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

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.

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

10.21203/rs.3.rs-72741/v1 ◽

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.

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

10.21203/rs.3.rs-72741/v2 ◽

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.

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

Biochemical Journal ◽

10.1042/bj3260617 ◽

1997 ◽

Vol 326 (2) ◽

pp. 617-624 ◽

Cited By ~ 31

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.

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

Plant Disease ◽

10.1094/pdis.2004.88.5.575c ◽

2004 ◽

Vol 88 (5) ◽

pp. 575-575 ◽

Cited By ~ 2

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.

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Exploring the Relationship Between Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17

Frontiers in Microbiology ◽

10.3389/fmicb.2019.02035 ◽

2019 ◽

Vol 10 ◽

Author(s):

Fangzhong Wang ◽

Mingyu Wang ◽

Qi Zhao ◽

Kangle Niu ◽

Shasha Liu ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Thermoanaerobacterium Thermosaccharolyticum ◽

The Relationship

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Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum

Applied Microbiology and Biotechnology ◽

10.1007/s00253-004-1767-3 ◽

2004 ◽

Vol 66 (6) ◽

pp. 635-640 ◽

Cited By ~ 19

Author(s):

P. J. Weimer ◽

R. G. Koegel ◽

L. F. Lorenz ◽

C. R. Frihart ◽

W. R. Kenealy

Keyword(s):

Clostridium Thermocellum ◽

Ruminococcus Albus ◽

Wood Adhesives ◽

Fiber Fermentation

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Symbiotic Behavior during Co-culturing of Clostridium thermocellum NKP-2 and Thermoanaerobacterium thermosaccharolyticum NOI-1 on Corn Hull

BioResources ◽

10.15376/biores.9.2.2471-2483 ◽

2014 ◽

Vol 9 (2) ◽

Cited By ~ 3

Author(s):

Suphavadee Chimtong ◽

Chakrit Tachaapaikoon ◽

Somphit Sornyotha ◽

Patthra Pason ◽

Rattiya Waeonukul ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Thermoanaerobacterium Thermosaccharolyticum

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

Applied and Environmental Microbiology ◽

10.1128/aem.67.1.6-14.2001 ◽

2001 ◽

Vol 67 (1) ◽

pp. 6-14 ◽

Cited By ~ 49

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.

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