General Microbiology - Schlegel H. 1987

Degradation of natural substances
Cellulose

Although green plants have been synthesizing Organic compounds from carbon dioxide for many millions of years, there has been no significant accumulation of organic matter over this period. Only a small fraction of it has been preserved under anaerobic conditions in the form of highly reduced carbon compounds, namely petroleum, natural gas, and hard coal. Under aerobic conditions, all substances of biological origin undergo decomposition. No matter how complex a given substance may be, nature always harbors a microorganism capable of breaking it down completely or partially, with the breakdown products subsequently utilized by other microorganisms. Thus, collectively, microorganisms are biochemically "omnipotent," which gives reason to speak of microbial "universality." Today, however, certain qualifications must be made regarding this statement. Many man-made low-molecular-weight substances (such as pesticides, detergents, etc.) and high-molecular-weight polymers have proven to be persistent and are not degraded by microorganisms (as far as long-term observations and experimental results suggest).

Information on microorganisms that affect individual natural substances by degrading and Processing them has been obtained mainly from experiments with enrichment cultures. Simple culture media containing the test natural substance as an energy source support the growth of only those microorganisms whose nutritional requirements are not overly complex; furthermore, in liquid enrichment cultures, only the organisms that grow most rapidly under the given conditions will survive. Consequently, it is questionable whether such forms can be considered typical Representatives of the organisms carrying out the same decomposition processes in nature. With this experimental setup, many species requiring different conditions remain undetected. There are still many gaps in our knowledge of the Degradation of natural substances, and the biochemical analysis of these degradation pathways is only just beginning.

Cellulose is the primary constituent of all plant material, and its synthesis surpasses that of any other natural compound in scale. Plant residues preserved in and returned to the soil consist of 40–70% cellulose. Such a large Abundance of cellulose in nature determines the vital role of cellulose-degrading microorganisms in mineralization processes and the carbon cycle (Section 1.3).

Cellulose consists of chains of ß-D-glucose with a degree of polymerization of about 14,000 (Section 2.2.3). The Physical Properties of cellulose fibrils—especially their mechanical strength and insolubility—do not depend on The Structure of individual chains. The chains must be linked together in such a way that hydrophilic groups are concealed (which enhances stability). According to X-Ray Diffraction Analysis, crystalline regions alternate with non-crystalline regions in cellulose. Cellulose fibers are bundles of fibrils enveloped in a common sheath containing wax and pectin.

The enzymatic breakdown of cellulose is carried out by cellulase. Experiments with Fungi have demonstrated that the cellulase system comprises at least three Enzymes: 1) endo-ß-1,4-glucanase simultaneously cleaves various ß-1,4-bonds within the macromolecule, leading to The formation of large fragments with free ends; 2) exo-ß-1,4-glucanase cleaves the disaccharide cellobiose from the end of the chain; 3) ß-glucosidase hydrolyzes cellobiose to yield glucose.

Under laboratory culture conditions, microorganisms typically synthesize these enzymes only when cellulose is the sole available substrate. Their synthesis is repressed both by other substrates and by cellobiose, the end product of cellulose degradation.

Cellulose decomposition under aerobic conditions. In well-aerated soils, cellulose is degraded and utilized by aerobic microorganisms (fungi, myxobacteria, and other eubacteria), whereas under anaerobic conditions, it is broken down primarily by clostridia.

Under aerobic conditions, fungi play a major role in cellulose degradation. They are more efficient in this regard than Bacteria, particularly in acidic soils and in The breakdown of lignin-impregnated cellulose (wood). Representatives of two genera, Fusarium and Chaetomium, play a prominent role in this process. Cellulose is also degraded by Aspergillus fumigatus, A. nidulans, Botrytis cinerea, Rhizoctonia solani, Trichoderma viride, Chaetomium globosum, and Myrothecium verrucaria. The last three species serve as test organisms for detecting cellulose breakdown and for evaluating agents used to impregnate various Materials to protect them against cellulose-degrading microorganisms. Fungi produce cellulases that can be isolated from both the mycelium and the culture medium.

