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


 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.


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 ber >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 ber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn ber, 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 ber 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 ber 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 nal solubilization increased from 67% to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn ber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn ber 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.

Full Text
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF.    Effect of different carbohydrates on C. thermocellum cellulase activity. 0.5 g/L initial Avicel was incubated with 10 mg/g C. thermocellum cellulase and activity was estimated by measuring the decrease in OD600 after 24h.The error bars show the SD for n=2. XOS = xylooligosaccharides, AX = arabinoxylan, Control Broth -see Materials and Methods. Glucomannan, galactomannan and xyloglucan used here are all oligosaccharide mixtures, prepared by partial enzymatic hydrolysis of the respective carbohydratesee Materials and Methods.

Figure 5
Effect of XOS addition on growth of C. thermocellum on Avicel. Fermentations were done in serum bottles with starting concentration of 10 g/L Avicel. a) Avicel remaining (g/L), b) ethanol produced (g/L) and c) cell growth estimated by total pellet nitrogen (g/L).

Figure 6
Effect of various carbohydrates on C. thermocellum growth on Avicel. C. thermocellum grown on 10 g/L Avicel with the addition of 10 g/L of various carbohydrates. Rate was calculated by differentiating a Boltzmann Sigmoidal t curve on the Avicel utilization timecourse data. R2 values of >0.98 were observed in all cases. Error bars show the standard deviation (n≥2). For the control n = 6. XOS = Xylooligosaccharides, AX = arabinoxylan.

Figure 7
Comparison of monoculture and coculture in fermenting corn ber. Fermentation data for C. thermocellum monoculture and its coculture with LL1355 and LL1548 on 40 g/L corn ber in bioreactors: a) Sugars remaining in solid (g/L); b) Rate of solubilization (g/L/h); c) Sugars in liquid (g/L); d) biocatalyst as determined by normalized CipA intensity. Cocultures were inoculated with LL1355 and LL1548 at 0 and again at 50 hours.

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