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. 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%. 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.

中文翻译：

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与半纤维素发酵微生物共培养逆转了热固梭菌在高固体含量下对玉米纤维增溶的抑制作用

纤维素分解嗜热纤维梭菌（Clostridium thermocellum）是重要的生物催化剂，因为它具有溶解木质纤维素原料的能力，而无需预处理或添加外源酶。在低浓度的底物下，热纤梭菌可以在5天内溶解> 95％的玉米纤维，但是当底物浓度高于20 g / L时，溶解度显着下降。这与模型纤维素（如Avicel）不同，在模型纤维素上，最大增溶速率与底物浓度成正比。这项研究的目的是检查玉米纤维浓度增加时的发酵，并研究降低性能的可能原因。由LC-MS / MS测定的CipA支架素水平推论，玉米纤维上热纤梭菌的生长速率随固形物含量的增加几乎没有增加。为了测试抑制作用，我们评估了废肉汤对生长和纤维素酶活性的影响。发现玉米纤维发酵后残留的液体强烈抑制纤维二糖（不需要纤维素水解的底物）上的生长。另外，通过添加废肉汤，热纤梭菌纤维素酶的水解活性也降低到不到一半。注意到在40 g / L玉米纤维发酵的废液中积累了> 15 g / L的半纤维素低聚糖，我们测试了各种模型碳水化合物对纤维二糖和Avicel生长的影响。一些化合物，例如低聚木糖，导致纤维素分解活性下降，对Avicel的最大增溶速率降低。然而，没有相关的模型化合物可以复制废肉汤对纤维二糖上热纤梭菌生长的强烈抑制作用。热纤梭菌与消耗半纤维素的伴侣Herbinix spp的共培养。菌株LL1355和解热嗜热厌氧杆菌展示了较低水平的未发酵半纤维素水解产物，最大溶解率增加了一倍，最终溶解度从67％增加到93％。该研究记录了随着玉米纤维浓度的增加而对热纤梭菌的抑制作用，并证明了木寡糖对纤维素酶活性的抑制作用，但是还需要进一步的工作来理解为什么玉米纤维发酵液会抑制纤维二糖的生长。我们的结果支持利用半纤维素的共培养合作伙伴增加C的重要性。