency of long and thin amylose molecules to line up next to each other and form insoluble aggregates via hydrogen bonds. Uridine diphosphate glucose (UDPC) can serve as a donor of glucose residues in the biosynthesis of amylose. For its formation in the reaction medium, the presence of a seed is necessary, which can be polysaccharides constructed from only 3-4 glucose residues connected by α (1-4) bonds.
Residual glucose is transferred to the acceptor (primer), where chain elongation occurs. The reaction follows the scheme:
UDPG + acceptor (G) k - UDP + acceptor (G) k + 1,
where Г - glucose residues.
The enzyme that catalyzes this reaction is called UDPG starch glucosyltransferase.
In most plants, the active donor of glucose is not UDP-glucose, but adenosine diphosphate glucose α (ADPG). The reaction of attachment of glucose residues from ADPG to a low-molecular-weight acceptor proceeds in a similar way and is catalyzed by the enzyme ADPG-starch-glucosyltransferase. The synthesis of a branched amylopectin molecule having α (1-6) -linkages occurs using the enzyme α-glucantransferase (Q-enzyme). The D-enzyme or glucosyltransferase, which forms α (1-4) -links and participates in the formation of the seed, is also involved in the synthesis of starch.
The decomposition of starch occurs with the participation of two processes - hydrolysis and phosphorolysis. Hydrolytic decomposition of starch is carried out under the action of four enzymes of the class of hydrolysis of α-amylase, catalyzes the cleavage of α (1-4) -bonds, and the bonds are broken randomly. The end product of this breakdown is maltose, glucose, dextrins. Under the action of β-amylase, α (1-4) -linkages are cleaved with the formation of maltose residues. The glucoamylase enzyme catalyzes the sequential cleavage of glucose residues from the starch molecule. Amylopectin-1,6-glucosidase or R-enzyme catalyzes the cleavage of α (1-6) -links in the amylopectin molecule, i.e. acts on branch points.
Phosphorolysis is the addition of phosphoric acid at the site of the cleavage of the glucosidic bond between the monosaccharide residues in the polysaccharide chain, with the formation of glucose-1-phosphate. This reaction is catalyzed by the enzyme gluconphosphorylase, which belongs to the class of transferases. The starch in a plant can undergo very rapid degradation, since degradation enzymes are found in all organs of the plant.
Cellulose synthesis
Cellulose is insoluble in water, it only swells in it. With acid hydrolysis (boiling in sulfuric acid) it turns into glucose, with a weaker hydrolysis - into cellobiose. Using X-ray diffraction analysis, it was found that the cellulose molecule has a threadlike shape. These filamentous molecules, due to hydrogen bonds, are connected in bundles of 40-60 pieces per micelle. In the cell walls of plants, cellulose micelles are linked by hydrogen bonds with various heteropolysaccharides. For example, in the white maple, they are xyloglucan, arabinogalactan, and rhamnogalacturon interconnected by glycosidic bonds. In addition, there is evidence that a special hydroxyproline-rich glycoprotein extensin is involved in the construction of the plant cell wall.
Cellulose is built from β-glucose residues. It is not free glucose that participates in the biosynthesis of cellulose, but its HDF derivative - guanosine diphosphate glucose with the participation of the cellulose synthetase enzyme according to the scheme:
HDF - glucose + (glucose) to → HDF + (glucose) to + 1
The decomposition of cellulose proceeds mainly by hydrolytic pathway under the action of the enzyme cellulase to cellobiose disaccharide.
Carbohydrates are transported in the form of sucrose. In the process of photosynthesis, a lot of carbohydrates are formed, and in this regard, the outflow of assimilates to other parts of the cell from chloroplasts is of great importance. The penetration of phosphorylated hexoses and sucrose through the chloroplast membrane is difficult; triose phosphates (PHA and PDA) penetrate most easily through the chloroplast membranes. It is assumed that the resulting complex carbohydrates break down into triose phosphates and in this form move into the cytoplasm, where they can serve as material for the resynthesis of hexoses, sucrose, and starch.
