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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

An integrated biorefinery approach to obtain xylo-oligosaccharides from corncob using lactic acid-rich fermentation broth

Although xylo-oligosaccharides production from lignocelluloses via lactic acid hydrolysis is advantageous in terms of high yields with less by-products, the use of high purity lactic acid inevitably increases the cost of xylo-oligosaccharides production. The utilization of lactic acid-rich fermentation broth for xylo-oligosaccharides production can avoid the separation and purification of lactic acid. However, the feasibility of lactic acid-rich fermentation broth for xylo-oligosaccharides production from lignocelluloses was unclear. In this work, the results indicated that the highest xylo-oligosaccharides yield of 72% was obtained from corncob by lactic acid-rich fermentation broth pretreatment and xylanase hydrolysis. Subsequently, monosaccharides-rich hydrolysate (90.9 g/L) was obtained from solid residues of corncob by cellulase hydrolysis. Lactic acid-rich fermentation broth was produced by Weizmannia coagulans fermentation of the monosaccharide-rich hydrolysate. Besides, the obtained xylo-oligosaccharides and lactic acid mixture might be used as feed additives, avoiding the purification and separation of lactic acid or xylo-oligosaccharides. Meanwhile, the production cost of feed grade xylo-oligosaccharides from this work was much lower than the current market price. Overall, this work proposed a new strategy for efficient and environmental-friendly production of xylo-oligosaccharides from corncob.

09 BIOMASS FUELS↗
DOE OSTI2013

A review of dark fermentative hydrogen production from biodegradable municipal waste fractions

Highlights: ► A large number of factors affect fermentative hydrogen production. ► Harmonization and systematic comparison of results from different literature sources are needed. ► More than 80 publications on H{sub 2} production from food waste and OFMSW have been examined. ► Experimental data from the reviewed literature were analyzed using statistical tools. ► For a reliable assessment of the process performance, the use of multiple parameters appears to be recommended. - Abstract: Hydrogen is believed to play a potentially key role in the implementation of sustainable energy production, particularly when it is produced from renewable sources and low energy-demanding processes. In the present paper an attempt was made at critically reviewing more than 80 recent publications, in order to harmonize and compare the available results from different studies on hydrogen production from FW and OFMSW through dark fermentation, and derive reliable information about process yield and stability in view of building related predictive models. The review was focused on the effect of factors, recognized as potentially affecting process evolution (including type of substrate and co-substrate and relative ratio, type of inoculum, food/microorganisms [F/M] ratio, applied pre-treatment, reactor configuration, temperature and pH), on the fermentation yield and kinetics. Statistical analysis of literature data from batch experiments was also conducted, showing that the variables affecting the H{sub 2} production yield were ranked in the order: type of co-substrate, type of pre-treatment, operating pH, control of initial pH and fermentation temperature. However, due to the dispersion of data observed in some instances, the ambiguity about the presence of additional hidden variables cannot be resolved. The results from the analysis thus suggest that, for reliable predictive models of fermentative hydrogen production to be derived, a high level of consistency between data is strictly required, claiming for more systematic and comprehensive studies on the subject.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2015

Converting the organic fraction of solid waste from the city of Abu Dhabi to valuable products via dark fermentation – Economic and energy assessment

Graphical abstract: Display Omitted - Highlights: • The cost and energy demand for dark fermentation using OFMSW were established. • Dark fermentation using OFMSW can produce a carbon source for bioprocesses of about 330 USD/t{sub COD}. • A maximum purification cost of VFAs from dark fermentation using OFMSW was established to 15 USD/m{sup 3}. • Replacing fossil fuel based products by dark fermentation will probably lead to net energy savings. - Abstract: Landfilling the organic fraction of municipal solid waste (OFMSW) leads to greenhouse gas emissions and loss of valuable resources. Sustainable and cost efficient solutions need to be developed to solve this problem. This study evaluates the feasibility of using dark fermentation (DF) to convert the OFMSW to volatile fatty acids (VFAs), fertilizer and H{sub 2}. The VFAs in the DF effluent can be used directly as substrate for subsequent bioprocesses or purified from the effluent for industrial use. DF of the OFMSW in Abu Dhabi will be economically sustainable once VFA purification can be accomplished on large scale for less than 15 USD/m{sup 3}{sub effluent}. With a VFA minimum selling price of 330 USD/t{sub COD}, DF provides a competitive carbon source to sugar. Furthermore, DF is likely to use less energy than conventional processes that produce VFAs, fertilizer and H{sub 2}. This makes DF of OFMSW a promising waste treatment technology and biorefinery platform.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2018

