Levels Of Cellulolytic Activity Research Articles

STUDY OF THE CELLULOLYTIC ACTIVITY OF A COLLECTION OF "FIELD" BACTERIAL CULTURES

Decomposition of organic substances is widespread in all types of soils and plays an important role in the carbon cycle. The decomposition of cellulose plays a special role, since cellulose is the most common polysaccharide in plant residues and can be used by new plants in the form of simpler compounds. At the same time, the rate of decomposition of plant residues in the soil takes a considerable time and largely depends on the type of soil and its microbial composition. The use of individual most active microorganisms in the composition of biological products can increase soil fertility, especially in those areas where the original composition of the microflora does not contribute to the active decomposition of plant residues. In this study, the cellulolytic activity of various strains of microorganisms isolated from the soil was studied and compared. The objects of the study were a collection of 56 strains. The study of cellulolytic activity was carried out on nutrient media containing a cellulose source using congo dye ed. The obtained results showed a different level of cellulolytic activity in the studied strains. In some cases, there was an increase in activity with a decrease in the number of bacterial cells. 18 of 56 strains demonstrated cellulose degradation only within the limits of bacterial colony growth. According to the results of the studies, 6 strains of microorganisms with the largest zone of cellulolytic activity, reaching a radius of 15 mm around the grown colony, were noted. Among the most active strains, only one was Gram-negative bacteria, while the rest were gram-positive microorganisms.

Efficiency of using Biosystem POWER, KS (BioSistem POWER, SC) preparation to accelerate the destruction of post-harvest residues

Disposal of post-harvest residues from agro-ecosystem remains one of the urgent issues of today for agricultural producers. Burning straw and stubble is an extremely radical and destructive method for biogeocenosis, because during combustion between straw and the soil layer due to water vapor, conditions are created that the burning temperature exceeds 600°С. As a result of such actions, microorganisms, worms, insects, small vertebrates die and the fertile layer of humus is destroyed. Modern biological preparations, which contain a complex of living agronomically useful microorganisms, help to solve the issue of postharvest residues. By accelerating the destruction of organic masses in the soil, the C:N ratio is equalized, organic substances are supplied in a form available to plants, the development of phytopathogens is inhibited, moisture is retained, and humus accumulation processes are active. Field studies were conducted to determine the effectiveness of the new biological preparation BioSistem POWER KS (BioSistem POWER, SC), which contains active bacterial strains of the genera Paenabacillus, Azotobacter, Enterobacter and micromycetes of the genus Trichoderma. Selection of soil samples was carried out 90 days after processing, laboratory tests were carried out in accordance with generally accepted microbiological methods. It was established that the use of BioSistem POWER KS biological preparation (BioSistem POWER, SC) compared to the control increases the level of carbon dioxide emission of the soil of the experimental variants by 2 times, the level of cellulolytic activity by 23–34% depending on the rate of use, the antifungal activity of the soil by 2.5-3 times. Therefore, the biopreparation BioSistem POWER KS (BioSistem POWER, SC) at the rate of 0.3 and 5 l/ha can be recommended to agricultural producers for use in the autumn-spring period to accelerate the decomposition of post-harvest residues.

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus.

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

Cellulolytic potential of gut bacterial biomass in silkworm Bombyx mori. L

Abstract Exploring bacterial populations with cellulolytic activity from the midgut of Bombyx mori was proposed in this study. As the initial efforts, cellulose degrading bacteria was harvested from the midgut of Bombyx mori by using selective CMC (carboxymethylcellulose) agar medium. The gut bacterial biomass was categorized by biochemical and molecular identification; they were validated through 16S rDNA analysis. All the gut isolates were subjected to plate assay separately, to display the efficient cellulose fabricator. Among them, Bacillus aryabhattai and Bacillus sp. has shown the highest level of cellulolytic activity, when compared to the zone of clearance developed by the other isolates. Zymographic analysis also revealed same, while treated with cellulolytic substrate and the cellulolytic activity was visualized in the gut isolate Bacillus aryabhattai. For futher adjudication, cellulose degradation study was performed with Bacillus aryabhattai and Bacillus sp in HPLC and FTIR. Both the results of HPLC and FTIR conferred the status that Bacillus aryabhattai is the superior in cellulolytic activity. The Productivity of cellulase in Bacillus aryabhattai was detected and optimized at different pH, temperature, incubation time and different concentration of cellulose and identified their peak outputs. The gut microbiome based cellulase can act as a novel biocatalyst in the field biofuels recovery and also can be utilized as Phytobezoars in treatment of cattle and humans.

Characterization of cellulolytic activity in the gut of the terrestrial land slug Arion ater: Biochemical identification of targets for intensive study.