Intercellular parenchymal transport is carried out in two ways - along the plasmodesmata (symplast) or through free space (apoplast). Sucrose formed in the cells of the leaf mesophyll is desorbed into the apoplast. Leaving the parenchymal cells into the apoplast, sucrose is cleaved into hexoses by invertase. Hexoses move along the apoplast to the transfer cells of the conducting beams along the concentration gradient. Upon contact with the transfer cells of the phloem, they are converted back to sucrose. Next, the sieve tubes are loaded, sucrose flows against the concentration gradient, and energy consumption (ATP) is required.
It is assumed that sucrose crosses the membrane with the help of a carrier in a complex with a proton. At the same time, due to the work of H + -ATP-ase, H + ions are pumped out of the phloem cells, and then come back along the pH gradient, dragging sucrose along with it against the gradient of its concentration. The main transport form of carbohydrates in the phloem is sucrose (C 12 H 22 O 11). In some species, along with sucrose, oligosaccharides (raffinose, stachyose), as well as some alcohols, serve as the transport form of carbohydrates.
In the Calvin-Benson cycle, fructose-6-phosphate (F-6-P) is formed, as noted above. This hexose phosphate can be converted by specific enzymes into other phosphorylated hexoses, namely, glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P). The reverse transformation also occurs easily.
From these three hexose phosphates, chains of carbohydrate molecules are then built, which are used for transport, storage, and synthesis reactions. In order for such transformations to occur, hexose phosphates must first be activated. This is usually achieved as a result of their attachment to nucleotides - complex circular structures similar to adenylic acid ATP. The product of such an addition reaction is nucleotide derivatives of monosaccharides, or nucleotide sugars. The most common uridine diphosphoglucose (UDPG) is produced by the reaction between uridine triphosphate (UTP) and glucose 1-phosphate (G-1-P). UTP itself is formed indirectly, as a result of the transfer of a phosphate group from ATP to UDP (uridine diphosphate).
The ATP and UTP nucleotides are present in all cells because they are used along with other nucleotides in DNA and RNA synthesis.
Sugars are transported through the plant in the form of sucrose, a disaccharide consisting of glucose and fructose residues (Figure 5.2). Sucrose is formed by the reaction between UDPG and F-6-P:
The equilibrium of this reaction is strongly shifted towards the synthesis of sucrose, which provides the possibility of accumulation of this disaccharide in significant concentrations. For subsequent use, sucrose must first undergo cleavage: the invertase enzyme catalyzes its hydrolysis with the formation of free glucose and fructose.
The energy of the glycosidic bond in such a reaction is wasted, being distributed between the two molecules. Therefore, if glucose and fructose are to decompose during respiration or participate (as raw materials) in the synthesis of polysaccharides, then they must first undergo phosphorylation again due to ATP. The processes of synthesis and decomposition of sucrose clearly show that often anabolic and catabolic reactions (reactions of synthesis and decomposition) follow different paths.
Synthesis of starch and cellulose
Long polymer chains of starch and cellulose are built from the same elementary links - glucose residues, only connected in different ways. This structural difference is responsible for the fact that the two considered glucose polymers (glucans) are significantly different in nature; starch, for example, is easily digested in the human body "and cellulose is not digested at all. Their main difference is that the 1st and 4th carbon atoms of two adjacent glucose residues are linked by α-bonds in starch, and in cellulose (β -links (Fig. 5.3). Starch is presented in two forms: a linear polymer, or amylose, which does not contain any other bonds, except for α-1,4-glycosidic, and a branched polymer, or amylopectin, in which, along with α-1,4 -glycosidic bonds also have 1,6-bonds. The difference in the nature of the bonds also determines the unequal spatial arrangement of polymer chains. Starch is the main reserve polysaccharide of the plant. It is insoluble in water and is deposited layer by layer in starch grains contained in chloroplasts (see Fig. 2.20) or in the chlorophyll-free leukoplasts of the storing tissues of the stem, roots and seeds.Sometimes the cells of the storing tissue are literally clogged with starch grains, which are easy to identify in them, since they are capable of staining turn blue with iodine. Being insoluble in water, starch, in contrast to sucrose and from hexoses, does not cause an osmotic effect in cells (see Chapter 6). Therefore, the formation of starch in leaf cells during periods of intense photosynthesis prevents the suppression of the latter, which occurs as a result of the accumulation of photosynthetic products. In the dark, starch is gradually hydrolyzed again to form glucose phosphate, which is then converted into sucrose, which is transported to other parts of the plant.