Cytophaga and Sporocytophaga are aerobic, cellulose-degrading bacteria. They are most easily isolated by the conventional method of liquid enrichment cultures. These two genera, closely related to myxobacteria, encompass numerous species. Little is known about the utilization of cellulose by myxobacteria or their primary action upon it. Neither extracellular cellulase nor any cellulose breakdown products have been detected in these organisms. The Cells of these bacteria adhere closely to cellulose fibers, lying parallel to the fiber axis. Apparently, they hydrolyze cellulose only upon close contact with the fiber, and the Hydrolysis products are immediately absorbed. On cellulose agar, Cytophaga colonies are never surrounded by a clear zone containing enzymatic breakdown products of cellulose.

In addition to Cytophaga species, myxobacteria of the genera Polyangium, Sporangium, and Archangium, which form fruiting bodies, are also capable of growing on cellulose.

Many of the aerobic bacteria that might be termed "omnivorous" can also utilize cellulose as a growth substrate. Some of these appear to use cellulose only when other carbon sources are absent; the synthesis and excretion of cellulases in such bacteria are regulated via catabolite repression. Certain forms resembling Pseudomonas were previously grouped into the genus Cellvibrio. They are now described as Pseudomonas fluorescens var. *cellulosa*. Among coryneform bacteria, Cellulomonas deserves mention; it has even been suggested that this bacterium might be used to produce protein from cellulose.

Only a few cellulose-degrading species have been described among actinomycetes: Micromonospora chalcea, Streptomyces cellulosae, and Streptosporangium.

Cellulose decomposition under anaerobic conditions. Under anaerobic conditions, cellulose is cleaved primarily by mesophilic and thermophilic clostridia. The thermophilic species Clostridium thermocellum grows on simple synthetic media, utilizing cellulose or cellobiose as a substrate and ammonium salts as a nitrogen source; this bacterium does not utilize glucose or many other sugars. The Fermentation products of cellulose include ethanol, acetic, formic, and lactic acids, molecular hydrogen, and CO₂. Extracellularly, cellulose is probably cleaved only as far as cellobiose. Fermentation of cellulose by the mesophilic species Clostridium cellobioparum yields similar products. The long rod Bacillus dissolvens behaves similarly to the aforementioned Cytophaga species: its cells adhere tightly to cellulose fibers and do not secrete cellulase into the medium.

Microbiological processes in the rumen of ruminants. In the rumen of ruminants, cellulose is also degraded predominantly by bacteria. Hay, straw, and grass serve as the primary sources of CARBOHYDRATES for ruminants. In dry grass, roughly half of the carbohydrates consist of fructosans and xylans, with cellulose accounting for approximately the same proportion. The cellulosic components of feed would be unavailable for utilization had ruminants not evolved a symbiotic relationship with microbes capable of degrading cellulose (Fig. 14.1).

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Fig. 14.1. Diagram of the transformations undergone by nutrients under the action of Microorganisms in the Stomach of ruminants. 1, rumen; 2, reticulum; 3, omasum; 4, abomasum. For explanation, see text.

The first two compartments of the ruminant stomach—the rumen and the reticulum—act essentially as a large fermentation chamber (with a capacity of 100 to 250 L) that provides ideal conditions for the growth of numerous microorganisms. They are guaranteed a constant Temperature (37–39 °C), a continuous supply of mineral solution (about 100–200 L of saliva per day) well-buffered with bicarbonate and phosphate (pH 5.8–7.3), a periodic influx of nutrients in the form of finely comminuted, cellulose-rich feed, and, finally, mechanical mixing driven by rumen motility. Thus, the rumen resembles a semi-continuous culture system for microorganisms.

Protozoa and bacteria predominate among the inhabitants of the rumen. 1 mL of rumen fluid contains several million protozoa, mainly Ciliates belonging to the genera Diplodinium and Entodinium. These species are specific to the rumen and rare elsewhere. By mass, they constitute 6 to 10% of the rumen contents, a portion of this mass being Polysaccharides stored by the protozoa themselves. However, protozoa may not play a vital role in the rumen, and it remains unclear whether they participate in cellulose degradation.