Dry anaerobic digestion of cow manure and agricultural products in a full-scale plant: Efficiency and comparison with wet fermentation

Highlights: • The first full-scale, agricultural dry fermentation plant of Italy was monitored. • Recirculation rate, leachate, biogas and energy production were monitored. • Dry fermentation showed biogas yields similar to wet. • Retention time, recirculation of digestate and leachate are key factors. • Plant operation required significant man labor (loading and unloading of reactors). - Abstract: For years, anaerobic digestion processes have been implemented for the management of organic wastes, agricultural residues, and animal manure. Wet anaerobic digestion still represents the most common technology, while dry fermentation, dedicated to the treatment of solid inputs (TS > 20%) can be considered as an emerging technology, not in terms of technological maturity, but of diffusion. The first agricultural dry anaerobic digestion plant constructed in Italy was monitored from the start-up, for over a year. The plant was fed with manure and agricultural products, such as corn silage, triticale, ryegrass, alfalfa, and straw. Three Combined Heat and Power units, for a total installed power of 910 kW{sub e}, converted biogas into thermal and electric energy. The monitoring included the determination of quality and quantity of input feedstocks, of digestate (including recirculation rate), of leachate, biogas quality (CH{sub 4}, CO{sub 2}, H{sub 2}S), biogas yield, energy production, labor requirement for loading, and unloading operations. The results of the monitoring were compared to performance data obtained in several full scale wet digestion plants. The dry fermentation plant revealed a start-up phase that lasted several months, during which the average power resulted in 641 kW{sub e} (70.4% of nominal power), and the last period the power resulted in 788 kW{sub e} (86.6% of installed power). Improving the balance of the input, the dry fermentation process demonstrated biogas yields similar to wet anaerobic digestion, congruent to the energy potential of the biomasses used in the process. Furthermore, the operation of the plant required significant man labor, mainly related to loading and unloading of the anaerobic cells.

09 BIOMASS FUELS↗
DOE OSTI2024

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

Abstract Background Cost-effective production of biofuels from lignocellulose requires the fermentation of d -xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD + to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae . Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans , which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis . Conclusion Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

09 BIOMASS FUELS↗
DOE OSTI2021

Diverse Profile of Fermentation Byproducts From Thin Stillage

The economy of biorefineries is influenced not only by biofuel production from carbohydrates but also by the production of valuable compounds from largely underutilized industrial residues. Currently, the demand for many chemicals that could be made in a biorefinery, such as succinic acid (SA), medium-chain fatty acids (MCFAs), and lactic acid (LA), is fulfilled using petroleum, palm oil, or pure carbohydrates as raw materials, respectively. Thin stillage (TS), the residual liquid material following distillation of ethanol, is an underutilized coproduct from the starch biofuel industry. This carbon-rich material has the potential for chemical upgrading by microorganisms. Here, we explored the formation of different fermentation products by microbial communities grown on TS using different bioreactor conditions. At the baseline operational condition (6-day retention time, pH 5.5, 35°C), we observed a mixture of MCFAs as the principal fermentation products. Operation of a bioreactor with a 1-day retention time induced an increase in SA production, and a temperature increase to 55°C resulted in the accumulation of lactic and propionic acids. In addition, a reactor operated with a 1-day retention time at 55°C conditions resulted in LA accumulation as the main fermentation product. The prominent members of the microbial community in each reactor were assessed by 16S rRNA gene amplicon sequencing and phylogenetic analysis. Under all operating conditions, members of the Lactobacillaceae family within Firmicutes and the Acetobacteraceae family within Proteobacteria were ubiquitous. Members of the Prevotellaceae family within Bacteroidetes and Lachnospiraceae family within the Clostridiales order of Firmicutes were mostly abundant at 35°C and not abundant in the microbial communities of the TS reactors incubated at 55°C. The ability to adjust bioreactor operating conditions to select for microbial communities with different fermentation product profiles offers new strategies to explore and compare potentially valuable fermentation products from TS and allows industries the flexibility to adapt and switch chemical production based on market prices and demands.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids

ABSTRACT Volatile organic acids are byproducts of fermentative metabolism, for example, anaerobic digestion of lignocellulosic biomass or organic wastes, and are often times undesired inhibiting cell growth and reducing directed formation of the desired products. Here, we devised a general framework for upgrading these volatile organic acids to high‐value esters that can be used as flavors, fragrances, solvents, and biofuels. This framework employs the acid‐to‐ester modules, consisting of an AAT (alcohol acyltransferase) plus ACT (acyl CoA transferase) submodule and an alcohol submodule, for co‐fermentation of sugars and organic acids to acyl CoAs and alcohols to form a combinatorial library of esters. By assembling these modules with the engineered Escherichia coli modular chassis cell, we developed microbial manufacturing platforms to perform the following functions: (i) rapid in vivo screening of novel AATs for their catalytic activities; (ii) expanding combinatorial biosynthesis of unique fermentative esters; and (iii) upgrading volatile organic acids to esters using single or mixed cell cultures. To demonstrate this framework, we screened for a set of five unique and divergent AATs from multiple species, and were able to determine their novel activities as well as produce a library of 12 out of the 13 expected esters from co‐fermentation of sugars and (C2‐C6) volatile organic acids. We envision the developed framework to be valuable for in vivo characterization of a repertoire of not‐well‐characterized natural AATs, expanding the combinatorial biosynthesis of fermentative esters, and upgrading volatile organic acids to high‐value esters. Biotechnol. Bioeng. 2016;113: 1764–1776. © 2016 Wiley Periodicals, Inc.

Layton, Donovan S.↗
DOE OSTI2023

Combined effects of lipase and Lactiplantibacillus plantarum 1‐24‐LJ on physicochemical property, microbial succession and volatile compounds formation in fermented fish product

Abstract BACKGROUND Studies have shown that either the addition of starter culture or enzyme can improve fermentation in fish or other products. However, little research has been carried out on the effects of coupling starter cultures with lipase on the microbial community and product quality. Suanzhayu is a Chinese fermented fish product that mainly relies on spontaneous fermentation, resulting in an unstable flavor and quality. The present study investigated the impact of lipase and Lactiplantibacillus plantarum 1‐24‐LJ on the quality of Suanzhayu . RESULTS Inoculation decreased pH and 2‐thiobarbituric acid reactive substances (TBARS) values, and also helped the dominance of the strain in the ecosystem, whereas lipase addition raised TBARS values and had little effect on pH, water activity ( a w ) and microbiota. The addition of lipase and/or Lpb. plantarum increased the content of alcohols, aldehydes, ketones, esters and umami amino acids. The co‐additions with the most significant effect and the total contents of volatile compounds (VCs) and free amino acids (FAAs) were 1801.92 g per 100 g and 21 357.05 mg per 100 g, respectively. Former‐ Lactobacillus was negatively correlated with pH, a w and Prevotella , but positively with VCs (ethyl ester of heptanoic acid, ethyl ester of octanoic acid) and FAAs (Tyr, Phe). Furthermore, adding Lpb. plantarum 1‐24‐LJ alone or in combination with lipase shortened the fermentation process. CONCLUSION The present study provides a recommended Suanzhayu process approach for improving product quality and flavor, as well as shortening fermentation time, by adding Lpb. plantarum 1‐24‐LJ with or without lipase. © 2023 Society of Chemical Industry.

Zhang, Zuoli↗
DOE OSTI2013

Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process

Wood pulping hydrolysate (WPH) containing mainly xylose and glucose as a potential substrate for acetone-butanol-ethanol (ABE) fermentation was studied. Due to the inhibitors present in the hydrolysate, several dilution levels and detoxification treatments, including overliming, activated charcoal adsorption, and resin adsorption, were evaluated for their effectiveness in relieving the inhibition on fermentation. Detoxification using resin and evaporation was found to be the most effective method in reducing the toxicity of WPH. ABE production in batch fermentation by Clostridium beijerinckii increased 68%, from 6.73 g/L in the non-treated and non-diluted WPH to 11.35 g/L in the resin treated WPH. With gas stripping for in situ product removal, ABE production from WPH increased to 17.73 g/L, demonstrating that gas stripping was effective in alleviating butanol toxicity by selectively separating butanol from the fermentation broth, which greatly improved solvents production and sugar conversion in the fermentation. (C) 2013 Elsevier Ltd. All rights reserved.