Figure: 5.3. Structure of starch (A) and cellulose (B) (Modified by J. Bonner, AW Galston. 1952. Principles of Plant Physiology, San Francisco, WH Freeman and Co.) Please note that the chemical formulas of starch and cellulose are the same, but differ these polysaccharides by the spatial orientation of their oxygen bridges. A. Starch, the main storage polysaccharide of the plant, is built from two distinct components: amylose, with its long unbranched glucose units, and amylopectin, which is made up of a large number of short branched chains. B. Cellulose, the main component of the primary cell wall, exists in the form of long polymer chains. The chains are combined into micellar strands, and the latter into microfibrils. Microfibrils large enough to be viewed with an electron microscope constitute the "base" and "weft" of the cell wall
The initial product for starch synthesis is adenosine diphosphoglucose (ADPG), which is formed from ATP and G-1-P:
A starch molecule is built by the gradual addition of one glucose residue after another in the reaction of ADPG with a preformed glucose chain:
With a low sucrose content, starch is broken down and. converted to sucrose. However, at first, it is split into glucose residues and a phosphoric acid residue is added to each of them, i.e., G-1-P is formed, which ensures the conservation of binding energy:
This G-1-P can then be used to synthesize sucrose, which we described above. In seeds and in some other organs, in which large amounts of starch are simultaneously decomposed, it breaks down to maltose disaccharide (G-G) under the action of aα-amylase. Maltose then breaks down to glucose, from which (for transport) sucrose is synthesized again. In this second pathway, unlike the first, the binding energy is not conserved, so here ATP is required to convert glucose into glucose-6-P.
Cellulose, the most abundant carbohydrate on earth, is the main component of the primary cell wall. Its molecules are built in the same way as starch molecules are built, with the difference, however, that the role of a glucose donor is played by another nucleotide derivative of a monosaccharide - guanosine diphosphoglucose (GDPG) - and that the bond between monomeric units belongs not to α-, but to β-type.
In some cases, UDPG can also be a glucose donor for cellulose synthesis.
In the body of higher plants, cellulose is rarely degraded (except for the decomposition caused by the activity of microbes). Two well-known exceptions to this rule relate to cells in the leaf separation zone, formed before the shedding of leaves, and xylem vessels, in which the transverse walls dissolve. In the separation zone of the leaf, the cellulase enzyme destroys the cell walls, splitting the cellulose contained in them into individual monomeric units, i.e., to glucose. The cell walls weakened by this process eventually rupture and the leaf is shed.
Cellulose microfibrils in the cell wall are held together by a matrix of mixed polysaccharide chains, mainly xyloglucans and arabinogalactans (see Fig. 2.31). (Xylose and arabinose are five-carbon sugars (pentoses), and galactose is hexose, akin to glucose.) These polysaccharides are also synthesized from precursors, nucleotides sugars, mainly in dictyosomes. The vesicles detached from the dictyosomes ultimately merge with the plasmalemma and in this way transfer their contents to the forming cell wall.
So, all polysaccharides easily pass one into another, but their synthesis always goes through the stage of nucleotide sugars, while the decay occurs in a more direct way.
Most plant products contain so-called fiber and pectin, which are not digested in the gastrointestinal tract. However, they are necessary for a person. If food is poor in them, intestinal atony and constipation occur. Thus, fiber is a regulator of intestinal motor function.
Fiber improves bowel function, slows down putrefaction and gas formation, and reduces the absorption of certain harmful substances. So, for example, for the prevention of occupational diseases in workers with metal salts and radioactive substances, it is recommended to eat red currants, which contain many pectins.
