scholarly journals Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

2020 ◽  
Author(s):  
Dhananjay Beri ◽  
Christopher D Herring ◽  
Sofie Blahova ◽  
Suresh Poudel ◽  
Richard Giannone ◽  
...  
Keyword(s):  
Fermentation Broth ◽  
Cellulase Activity ◽  
Corn Fiber ◽  
Cellulolytic Activity ◽  
Fiber Concentration ◽  
Exogenous Enzyme ◽  
Thermoanaerobacterium Thermosaccharolyticum ◽  
Solids Loading ◽  
Lignocellulosic Feedstocks ◽  
Fiber Fermentation

Abstract BackgroundThe cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber >95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance.ResultsThe rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC-MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less than half by adding spent broth. Noting that >15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose consuming partners - Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum –exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67% to 93%. ConclusionsThis study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

scholarly journals Coculture with Hemicellulose-Fermenting Microbes Reverses Inhibition of Corn Fiber Solubilization by C. Thermocellum at Elevated Solids Loadings

2020 ◽  
Author(s):  
Dhananjay Beri ◽  
Christopher D Herring ◽  
Sofie Blahova ◽  
Suresh Poudel ◽  
Richard Giannone ◽  
...  
Keyword(s):  
Fermentation Broth ◽  
Cellulase Activity ◽  
Corn Fiber ◽  
Cellulolytic Activity ◽  
Fiber Concentration ◽  
Exogenous Enzyme ◽  
Thermoanaerobacterium Thermosaccharolyticum ◽  
Solids Loading ◽  
Lignocellulosic Feedstocks ◽  
Fiber Fermentation

Abstract Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber >95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC-MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less than half by adding spent broth. Noting that >15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose consuming partners - Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum –exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67% to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

scholarly journals Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Dhananjay Beri ◽  
Christopher D. Herring ◽  
Sofie Blahova ◽  
Suresh Poudel ◽  
Richard J. Giannone ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Fermentation Broth ◽  
Cellulase Activity ◽  
Corn Fiber ◽  
Cellulolytic Activity ◽  
Exogenous Enzyme ◽  
Thermoanaerobacterium Thermosaccharolyticum ◽  
Solids Loading ◽  
Lignocellulosic Feedstocks ◽  
Fiber Fermentation

Abstract Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

scholarly journals First Report of Black Soft Rot of Indian Gooseberry Caused by Syncephalastrum racemosum

Plant Disease ◽  
2004 ◽  
Vol 88 (5) ◽  
pp. 575-575 ◽  
Author(s):  
Neelima Garg ◽  
Om Prakash ◽  
B. K. Pandey ◽  
B. P. Singh ◽  
G. Pandey
Keyword(s):  
Cellulase Activity ◽  
Basal Medium ◽  
Petri Dish ◽  
Soft Rot ◽  
Cellulolytic Activity ◽  
Academic Press ◽  
First Report ◽  
Syncephalastrum Racemosum ◽  
Indian Gooseberry ◽  
Pathogenicity Tests

Indian gooseberry (Emblica officinalis Gaertn.) is a medicinal plant with high nutraceutical value. During November and December 2003, soft rot was noticed on harvested and stored (20 ± 5°C and 65 ± 5% relative humidity) fruits at the experimental farm in Rehmanhera, Lucknow, India (26°50′N, 80°54′E). These fruits had numerous, minute brown necrotic lesions showing white mycelial growth. A pronounced halo of water-soaked, faded tissue surrounded the lesion between the fringe of mycelium and healthy tissues. The rotted surface was covered with a black, powdery layer of spores. On Czapek yeast extract agar, fungal colonies were blackish grey, moderately dense, and covered the entire petri dish. The fungus produced aseptate mycelium. The sporangial heads were 30 to 50 μm in diameter with sporangiospores found linearly within cylindrical sacs (merosporangia) borne on spicules around the columella. Sporangiospores, spherical to cylindrical in shape and borne in chains, measured 3.0 to 5.0 μm long. The fungus was morphologically and physiologically identified as Syncephalastrum racemosum Schr. (2). For pathogenicity tests, healthy fruits (10 replicates) were surface sterilized and punctured inoculated aseptically with 1.0 × 106 conidia and incubated at 20 ± 5°C Typical symptoms of the disease appeared after 4 days. The fungus exhibited a strong level of cellulolytic activity as indicated by prolific growth on Indian gooseberry fiber waste under solid-state fermentation conditions. The level of cellulase activity (1) was 21 filter paper activity unit per ml at 72 hr in culture supernatant of basal medium having carboxymethyl cellulose as the carbon source. The fungus showed resistance to tannins (as much as 2%), since it could grow well in liquid growth medium (Czapek Dox broth) with 2% tannins and aonla juice with 1.8% tannins. Since Indian gooseberry is rich in fiber (2.5 to 3.4%) and tannins (1.5 to 2.0%), this may be an important pathogen. To our knowledge, this is the first report of the occurrence of Syncephalastrum racemosum on Indian gooseberry fruits. References: (1) T. K. Ghose. Pure Appl. Chem. 59(2):257, 1987. (2) J. I. Pitt and A. D. Hocking. Fungi and Food Spoilage. Academic Press. North Ryde, Australia, 1985.