Lu, CC↗
DOE OSTI2015

Early fermentation volatile metabolite profile of non-Saccharomyces yeasts in red and white grape must: A targeted approach

Saccharomyces cerevisiae is the main driver of alcoholic fermentation. It is typically inoculated at high levels to ensure successful implantation as well as reduce the risks of stuck fermentations and off-flavor production. However, winemakers have found that wines produced with only S. cerevisiae can be lacking in complexity compared to fermentations where non-Saccharomyces yeasts are more active. This paper sought to understand the early fermentation characteristics of Kazachstania gamospora, Lachancea thermotolerans, Metschnikowia pulcherrima, Torulaspora delbrueckii and Zygosaccharomyces kombuchaensis in both Sauvignon blanc and Syrah musts. S. cerevisiae was used as a control. Solid-phase microextraction coupled to GC–MS was used to evaluate the musts once they reached 2% ethanol concentration. The method targeted 90 different compounds known to occur in wine and/or be produced by yeast during fermentation. For the first time, K. gamospora and Z. kombuchaensis have been studied in the context of wine. While the other yeasts are commercially available starter cultures, they have never been profiled this extensively. Analysis showed that each yeast profile was unique and different based on the must. Finally, the non-Saccharomyces yeasts produced lower concentrations of esters, alcohols and terpenes with the exception of K. gamospora which produced more total esters than the control.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Enhanced, continuous, liquid-liquid extraction and in-situ separation of volatile fatty acids from fermentation broth

In 2018 alone, the US landfilled 35.3 million tons of food waste, about 24% of the total landfilled mass. In addition to the negative impacts landfills have demonstrated on the environment and human health, some states have begun to outlaw or dissuade the disposal of food waste and sewage sludge into landfills altogether. An urgent need has thus been created for the development of digestion processes like anaerobic digestion (AD) and arrested methanogenesis (AM) to convert food waste into valuable chemical products. Unfortunately, the buildup of volatile fatty acids (VFAs) during these processes eventually halts the reaction, and energy efficient methodologies for VFA removal are critical for the operation of fermenters. Additionally, VFAs themselves can serve as valuable chemical precursors, and recently AD processes have been modified to increase VFA production during fermentation. However, even with significant research over the past three decades, the separation of VFAs from the fermenter broth has remained expensive. Moreover, the separation of these VFAs from the fermenter broth may cost up to 50% of the entire process budget, hindering the widespread commercial adoption of AD and AM. Here we present a novel liquid-liquid extraction process termed CLEANS (Continuous Liquid-liquid Extraction And iN-situ Separation) as a highly efficient method for continuously separating VFAs from a real fermentation broth solely under gravity. Our optimized process (using an aqueous broth feed pH of 2.5, tri-noctylamine as an extractant, and a 10:1 ratio of aqueous broth to organic extractant), achieved a VFA distribution constant K D = 44.5 ± 7.9, a single-pass recovery = 81.3 ± 2.5%, and an extraction factor = 8.1 ± 0.3. These KD values are over an order of magnitude higher than what has been previously reported for comparable processes. A high aqueous-to-organic flowrate ratio, enabled for the first time by CLEANS, was found to be particularly crucial for achieving optimal extraction. Our separation process demonstrates excellent reproducibility and potential for scalability. The economic and environmental implications of this work are briefly discussed.

42 ENGINEERING↗
DOE OSTI2024

Compositional and temporal division of labor modulates mixed sugar fermentation by an engineered yeast consortium

Abstract Synthetic microbial communities have emerged as an attractive route for chemical bioprocessing. They are argued to be superior to single strains through microbial division of labor (DOL), but the exact mechanism by which DOL confers advantages remains unclear. Here, we utilize a synthetic Saccharomyces cerevisiae consortium along with mathematical modeling to achieve tunable mixed sugar fermentation to overcome the limitations of single-strain fermentation. The consortium involves two strains with each specializing in glucose or xylose utilization for ethanol production. By controlling initial community composition, DOL allows fine tuning of fermentation dynamics and product generation. By altering inoculation delay, DOL provides additional programmability to parallelly regulate fermentation characteristics and product yield. Mathematical models capture observed experimental findings and further offer guidance for subsequent fermentation optimization. This study demonstrates the functional potential of DOL in bioprocessing and provides insight into the rational design of engineered ecosystems for various applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol

Significance Future production of renewable transportation fuels such as ethanol must rely on abundant nonfood plant sources also known as lignocellulosic biomass. However, a major historical barrier to low-cost production of ethanol from biomass is the low ethanol yields and titers that result from fermentation of biomass solids at high solids when compared with simple sugar fermentations. Here, we show that combining a cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment process with subsequent simultaneous saccharification and fermentation (SSF) can achieve similar high ethanol yields and titers that match that of separate pure glucose fermentations. We demonstrate a strategy whereby direct fermentation of biomass to ethanol is now limited by the microbe rather than by the process.