Polysaccharide: Fiber
Fiber (cellulose) - a polysaccharide that makes up the bulk of plant cell walls. Fiber is insoluble in water, it only swells in it. Fiber makes up over 50% of wood. In cotton fibers, it is more than 90%. When boiled with strong sulfuric acid, the fiber is completely converted into glucose. With weaker hydrolysis, cellobiose is obtained from fiber.
In the fiber molecule, cellobiose residues are linked by glycosidic bonds in the form of a long chain. The molecular weight of fiber has not been precisely established. It is believed that fiber is not an individual substance, but a mixture of homologous substances. The molecular weights of cellulose obtained from various sources fluctuate quite strongly: cotton - 330,000 (in the chain of 2020 glycosidic residues); ramie - 430,000 (2660 remains), spruce wood - 220,000 (1360 remains). With the help of X-ray diffraction analysis, it was found that the fiber molecules have a threadlike shape. These filamentous molecules are combined into bundles - micelles. Each micelle contains approximately 40-60 fiber molecules.
The combination of individual fiber molecules into micelles occurs due to hydrogen bonds, which are carried out both due to the hydrogen atoms of the hydroxyl groups of the fiber, and due to the water molecules adsorbed by the fiber. In plant cell walls, cellulose micelles are hydrogen bonded to various heteropolysaccharides. For example, in the white maple, they are xyloglucan interconnected by glycosidic bonds, consisting of residues of glucose, xylose, galactose and fucose; arabinogalactan, built from the remains of arabinose and galactose; rhamnogalacturonan, formed by residues of galacturonic acid and rhamnose. In addition, there is evidence that a special, hydroxyproline-rich glycoprotein extensin is also involved in the construction of the plant cell wall, especially in the early stages of its formation. When lignification of cell walls, lignin also accumulates in them.
Fiber is not digested in the human gastrointestinal tract. It is digested only by ruminants, in the stomach of which there are special bacteria that hydrolyze fiber with the help of the enzyme cellulase secreted by them.
Hemicellulose (semi-cellulose). Under this name, a large group of high molecular weight polysaccharides that do not dissolve in water, but are soluble in alkaline solutions, are united. Hemicelluloses are found in significant quantities in the lignified parts of plants: straw, seeds, nuts, wood, corn cobs. A large amount of hemicellulose is found in bran. Hemicelluloses are more easily hydrolyzed by acids than fiber. At the same time, they form mannose, galactose, arabinose or xylose, and therefore are respectively named - mannans, galactans and pentosans (araban or xylan).
1The process of biosynthesis of bacterial cellulose (BC) on the enzymatic hydrolyzate of the lignocellulose material of miscanthus was studied. Lignocellulosic material was obtained by treating miscanthus with a dilute solution of nitric acid in a pilot plant. Enzymatic hydrolysis was carried out in an 11 l fermenter. BC biosynthesis was carried out using a symbiotic culture of Medusomyces gisevii Sa-12. It has been established that the number of acetic acid bacteria during cultivation is 1.2 times less than that of yeast. The main utilization of the substrate occurs in 6 days of cultivation, the constant of the substrate utilization is 0.234 day-1. It was shown that the enzymatic hydrolyzate of miscanthus lignocellulosic material is not a benign nutrient medium for BC biosynthesis, the BC yield was 5.6%, which is 1.6 times less than the yield on a synthetic nutrient medium. It has been established that bacterial cellulose obtained on this medium is chemically pure.
bacterial cellulose
Medusomyces gisevii
infrared spectroscopy
enzymatic hydrolyzate
miscanthus
1. Bacterial cellulose synthesized by Gluconacetobacter hansenii for use in medicine / T.I. Gromovs and [others] // Applied Biochemistry and Microbiology. - 2017. - T. 53, No. 1. - P. 69–75.
2. Prospects for the use of bacterial cellulose in meat products / T.I. Thunder and [others] // Meat Industry. - 2013. - No. 4. - P. 32–35.