scholarly journals Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in EthanologenicKlebsiella oxytoca P2

2001 ◽  
Vol 67 (1) ◽  
pp. 6-14 ◽  
Author(s):  
Shengde Zhou ◽  
F. C. Davis ◽  
L. O. Ingram
Keyword(s):  
Fermentation Broth ◽  
Klebsiella Oxytoca ◽  
Cellulase Activity ◽  
Erwinia Chrysanthemi ◽  
Crystalline Cellulose ◽  
Fungal Enzymes ◽  
Extracellular Milieu ◽  
Extracellular Secretion ◽  
Genes Encoding ◽  
Fuels And Chemicals

ABSTRACT The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) ofKlebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. GenescelY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.

scholarly journals Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes

1997 ◽  
Vol 326 (2) ◽  
pp. 617-624 ◽  
Author(s):  
Irina KATAEVA ◽  
Gérard GUGLIELMI ◽  
Pierre BÉGUIN
Keyword(s):  
Clostridium Thermocellum ◽  
Analytical Ultracentrifugation ◽  
Cellulase Activity ◽  
Hydrolytic Activity ◽  
Binding Domain ◽  
Cellulolytic Activity ◽  
Cellulose Binding Domain ◽  
Optimal Ratio ◽  
Cellulose Binding

Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD–CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.

Ultrastructural Localization of Cellulase in Diplodia Maydis and in D. Maydis-Infected Com Stalk Tissue

1977 ◽  
Vol 35 ◽  
pp. 454-455
Author(s):  
Judith A. Murphy ◽  
Mary R. Thompson ◽  
A.J. Pappelis
Keyword(s):  
Culture Medium ◽  
Cupric Oxide ◽  
Cellulase Activity ◽  
Growth Period ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Low Ph ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Activity ◽  
X Ray

BeMiller et.al.(l) found that D. maydis did not have the solubilizing enzyme C1. They reported that D- maydis exhibited cellulolytic activity constitutively, and hypothesized that the cellulolytic enzymes were attached to fungal hyphal surfaces because they found cellulase released to the culture medium only after the growth period, when available cellulose had been used up.The purpose of this study was to determine the location of cellulolytic enzymes (EC 3.2.1.4; beta-1,4-glucan glucanohydrolase) in D. maydis and D. maydis-infected corn tissue at the ultrastructural level.Cellulase activity produces glucose as an end product which will reduce cupric oxide and can be visualized with an EM because it is electron dense and the Cu component can be verified with x-ray analysis(Figs.l,2). After thorough washing, samples fixed in aldehydes are incubated in a substrate mixture at a low pH. The enzyme is activated and reducing sugar is released. The sample is then reacted with Benedict's solution at a high temperature, allowing CuO crystals to be deposited at the site of reaction.

In Situ Corn Fiber Conversion for Ethanol Improvement by The Addition of Novel Lignocellulolytic Enzyme Cocktail

2020 ◽  
Author(s):  
Le Gao ◽  
Shulin Chen ◽  
Dongyuan Zhang
Keyword(s):  
Batch Fermentation ◽  
Ethanol Yield ◽  
Fermentation Broth ◽  
High Viscosity ◽  
Corn Fiber ◽  
Yeast Fermentation ◽  
Lignocellulolytic Enzymes ◽  
High Sugar ◽  
Corn Mash

Abstract Background: The technology of converting corn mashes to ethanol has been mature, but corn mashes has high-viscosity and high-sugar characteristics which hindered cellulose utilization and yeast-fermentation efficiency. The excessive viscosity of corn mash is caused by the presence of non-starch polysaccharides, such as cellulose in cereal grains. Corn kernel fiber (mostly cellulose) is typically unconverted in the process. Results: A novel lignocellulolytic enzymes cocktail with strong substrate specificity was prepared for high-viscosity, high-sugar corn mash. The in situ conversion of corn mashes with novel lignocellulolytic enzymes at the optimum cellulase dosage of 50 FPU/L resulted in 12.4%, 12.0%, 11.8%, and 12.9% increased ethanol concentration compared with the reference mash at 0.3, 1, 5, and 70 L batch-fermentation scales, respectively. The highest yield of ethanol from corn mash digested with the prepared novel lignocellulolytic enzyme reached 117.0 ± 0.1g/L at the 70 L batch fermentation, which was a 12.9% increase in ethanol yield. Adding the lignocellulolytic enzymes caused the greatest decrease in viscosity of corn mash by 40.9% compared with the reference mash (33.5 ± 1.5 Pa·s), whereas the residual sugars decreased by 56.3%. Simultaneously, the application of novel lignocellulolytic enzymes increased the value of dried distiller’s grain with solubles by increasing the protein content and decreasing the residual cellulose and starch content.Conclusion: The application of novel lignocellulolytic enzymes significantly improved the alcohol concentration, productivity, and yield. With the same amount of material, the application of the novel enzymes cocktail can enhance the ethanol yield by more than 10%. The in situ conversion of cellulose promoted the release of contents, including starch and protein, which can decrease the fermentation broth viscosity and improve the rheological property, thereby improving the ethanol yield. Thus, this technology can increase the net revenue of fuel-ethanol industrialization and promote the technological progress of renewable energy.