Nguyen, Thanh Yen↗
DOE OSTI2025

Switchgrass Steroidal Saponins Reduce Fungal Disease but Decrease Yeast Fermentation Yield

Increasing the production of bioproducts from lignocellulosic feedstocks requires improvement in both field production and biorefinery efficiency. When plant traits arise that improve field production but decrease biofuel yield, these trade-offs can represent challenges in the entire production process. To examine trade-offs between field and production traits, we examined factors underlying switchgrass resistance to fungal rust pathogens in field conditions and factors that impede yeast fermentation in the lab using repeated measurements on a switchgrass genetic diversity panel. We found that the same switchgrass genotypes that showed high fungal pathogen resistance also showed recalcitrance to yeast fermentation. These switchgrass genotypes were mostly from the Atlantic genetic group, which had high levels of specialized metabolites of the saponin class. Among 1589 metabolites identified through metabolomics, we found that saponins were among the most likely to explain variation in both rust infection and fermentation yield using random forest feature selection, and that only four of these were sufficient to explain 57.9% of the variation in rust susceptibility. Through follow-up testing in recalcitrant biomass, we found that the bacterium Zymomonas mobilis does not suffer the same inhibition as the yeast Saccharomyces cerevisiae, and that the addition of ergosterol (thought to be the fungal cellular target of saponin inhibition) rescues yeast fermentation. Several lines of evidence point to a central role for saponins as key metabolites protecting switchgrass from fungal pathogens and interfering with yeast fermentation, underscoring an ongoing need for collaboration between plant breeders and biofuel production scientists.

VanWallendael, Acer [North Carolina State Universi↗
DOE OSTI2019

Fluctuant storage temperature increased the heterogeneous distributions of pH and fermentation products in large round bale silage

Abstract In practical of silage production, variations in the fermentation parameters of silage might be attributable to changes in weather data, especially for ambient temperature. The objective of this study was to investigate how the changed temperature of outside weather condition affects distributions of pH and fermentation products in large round bale silage. The bale silages (1.22 m × 1.25 m ø; density, about 168.9 kg dry matter [DM]/m 3 ) were produced by BR6090 Combi from New Holland Agriculture, wrapped with six layers of stretch film, stored at ambient temperature (2–20°C) and constant temperature (10°C; 15°C; 25°C) for 3, 9, 15, 45 and 90 days. Compared with other silages, silage stored at 25°C had higher ( p < 0.05) lactic acid content, lower ( p < 0.05) pH, butyric acid content and ammonia‐nitrogen (N) ratio of total N. Fluctuant storage temperature limited lactic acid fermentation process with delayed pH decline and continuous butyric production and crude protein (CP) degradation. The differences of pH, lactic acid, propionic acid and ammonia‐N were considerably less evident in the centre of silages stored at different temperatures, and they increased towards the boundaries. The data shows that fluctuant storage temperature had undesirable effect on the fermentation quality of silage, especially for the external part of silo; relatively high and stable storage temperature for initial stage of ensiling process was necessary, but did not promote a positive response to the homogeneous distributions of pH and fermentation products in large round bale silage.

Li, Ping↗
DOE OSTI2018

The effect of litchi juice on exopolysaccharide production in milk fermented by Lactobacillus casei

Summary In this paper, Lactobacillus casei growth, sugar utilisation, exopolysaccharides ( EPS s) formation and EPS s characterisation during fermentation of milk with or without litchi juice were evaluated. During L. casei fermentation of litchi‐milk samples, the EPS s content increased with the increase of litchi juice levels (0–25%, v/v), and the both relationship appeared in S ‐type curve. The viscosity of fermented milk also significantly increased with the formation of EPS s, and which positively correlated with the EPS s content ( R 2 = 0.9782). The EPS s from the fermented litchi‐milk samples was isolated for further structural characterisation. Monosaccharide and molecular weight analysis of purified EPS s sample proved that it contained no carbohydrate other than glucose, and its molecular weight was calibrated as 3.03 × 10 6 Da. FT ‐ IR and NMR ( 1 H and 13 C) spectroscopy analysis confirmed the synthesised EPS s sample was α‐glucans (dextrans) with mainly α‐(1→6) linkages. To the best of our knowledge, this is the first study to report that litchi juice can enhance the EPS s (dextrans, mainly α‐(1→6) linkages) production in fermented milk.

Yu, Yuanshan↗
DOE OSTI2014

Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose

Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae. Yeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-β-D-glucopyranosyl-D-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular β-glucosidase. Xylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

09 BIOMASS FUELS↗
DOE OSTI2021

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.

09 BIOMASS FUELS↗