3. Octave S. Biorefinery: toward an industrial metabolism / S. Octave, D. Thomas // Biochimie. - 2009. - No. 91. - P. 659–664.
4. Gismatulina Y.A., Budaeva V.V., Sakovich G.V., Veprev S.G., Shumny V.K. Cellulose from various parts of soranovskii miscanthus // Russian Journal of Genetics: Applied Research. - 2015. - Vol. 5, no. 1. - P. 60–68.
5. Gismatulina Yu.A. Comparison of the physicochemical properties of celluloses obtained by the combined method from the leaf and stem of miscanthus / Yu.A. Gismatulina / Bulletin of Altai Science. - 2014. - No. 1 (19). - S. 302–307.
6. Makarova E.I. Bioconversion of non-food cellulose-containing raw materials: energy plants and agricultural waste: dis. ... Cand. technical sciences. - Shchelkovo, 2015 .-- 161 p.
7. Gladysheva E.K. Features of the structural characteristics of bacterial cellulose synthesized on an enzymatic hydrolyzate of lignocellulose material of oat fruit shells / E.K. Gladysheva, E.A. Skiba, L.A. Aleshina // Polzunovsky Bulletin. - 2016. - No. 4–1. - S. 152-156.
8. Skiba E.A., Budaeva V.V., Baibakova O.V., Udoratina E.V., Shakhmatov E.G., Shcherbakova T.P., Kuchin A.V., Sakovich G.V. Enzymatic Hydrolysis of Lignocellulosic Materials in Aqueous Media and the Subsequent Microbiological Synthesis of Bioethanol // Catalysis in Industry. - 2016. - Vol. 8, no. 2. - P. 168–175.
9. Skiba E.A. Method for determining the biological good quality of hydrolysates from cellulose-containing raw materials using the Saccharomyces cerevisiae strain VKPM Y-1693 / E.A. Skiba // Izvestiya vuzov. Applied chemistry and biotechnology. - 2016. - No. 1 (16). - S. 34–44.
10. Gladysheva E.K. Biosynthesis of bacterial cellulose by the culture of Medusomyces gisevii / E.K. Gladysheva, E.A. Skiba // Bulletin of the Voronezh State University of Engineering Technologies. - 2015. - No. 3. - P. 149-156.
11. Gladysheva E.K. Investigation of the effect of temperature on the synthesis of bacterial cellulose by a producer of Medusomyces gisevii / E.K. Gladysheva // Modern high technologies. - 2016. - No. 8–1. - S. 36-40.
12. Yurkevich D.I. Medusomycete (Kombucha): scientific history, composition, features of physiology and metabolism / D.I. Yurkevich, V.P. Kutyshenko // Biophysics. - 2002. - No. 6. - P. 1116–1129.
13. Yang, X.-Y., Huang C., Guo H.-J., Xiong L., Li Y.-Y., Zhang H.-R., Chen X.-D. Bioconversion of elephant grass (Pennisetum purpureum) acid hydrolysate to bacterial cellulose by Gluconacetobacter xylinus // Journal of Applied Microbiology. - 2013. - No. 115. - P. 995-1002.
14. Yarovenko V.L. Alcohol technology / V.L. Yarovenko, V.A. Marinchenko, V.A. Smirnov. - M .: Kolos, 1999 .-- 464 p.
15. New handbook of chemist and technologist. Raw materials and products of the industry of organic and inorganic substances. Ch. ΙΙ. - SPb .: NPO Professional, 2006. - 1142 p.
Scaling up the process and implementing in practice biotechnological production depends on factors such as the availability of reproducible mass cheap raw materials; ease of transformation of raw materials into a nutrient medium; the possibility of hardware design of production with standard or new efficient equipment; high yield of the target product and ensuring the standard of its quality. In view of the prospects of using BC in various fields, it is necessary to create its industrial production, while an important task is to find suitable carbon sources that have a low cost and do not compete with food products. The actual direction of obtaining BC is the use of cellulose-containing raw materials to obtain alternative nutrient media from it.