Cellulolytic activity in municipal solid waste composting

1970 ◽  
Vol 16 (7) ◽  
pp. 553-560 ◽  
Author(s):  
F. J. Stutzenberger ◽  
A. J. Kaufman ◽  
R. D. Lossin
Keyword(s):  
Municipal Solid Waste ◽  
Solid Waste ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Hydrolysis Rate ◽  
Bacillus Species ◽  
Cellulose Content ◽  
Cellulolytic Activity ◽  
Cellulose Decomposition ◽  
Ph Values

A study was made of the open windrow method of municipal solid waste composting as related to those factors that might influence cellulose decomposition. Composting temperatures reached a maximum (55–65 °C) at 3 weeks and then gradually decreased during the rest of the 49-day process. The pH values of compost samples homogenized in distilled water decreased slightly during the initial stages of the process and then increased gradually to final values of 7.0–8.5. Clarified compost extracts were assayed for cellulase activity by measuring the hydrolysis rate of carboxymethylcellulose (CMC). Maximal cellulase activity occurred at 65 °C, pH 6.0, with a CMC concentration of 2.5%. The cellulase activity of compost increased 10-fold at a logarithmic rate while the cellulose content decreased 50%. In a preliminary search for microorganisms active in cellulose degradation during the composting process, three cellulolytic species were isolated; these were identified as Aspergillus fumigatus, a Bacillus species, and a Thermoactinomyces species of the Actinobifida group.

scholarly journals Culturing-Enriched Metabarcoding Analysis of the Oryctes rhinoceros Gut Microbiome

Insects ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 782
Author(s):  
Matan Shelomi ◽  
Ming-Ju Chen
Keyword(s):  
Bacillus Cereus ◽  
Fat Body ◽  
Cellulase Activity ◽  
Gut Contents ◽  
Cellulolytic Activity ◽  
Gut Microbes ◽  
Oryctes Rhinoceros ◽  
Rhinoceros Beetle ◽  
Unculturable Bacteria ◽  
Insect Gut

Wood-feeding insects should have a source of enzymes like cellulases to digest their food. These enzymes can be produced by the insect, or by microbes living in the wood and/or inside the insect gut. The coconut rhinoceros beetle, Oryctes rhinoceros, is a pest whose digestive microbes are of considerable interest. This study describes the compartments of the O. rhinoceros gut and compares their microbiomes using culturing-enriched metabarcoding. Beetle larvae were collected from a coconut grove in southern Taiwan. Gut contents from the midgut and hindgut were plated on nutrient agar and selective carboxymethylcellulose agar plates. DNA was extracted from gut and fat body samples and 16S rDNA metabarcoding performed to identify unculturable bacteria. Cellulase activity tests were performed on gut fluids and microbe isolates. The midgut and hindgut both showed cellulolytic activity. Bacillus cereus, Citrobacter koseri, and the cellulolytic fungus Candida xylanilytica were cultured from both gut sections in most larvae. Metabarcoding did not find Bacillus cereus, and found that either Citrobacter koseri or Paracoccus sp. were the dominant gut microbes in any given larva. No significant differences were found between midgut and hindgut microbiomes. Bacillus cereus and Citrobacter koseri are common animal gut microbes frequently found in Oryctes rhinoceros studies while Candida xylanilytica and the uncultured Paracoccus sp. had not been identified in this insect before. Some or all of these may well have digestive functions for the beetle, and are most likely acquired from the diet, meaning they may be transient commensalists rather than obligate mutualists. Broader collection efforts and tests with antibiotics will resolve ambiguities in the beetle–microbe interactions.

Extracellular cellulase production by tropical isolates of Aureobasidium pullulans

2005 ◽  
Vol 51 (9) ◽  
pp. 773-776 ◽  
Author(s):  
T Kudanga ◽  
E Mwenje
Keyword(s):  
Carboxymethyl Cellulose ◽  
Cell Walls ◽  
Aureobasidium Pullulans ◽  
Plant Cell Wall ◽  
Synthetic Medium ◽  
Cellulase Activity ◽  
Nitrogen Sources ◽  
Cellulase Production ◽  
Cellulolytic Activity ◽  
Specific Activities

Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and α-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 µmol glucose·(mg protein)–1·h–1, while activities on α-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 µmol glucose·(mg protein)–1·day–1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.Key words: Aureobasidium pullulans, plant cell wall, cellulases, endoglucanase, exoglucanase.

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