The level of cellulolytic activity in different areas of the gut of the terrestrial slug Arion ater was assayed at different temperatures and pH values. To do this, crude gut proteins were isolated and assayed using modified dinitrosalicylic acid reducing sugar assay. Crude protein samples were also separated and cellulolytic activity identified using in gel CMC zymography and esculin hydrate activity gel assays. pH and temperature profiling revealed optimum cellulolytic activity between pH5.0 and 6.0 for different gut regions and retention of up to 90% of activity at temperatures up to 50°C. Zymograms and activity gels revealed multiple endoglucanase and β-glucosidase enzymes. To further investigate the source of this cellulolytic activity bacterial isolates from the gut were tested for endoglucanase and β-glucosidase activity using growth plate assays. 12 cellulolytic microbes were identified using 16S rDNA gene sequencing. These include members of the genera Buttiauxella, Enterobacter, Citrobacter, Serratia and Klebsiella. Gut metagenomic DNA was then subjected to PCR, targeting a 400bp region of the 16SrDNA gene which was subsequently separated and individuals identified using DGGE. This identified members of the genera Citrobacter, Serratia, Pectobacterium, Acinetobacter, Mycoplasma, Pantoea and Erwinia. In summary, multiple glycoside hydrolase enzymes active over a broad range of temperature and pH values in a relatively under studied organism were detected, indicating that the gut of A. ater is a viable target for intensive study to identify novel carbohydrate active enzymes that may be used in the biofuel industry.

Degradation of cellulose by the bean-pathogenic fungus Colletotrichum lindemuthianum. Production of extracellular cellulolytic enzymes by cellulose induction

Colletotrichum lindemuthianum was able to grow and produce extracellular cellulolytic activity in a defined medium containing cellulose as the main carbon substrate. As measured either by the hydrolysis of 4-methylumbelliferyl-beta-D -cellotrioside or the release of glucose from carboxymethylcellulose, activity reached a peak after 13 days of incubation and then declined whereas growth markedly increased afterwards. Detection of glucose in carboxymethylcellulose hydrolysates suggested the concerted operation of endo-1,4-beta-glucanase, cellobiohydrolase (exo-1,4-beta-glucanase) and beta-glucosidase activities. The highest levels of cellulolytic activity were obtained in media supplemented with cellulose and glutamate. Other carbon and nitrogen sources markedly influenced growth and enzyme production. Oligonucleotides homologous to specific regions of the cellobiohydrolase-encoding cbhII gene from Trichoderma reesei were used to isolate a C. lindemuthianum cbhII-DNA fragment whose sequence revealed homologies of 98% and 92% with the nucleotide and the deduced amino acid sequences of the corresponding cbhII-DNA of T. reesei, respectively. RT-PCR and Southern blot analyses of total RNA samples obtained from cellulose-grown but not from glucose-grown mycelium revealed the expression of the corresponding cbhII transcript. The cbhII-cDNA fragment was cloned and sequenced.

Production of pectate lyases and cellulases by Chryseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures.

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14 degrees C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24 degrees C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20 degrees C, and then it increased dramatically until the temperature was 28 degrees C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.

Soyabean meal protein degradabiuty in the sheep rumen

Measurement of N disappearance during the incubation of a feed in polyester bags in the rumen is a widely used procedure for determining the ruminal protein degradability characteristics of the feed. These can then be used in conjunction with measurements of rumen outflow rate to calculate the expected extent of degradation in the rumen, which is a necessary prerequisite for diet formulation. Using the polyester bag procedure rates of feedstuff protein degradation have been found to be lower when cereal diets are given than when forage diets are given. Whilst this has been attributed to lower levels of cellulolytic activity in the rumen of cereal-fed animals it has also been suggested that it may be due to the physical nature of the rumen contents causing blockage of the bag pores. In the present study the effect of feeding either an all-forage or a high-cereal diet on polyester bag-derived and in vivo estimates of soyabean meal (SBM) protein degradability in the sheep rumen was examined.

The production of cellulolytic enzymes by fungal cultures

Twelve fungal cultures belonging to the genera ofAspergillus, Tricboderma, Chaetomium, Stachybotrys, andHypocrea were screened for the production of cellulolytic activity. All twelve were found to degrade xylan, avicel, and carboxymethylcellulose, More cellulolytic activity was obtained with shaken cultures than with still cultures and the addition of citrate-phosphate buffer to the media greatly depressed the levels of cellulolytic activity. Varying the composition of the mineral salts in the medium had no effect on the cellulolytic activity. The growth ofAspergillus wentii under controlled conditions in a bioreactor showed that the cellulolytic activity was not affected by the aeration rate or the type of stirrer. The rate of stirring, however, did effect the cellulolytic activity, as at lower stirring speeds considerable wall growth occurred which resulted in low levels of cellulolytic activity. Culture supernatant fromAspergillus wentii was found to hydrolyze from 30–32% of Solka-Floc and from 2–10% of corn cobs, wheat straw, and newsprint. The extensive hydrolysis of the Solka-Floc indicates that with suitably treated cellulosic wastes and appropriate enzymes, appreciable amounts of sugars could be obtained.

THALASSIOMYCETES: III. THE GENUS ZALERION

On the basis of morphological and physiological studies, including analyses of cellulolytic activity, the thalassic deuteromycete genus Zalerion, an abundant taxon in northern and temperate marine areas, is erected. Included in this genus are four heretofore undescribed species. Different colonial morphologies and levels of cellulolytic activity, the latter characterized on CMC 50 T and Walseth cellulose, are evident among these taxa.