The massive use of fossil resources over the past century and the associated pollution problem have caused a significant number of environmental and economic problems. Presumably, these resources will be depleted in the near future. These reasons contribute to the progressive transition to an economy based on renewable materials (biomass) as raw materials for the production of chemicals, materials, fuels and energy within the so-called concept of bioconversion. Cellulose is one of the most abundant polysaccharides and is regarded as an inexhaustible and versatile source. It is promising to use the so-called energy, i.e. fast growing plants: miscanthus, millet, sorghum, etc. ... Miscanthus is a genus of perennial herbaceous plants of the bluegrass family. Represents a plant up to 200 cm high, erect stems, simple lamellar leaves, sharp top, wedge-shaped base, inflorescences in the form of panicles. The plant is a perennial cereal and, starting from the third year of cultivation, it can annually produce 10-15 t / ha / year of dry biomass in one field for 15-20 years, which corresponds to 4-6 t / ha of pure cellulose.
The IPCET SB RAS has developed a technology for obtaining enzymatic hydrolysates from miscanthus. Miscanthus is preliminarily subjected to chemical treatment with dilute solutions of acid and / or alkali, and then to enzymatic hydrolysis. Investigation of the process of BC biosynthesis on an enzymatic hydrolyzate of the lignocellulose material of oat hulls showed that for a successful microbiological synthesis the enzymatic hydrolyzate must have biological good quality.
In this work, the lignocellulosic material (LCM) of miscanthus was chosen as a substrate for enzymatic hydrolysis. LMC of miscanthus is obtained by processing raw materials in one stage with a dilute solution of nitric acid at atmospheric pressure in standard equipment. It has been shown that the enzymatic hydrolyzate obtained from the LMC of miscanthus is biologically benign for the biosynthesis of ethanol and does not need additional processing to free it from harmful impurities.
The aim of this work was to study the process of BC biosynthesis on the enzymatic hydrolyzate of the LCM miscanthus and to study the structure of the obtained samples by infrared spectroscopy. It should be noted that this problem is ambiguous, since BC producers are more demanding on the composition of nutrient media, therefore, data on the quality of the medium for yeast cannot be extrapolated to cellulose-synthesizing microorganisms.
Materials and research methods
LMC of miscanthus was obtained by treatment with a dilute solution of nitric acid in the pilot production of IPCET SB RAS and had the following composition (%, in terms of dry matter): mass fraction of acid-insoluble lignin - 10.6, mass fraction of ash - 4.8, mass fraction of cellulose according to Kurschner - 86.7, mass fraction of pentosans - 7.9.
Enzymatic hydrolysis of miscanthus LCM was carried out in an 11 L fermenter in an aqueous medium at 47 ± 2 ° C for 72 h using enzyme preparations Cellolux-A (0.04 g / g of substrate) and Bruzheim BGX (0.1 ml / g of substrate ), active acidity was maintained at the level of 4.7 ± 0.2 with the help of ammonium hydroxide and orthophosphoric acid, the initial concentration of the substrate was 60 g / L, the method is described in more detail in the work.
The resulting enzymatic hydrolyzate was filtered from the substrate residues under vacuum. The hydrolyzate was a transparent red liquid with a sour odor, active acidity 4.7 units. pH. The total amount of reducing substances (RS) was 49.0 g / l, of which xylose was 2.8 g / l. A chilled tea infusion was added to the filtered LMC enzymatic hydrolyzate from miscanthus (1 L of distilled water was brought to a boil, dry black tea was added, extraction was carried out for 15 min, cooled and filtered). At the same time, the nutrient medium was standardized in terms of the RS content from 20 to 25 g / l and the content of tea extractives from 1.6 g / l to 4.8 g / l.
A symbiotic culture Medusomyces gisevii Sa-12 was used as a producer for BC biosynthesis. Preliminarily, the culture was adapted on the studied nutrient medium. The inoculum was introduced into nutrient media in an amount of 10% of the volume of the nutrient medium; cultivation was carried out under static conditions at 27 ° C for 24 days. The cultivation conditions were selected on the basis of previous work.
