skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two‐stage fine chemical production with Kluyveromyces marxianus
Title: Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two‐stage fine chemical production with Kluyveromyces marxianus
Abstract Development of the bioeconomy is driven by our ability to access the energy‐rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value‐added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two‐stage bioprocessing system pairing the anaerobic fungusPiromyces indianaewith the yeastKluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose.P.indianaeefficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, whichK.marxianusconsumed while producing 2.4 g/L ethyl acetate. An engineered strain ofK.marxianuswas also able to produce 550 mg/L 2‐phenylethanol and 150 mg/L isoamyl alcohol fromP.indianaehydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.  more » « less
Award ID(s):
1836654
PAR ID:
10369840
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biotechnology Progress
Volume:
37
Issue:
5
ISSN:
8756-7938
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; (, Frontiers in Bioengineering and Biotechnology)
    Strik, David (Ed.)
    The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues. 
    more » « less
  2. ; ; (, Scientific Reports)
    Abstract In situdetoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression ofCbei_3974 andCbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, inClostridium beijerinckiiNCIMB 8052 (Cb), resulting in two strains:Cb_3974 andCb_3904. Expression ofCbei_3974 led to 2-fold increase in furfural detoxification relative toCb_3904 andCb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures ofCb_3974 relative toCb_3904 andCb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation,Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared toCb_3904 andCb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains ofCbtested. Overall,Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development ofCb_3974 represents a significant progress towards engineering solventogenicClostridiumspecies that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation. 
    more » « less
  3. ; ; ; ; ; ; (, Biotechnology for Biofuels)
    Abstract BackgroundFuture expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production betweenS. cerevisiae,O. polymorpha, andK. marxianusin glucose and cellulosic media at different temperatures. ResultsIn this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeastsKluyveromyces marxianusandOgataea polymorpha, as well asSaccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. ConclusionsThe eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole. 
    more » « less
  4. ; ; ; ; ; ; (, Biotechnology for Biofuels)
    null (Ed.)
    Abstract Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus . First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4 , KmARO7 , and KmPHA2 , each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2 , with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway. 
    more » « less
  5. ; ; ; ; (, Bioengineering)
    Microbial cell factories offer an eco-friendly alternative for transforming raw materials into commercially valuable products because of their reduced carbon impact compared to conventional industrial procedures. These systems often depend on lignocellulosic feedstocks, mainly pentose and hexose sugars. One major hurdle when utilizing these sugars, especially glucose, is balancing carbon allocation to satisfy energy, cofactor, and other essential component needs for cellular proliferation while maintaining a robust yield. Nearly half or more of this carbon is inevitably lost as CO2 during the biosynthesis of regular metabolic necessities. This loss lowers the production yield and compromises the benefit of reducing greenhouse gas emissions—a fundamental advantage of biomanufacturing. This review paper posits the perspectives of using CO2 from the atmosphere, industrial wastes, or the exhausted gases generated in microbial fermentation as a feedstock for biomanufacturing. Achieving the carbon-neutral or -negative goals is addressed under two main strategies. The one-step strategy uses novel metabolic pathway design and engineering approaches to directly fix the CO2 toward the synthesis of the desired products. Due to the limitation of the yield and efficiency in one-step fixation, the two-step strategy aims to integrate firstly the electrochemical conversion of the exhausted CO2 into C1/C2 products such as formate, methanol, acetate, and ethanol, and a second fermentation process to utilize the CO2-derived C1/C2 chemicals or co-utilize C5/C6 sugars and C1/C2 chemicals for product formation. The potential and challenges of using CO2 as a feedstock for future biomanufacturing of fuels and chemicals are also discussed. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email