skip to main content


Title: Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts
Abstract Background

Future 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.

Results

In 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.

Conclusions

The 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
Award ID(s):
1751840
NSF-PAR ID:
10276374
Author(s) / Creator(s):
; ; ; ; ; ;
Publisher / Repository:
Springer Science + Business Media
Date Published:
Journal Name:
Biotechnology for Biofuels
Volume:
14
Issue:
1
ISSN:
1754-6834
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Kluyveromyces marxianusis a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. UnlikeSaccharomyces cerevisiae, K. marxianusgrows on low‐cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluatedK. marxianusfor the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2‐pyrone synthase (2‐PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high‐stability 2‐PS mutant and a promoter substitution increased titer four‐fold. 2‐PS expression from a multi‐copy pKD1‐based plasmid improved TAL titers a further five‐fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed‐batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

     
    more » « less
  2. 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
  3. Abstract

    ‘Marginal lands’ are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bioenergy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to induce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioethanol. Five marginal land sites that span a latitudinal gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018–2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel production metrics (switchgrass biomass yields and composition and ethanol production). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central–Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the challenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions.

     
    more » « less
  4. Abstract

    Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of fourKluyveromyces marxianusstrains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties ofK. marxianusmetabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.

     
    more » « less
  5. null (Ed.)
    ABSTRACT Microbial fermentation is a common form of metabolism that has been exploited by humans to great benefit. Industrial fermentation currently produces a myriad of products ranging from biofuels to pharmaceuticals. About one-third of the world’s food is fermented, and the brewing of fermented beverages in particular has an ancient and storied history. Because fermentation is so intertwined with our daily lives, the topic is easily relatable to students interested in real-world applications for microbiology. Here, we describe the curriculum for a guided inquiry-based laboratory course that combines yeast molecular ecology and brewing. The rationale for the course is to compare commercial Saccharomyces cerevisiae yeast strains, which have been domesticated through thousands of generations of selection, with wild yeast, where there is growing interest in their potentially unique brewing characteristics. Because wild yeasts are so easy to isolate, identify, and characterize, this is a great opportunity to present key concepts in molecular ecology and genetics in a way that is relevant and accessible to students. We organized the course around three main modules: isolation and identification of wild yeast, phenotypic characterization of wild and commercial ale yeast strains, and scientific design of a brewing recipe and head-to-head comparison of the performance of a commercial and wild yeast strain in the brewing process. Pre- and postassessment showed that students made significant gains in the learning objectives for the course, and students enjoyed connecting microbiology to a real-world application. 
    more » « less