Microbiological indicators (the number of yeast and acetic acid bacteria) were monitored using a B-150 OPTIKA microscope. The growth of the BC film was assessed by the gravimetric method (laboratory analytical balance Explorer EX-224), the level of active acidity was monitored using an ionomer (I-160 MI ionometer). The concentration of radioactive substances was controlled by the spectrophotometric method (spectrophotometer "UNICO-2804", USA) using a dinitrosalicylic reagent, the concentration of xylose was determined by the standard method, which is based on the formation of furfural from pentosans.
The structure of bacterial cellulose was investigated on an infrared spectrophotometer "Infralum FT-801" in KBr tablets.
Research results and their discussion
The change in the number of yeast and acetic acid cells during the cultivation of Medusomyces gisevii Sa-12 on the enzymatic hydrolyzate of the Miscanthus LCM is shown in Fig. 1, the change in the level of active acidity during the cultivation of Medusomyces gisevii Sa-12 is shown in Fig. 2.
The concentration of yeast cells in the nutrient medium during cultivation was found to be an order of magnitude higher than that of acetic acid bacteria. For yeast, the lag phase was not observed, an increase in the cell concentration occurred from 0 to 12 days, after 12 days the death phase occurred. For acetic acid bacteria, a lag phase was observed, up to 8 days their number increased, from 8 to 10 days the number of cells remained constant, after 10 days the death phase occurred.
Figure: 3. Dependence of the concentration of radioactive substances and the yield of BC on the duration of cultivation
During the cultivation of the symbiotic culture of Medusomyces gisevii in the nutrient medium, as a result of the action of the protective mechanism, intermediate products of glycolysis accumulate: acetic, gluconic acids, ethanol and glycerol; indirectly, their accumulation can be judged by changes in pH. The initial active acidity of the nutrient medium was 4.0; by the sixth day of cultivation, the pH value dropped to 3.8. Further, in the process of cultivation, the value of the active acidity of the medium increased to 5.9. An increase in active acidity is not typical for this producer; however, a similar dependence was described when the producer of Gluconacetobacter xylinus CH001 was cultivated on an acid hydrolyzate of miscanthus.
In fig. 3 shows the dependence of the RS concentration and BC yield on the duration of cultivation.
The rate constant of substrate utilization is calculated by the formula:
where Ku.with. - constant of substrate utilization, day-1; S1, S2 - concentration of radioactive substances at the initial and final moments of time; t1, t2 - initial and final moments of time, days.
Figure: 4. IR spectrum of the BC sample
Utilization of the substrate occurred in two periods: from 0 to 6 days of cultivation, the rate constant for the utilization of the substrate was 0.234 day-1, from 6 to 24 the value decreased 12 times and amounted to 0.020 day-1. Rapid utilization of radioactive substances from 0 to 6 days is associated with the consumption of the substrate by microorganisms and their active reproduction. From 6 to 24 days, RV are slowly spent on maintaining the vital activity of microorganisms.
The miscanthus LCM hydrolyzate predominantly consists of glucose, the xylose concentration at time zero was 1.2 g / l. On the 7th day of cultivation, the total concentration of RS was 4.9 g / L, while the amount of xylose in the hydrolyzate remained practically unchanged and amounted to 0.8 g / L. After 24 days of cultivation, the concentration of RV in the nutrient medium was 3.4 g / l, and xylose - 0.3 g / l.
The rate of synthesis of the product (bacterial cellulose) is calculated by the formula
where Ks.p. - constant of synthesis of the product, day-1; C1, C2 - the mass of the product at the initial and final moment of time; t1, t2 - initial and final moments of time, days.
On the first day of cultivation, a clearly pronounced BC gel film was not observed on the surface of the nutrient medium. On the second day of cultivation, a thin BC gel film was formed. The main increase in biomass occurred from 2 to 6 days of cultivation - the BC output increased from 1.1% to 4.7%; the rate constant for the synthesis of the product during this period was 0.363 day-1. From 6 to 10 days, the BC yield increased to 5.6%, the rate constant of the product synthesis during this period decreased to 0.044 day-1. Further, the rate of BC synthesis decreases, tending to zero.
