Hydrothermal conditioning of oleaginous yeast cells to enable recovery of lipids as potential drop-in fuel precursors.

BACKGROUND: Lipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery. RESULTS: Hydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids. CONCLUSION: This work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration.

Draft genome sequence of Yarrowia lipolytica NRRL Y-64008, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.

Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass.

Rhodotorula toruloides is being developed for the use in industrial biotechnology processes because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of intracellular lipid bodies. Nineteen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel polymerase chain reaction (PCR)-based assay, which was validated using the classical microscopic test. Three of the strains were heterozygous for mating type (A1/A2). Ploidy analysis revealed a complex pattern. Two strains were triploid, eight haploid, and eight either diploid or aneuploid. Two of the A1/A2 strains were compared to their parents for growth on 75%v/v concentrated SGH. The A1/A2 strains were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33-9.40 g/L with a median of 6.12 g/L, excluding the two strains that did not grow. Lipid yields were 0.032-0.131 (g/g) and lipid contents were 13.5-53.7% (g/g). Four strains had significantly higher lipid yields and contents. One of these strains, which had among the highest lipid yield in this study (0.131 ± 0.007 g/g), has not been previously described in the literature. SUMMARY: The yeast Rhodotorula toruloides was used to produce oil using sugars extracted from a bioenergy grass.

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

Production of Designer Xylose-Acetic Acid Enriched Hydrolysate from Bioenergy Sorghum, Oilcane, and Energycane Bagasses.

Xylan accounts for up to 40% of the structural carbohydrates in lignocellulosic feedstocks. Along with xylan, acetic acid in sources of hemicellulose can be recovered and marketed as a commodity chemical. Through vibrant bioprocessing innovations, converting xylose and acetic acid into high-value bioproducts via microbial cultures improves the feasibility of lignocellulosic biorefineries. Enzymatic hydrolysis using xylanase supplemented with acetylxylan esterase (AXE) was applied to prepare xylose-acetic acid enriched hydrolysates from bioenergy sorghum, oilcane, or energycane using sequential hydrothermal-mechanical pretreatment. Various biomass solids contents (15 to 25%, w/v) and xylanase loadings (140 to 280 FXU/g biomass) were tested to maximize xylose and acetic acid titers. The xylose and acetic acid yields were significantly improved by supplementing with AXE. The optimal yields of xylose and acetic acid were 92.29% and 62.26% obtained from hydrolyzing energycane and oilcane at 25% and 15% w/v biomass solids using 280 FXU xylanase/g biomass and AXE, respectively.

Near-Complete Genome Sequence of Zygosaccharomyces rouxii NRRL Y-64007, a Yeast Capable of Growing on Lignocellulosic Hydrolysates.

The halotolerant and osmotolerant yeast Zygosaccharomyces rouxii can produce multiple volatile compounds and has the ability to grow on lignocellulosic hydrolysates. We report the annotated genome sequence of Z. rouxii NRRL Y-64007 to support its development as a platform organism for biofuel and bioproduct production.

Cellulosic Ethanol Production Using a Dual Functional Novel Yeast.

Reducing the cost of cellulosic ethanol production, especially for cellulose hydrolytic enzymes, is vital to growing a sustainable and efficient cellulosic ethanol industry and bio-based economy. Using an ethanologenic yeast able to produce hydrolytic enzymes, such as Clavispora NRRL Y-50464, is one solution. NRRL Y-50464 is fast-growing and robust, and tolerates inhibitory compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) associated with lignocellulose-to-fuel conversion. It produces three forms of ß-glucosidase isozymes, BGL1, BGL2, and BGL3, and ferment cellobiose as the sole carbon source. These ß-glucosidases exhibited desirable enzyme kinetic parameters and high levels of enzyme-specific activity toward cellobiose and many oligosaccharide substrates. They tolerate the product inhibition of glucose and ethanol, and are stable to temperature and pH conditions. These characteristics are desirable for more efficient cellulosic ethanol production by simultaneous saccharification and fermentation. NRRL Y-50464 provided the highest cellulosic ethanol titers and conversion rates at lower cellulase loadings, using either pure cellulose or agricultural residues, as so far reported in the literature. This review summarizes NRRL Y-50464 performance on cellulosic ethanol production from refined cellulose, rice straw, and corn stover processed in various ways, in the presence or absence of furfural and HMF. This dual functional yeast has potential to serve as a prototype for the development of next-generation biocatalysts. Perspectives on continued strain development and process engineering improvements for more efficient cellulosic ethanol production from lignocellulosic materials are also discussed.

Controlling autohydrolysis conditions to produce xylan-derived fibers that modulate gut microbiota responses and metabolic outputs.

Autohydrolysis is used for producing xylan-derived oligosaccharides from lignocellulosic biomass. Although numerous studies report optimized autohydrolysis conditions for various plants, few of these studies correlate process parameters with the resulting structural properties to their impact on intestinal bacterial communities. Thus, to further clarify these relationships, beechwood xylan (BWX)-derived substrates, processed under five conditions, were fermented in vitro by human gut microbiota. Autohydrolysis reduced the mean molecular size and substitutions of BWX. Distinct fermentation kinetics were observed with differing processing of BWX substrates, which correlated with impacts on community species evenness. The relative abundances of Bacteroides, Fusicatenibacter, Bifidobacterium, and Megasphaera within the fermentations varied with processing conditions. While the total short-chain fatty acid concentrations were the same among the treatments, processing conditions varied the extent of propionate and butyrate generation. Autolysis parameters may be an important tool for optimizing beneficial effects of xylan-derived fibers on human gut microbiota structure and function.

High solids loading biorefinery for the production of cellulosic sugars from bioenergy sorghum.