From a functional standpoint, the most important inhabitants of the rumen are bacteria. 1 mL of rumen fluid contains from 10⁹ to 10¹⁰ bacterial cells, which account for 5–10% of the dry mass of the rumen contents. Rumen-specific bacteria are strict anaerobes. Yeasts and other fungi are present here only in small numbers.

Bacteria convert polymeric feed carbohydrates into simple compounds such as Fatty acids and alcohols. Fatty acids are formed mainly from cellulose, starch, fructosans, and xylans. According to available data, about 90% (by weight) of all ingested cellulose undergoes decomposition. This process yields large quantities of acids, predominantly acetic (50–70 vol. %), propionic (17–21 vol. %), and butyric (14–20 vol. %), along with minor amounts of valeric and formic acids. In addition, up to 900 L of gas is produced daily, with approximately the following composition (by volume): 65% CO₂, 27% methane, 7% N₂, 0.18% H₂, and trace amounts of hydrogen sulfide. Relatively recently, bacteria were isolated from rumen contents that, under laboratory conditions, fermented cellulose into the same acids and in the same proportions as in the rumen. It can therefore be inferred that organic acids in the rumen are produced As a result of bacterial cellulose degradation.

Species capable of cleaving cellulose in the rumen include Ruminococcus albus and R. flavefaciens, which are Gram-negative cocci; Bacteroides succinogenes, a Gram-negative non-motile rod that produces primarily acetic and succinic acids; Butyrivibrio fibrisolvens; and Clostridium cellobioparum.

The absence of lactic acid in the rumen is attributed to The activity of the bacterium Veillonella alcalescens (Micrococcus lactilyticus), which ferments lactate to yield propionate, acetate, molecular hydrogen, and CO₂. Methane is not a direct product of cellulose degradation; it is of secondary origin, formed from fatty acids as well as molecular hydrogen and CO₂ (Section 9.4). HYDROGEN SULFIDE PRODUCTION in the rumen is linked to sulfate reduction by the bacterium Desulfotomaculum ruminis. Selenomonas ruminantium (Fig. 2.36, B) ferments glucose into lactic, acetic, and propionic acids.

In the natural habitats of ruminants—savannas and steppes—their forage is very poor in nitrogen and Proteins. Protein Synthesis is ensured by the symbiotic microflora of the rumen. Specifically, ruminants have developed a highly efficient "enterohepatic Urea Cycle." Urea produced in the Liver during ammonia detoxification is only partially excreted in the urine; the remainder enters the forestomachs via the Salivary Glands and the rumen wall, where it can be utilized by rumen microorganisms for protein synthesis (Fig. 14.1). Thanks to their symbiotic relationship with rumen microorganisms, ruminants are independent of exogenous protein sources. It has been demonstrated repeatedly that cows can be maintained on a protein-free diet.

Bacteria play a dual role in the Nutrition of ruminants. The acids formed during polysaccharide breakdown are absorbed directly in the rumen. The bacteria themselves are digested when the rumen contents pass into the intestine, so their cellular matter is also degraded and assimilated by the animal. Because rumen bacteria utilize inorganic nitrogen sources as well, this significantly increases the total protein intake available to the Organism.

Rumen bacteria hydrogenate plant Lipids. The resulting saturated fatty acids are absorbed in the intestine and subsequently incorporated into the body fat of cattle, which makes up meat, milk, and butter. Animals lacking a rumen do not exhibit such an increase in fat firmness. Fats stored in the bodies of pigs or rodents therefore have a softer consistency (a lower melting point) than ruminant fats; they contain Unsaturated fatty acids and shorter-chain fatty acids—namely those derived directly from plant feed. Considering that not only fats are modified by rumen bacteria, but 60–90% (by weight) of cattle proteins are also of bacterial origin, one can truly feel like a guest of bacteria when enjoying a beefsteak, and a guest of forage plants when savoring a pork schnitzel!