From 10 to 24 days, the BC yield decreased to 1%, which indicates the ongoing destruction processes, this period coincides with the phase of dying off of yeast and acetic acid bacteria. Thus, in practice, the beginning of the phase of dying off of microorganisms can serve as a criterion for the end of the process of BC biosynthesis.
The enzymatic hydrolyzate of miscanthus LCM is not a favorable nutrient medium for BC biosynthesis, the highest BC yield was 5.6%, which is 1.6 times less than the BC yield on a synthetic nutrient medium when Medusomyces gisevii Sa-12 is cultivated under similar conditions - 9, 0%. Presumably, this can be explained by the method of pretreatment of the feedstock and the presence of impurities in the enzymatic hydrolyzate of the miscanthus LCM, which can inhibit the biosynthesis of BC. Thus, the good quality of the enzymatic hydrolyzate of miscanthus LCM for ethanol biosynthesis is not a guarantee of good quality for the biosynthesis of BC, which is due to the high demands on the quality of nutrient media of symbiotic producers Medusomyces gisevii Sa-12 in comparison with Saccharomyces cerevisiae. It can be assumed that for successful BC biosynthesis, purer substrates should be used, for example, commercial cellulose of miscanthus.
In fig. 4 shows the IR spectrum of the BC sample synthesized on the enzymatic hydrolyzate of the LMC Miscanthus.
In the infrared spectrum of the BC sample, there is an intense band at 3381 cm-1, which indicates the stretching vibrations of OH groups. The less intense band at 2917 cm-1 is due to the stretching vibrations of the CH2, CH groups. In the BC spectrum, bands in the 2000-1500 cm-1 range belong to bending vibrations of OH groups of strongly bound water. Weak absorption bands in the range: 1430-1370 cm-1 are due to bending vibrations of CH2 groups; 1360-1320 cm-1 - bending vibrations of OH groups in CH2OH. The bands at 1281 and 1235 cm-1 indicate bending vibrations of OH groups in alcohols. The band at 1204 cm-1 indicates bending vibrations of OH groups. The absorption bands in the range 1000-1200 cm-1 are mainly due to the stretching vibrations of C-O-C and C-O in alcohols. Thus, it was confirmed by the IR method that BC obtained on the enzymatic hydrolyzate of LCM is a pure compound containing only cellulose.
The process of BC biosynthesis by the symbiotic culture Medusomyces gisevii Sa-12 on the enzymatic hydrolyzate of the Miscanthus LCM was studied. The main utilization of the substrate occurs in 6 days of cultivation, the constant of the substrate utilization is 0.236 day-1. It was found that the number of acetic acid bacteria during cultivation is an order of magnitude less than that of yeast, and after 10 days is 1.1 CFU / ml. It has been shown that, in practice, the onset of the death phase of symbiotic microorganisms can serve as a criterion for the end of the biosynthesis process, since this phase coincides with the process of BC destruction. It was shown that the enzymatic hydrolyzate of miscanthus LCM is not a benign nutrient medium for BC biosynthesis: the BC yield on the 10th day of cultivation is 5.6%, which is 1.6 times less than the BC yield on a synthetic nutrient medium, and on the 24th day the yield decreases up to 1.0%, that is, BC undergoes destruction. Infrared spectroscopy showed that BC is a pure compound containing only cellulose.
The study was supported by a grant from the Russian Science Foundation (project No. 17-19-01054).
Bibliographic reference
Gladysheva E.K. BIOSYNTHESIS OF BACTERIAL CELLULOSE ON ENZYMATIVE HYDROLYSATE OF LIGNOCELLULOSE MATERIAL MISCANTHUS // Fundamental research. - 2017. - No. 9-2. - S. 290-294;URL: http://fundamental-research.ru/ru/article/view?id\u003d41742 (date of access: 13.12.2019). We bring to your attention the journals published by the "Academy of Natural Sciences"