A novel process applying high solids loading in chemical-free pretreatment and enzymatic hydrolysis was developed to produce sugars from bioenergy sorghum. Hydrothermal pretreatment with 50% solids loading was performed in a pilot scale continuous reactor followed by disc refining. Sugars were extracted from the enzymatic hydrolysis at 10% to 50% solids content using fed-batch operations. Three surfactants (Tween 80, PEG 4000, and PEG 6000) were evaluated to increase sugar yields. Hydrolysis using 2% PEG 4000 had the highest sugar yields. Glucose concentrations of 105, 130, and 147 g/L were obtained from the reaction at 30%, 40%, and 50% solids content, respectively. The maximum sugar concentration of the hydrolysate, including glucose and xylose, obtained was 232 g/L. Additionally, the glucose recovery (73.14%) was increased compared to that of the batch reaction (52.74%) by using two-stage enzymatic hydrolysis combined with fed-batch operation at 50% w/v solids content.

Effect of using a nitrogen atmosphere on enzyme hydrolysis at high corn stover loadings in an agitated reactor.

A comprehensive review of the literature shows that enzyme hydrolysis efficiency decreases with increased solids loadings at constant enzyme:cellulose ratios for pretreated lignocellulosic substrates. In seeking a mechanistic explanation for this phenomenon, we found that a nitrogen atmosphere enhances enzyme hydrolysis and minimizes the decrease in glucose yields as solids loadings are increased in an agitated bioreactor. For liquid hot water pretreated corn stover, at solids loadings of both 100 and 200 g/L and hydrolyzed for 72 hr in a 1 L bioreactor at pH 5.0 with 3.6 mg protein per g biomass, glucose yields were 55% in a nitrogen atmosphere versus 45% in air with agitation and about 34% without agitation. While mixing promotes biomass/enzyme contact and disperses sugars released during hydrolysis that would otherwise cause product inhibition, nitrogen gas displaces air, avoiding deactivation of cellulases by oxygen. The nitrogen effect points to a facile approach of enhancing hydrolysis at high solids loadings.

Screening for Oily Yeasts Able to Convert Hydrolysates from Biomass to Biofuels While Maintaining Industrial Process Relevance.

Research has recently intensified to discover new oleaginous yeast strains able to function quickly and efficiently in low-cost lignocellulosic hydrolysates to produce high-quality lipids for use in biodiesel and chemicals. Detailed techniques are given here for ranking candidate yeast strains based on conversion of hydrolysate sugars to lipids and then optimizing cultivation conditions for best performers in a 96-well aerobic microcultivation format. A full battery of assays applicable to high throughput of small-volume samples are described for efficiently evaluating cell biomass production, lipid accumulation, fatty acid composition, and sugar utilization. Original data is additionally presented on the validation of the microtechnique for GC analysis of lipid composition in yeast since this application involved modification of a previously published assay for microalgae.

Sugar production from bioenergy sorghum by using pilot scale continuous hydrothermal pretreatment combined with disk refining.

Chemical-free pretreatments are attracting increased interest because they generate less inhibitor in hydrolysates. In this study, pilot-scaled continuous hydrothermal (PCH) pretreatment followed by disk refining was evaluated and compared to laboratory-scale batch hot water (LHW) pretreatment. Bioenergy sorghum bagasse (BSB) was pretreated at 160-190 °C for 10 min with and without subsequent disk milling. Hydrothermal pretreatment and disk milling synergistically improved glucose and xylose release by 10-20% compared to hydrothermal pretreatment alone. Maximum yields of glucose and xylose of 82.55% and 70.78%, respectively were achieved, when BSB was pretreated at 190 °C and 180 °C followed by disk milling. LHW pretreated BSB had 5-15% higher sugar yields compared to PCH for all pretreatment conditions. The surface area improvement was also performed. PCH pretreatment combined with disk milling increased BSB surface area by 31.80-106.93%, which was greater than observed using LHW pretreatment.

Improving ethanol yields with deacetylated and two-stage pretreated corn stover and sugarcane bagasse by blending commercial xylose-fermenting and wild type Saccharomyces yeast.

Corn stover and sugarcane bagasse are the most widely available agriculture processing biomass and could serve as feedstocks for production of biofuel. In this study, three different technologies are combined to develop a more efficient conversion process for each of these feedstocks. The three technologies are diluted alkaline deacetylation process, combined thermochemical and mechanical shear pretreatment, and fermentation using a combined inoculum of two commercial Saccharomyces yeast strains. The two yeast strains used were a non-GMO and GMO strain engineered for xylose fermentation. The final ethanol concentrations obtained were 35.7 g/L from deacetylated corn stover and 32.9 g/L from sugarcane bagasse. Blending the two yeast reduced residual xylose content from 1.24 g/L to 0.48 g/L and increased ethanol production by 6.5% compared to solely using the C5/C6 yeast. The optimized yeast blend also lowered the amount of C5/C6 yeast required for inoculation by 80%.

Overexpression of the Sorghum bicolor SbCCoAOMT alters cell wall associated hydroxycinnamoyl groups.

Sorghum (Sorghum bicolor) is a drought tolerant crop, which is being developed as a bioenergy feedstock. The monolignol biosynthesis pathway is a major focus for altering the abundance and composition of lignin. Caffeoyl coenzyme-A O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase that methylates caffeoyl-CoA to generate feruloyl-CoA, an intermediate required for the biosynthesis of both G- and S-lignin. SbCCoAOMT was overexpressed to assess the impact of increasing the amount of this enzyme on biomass composition. SbCCoAOMT overexpression increased both soluble and cell wall-bound (esterified) ferulic and sinapic acids, however lignin concentration and its composition (S/G ratio) remained unaffected. This increased deposition of hydroxycinnamic acids in these lines led to an increase in total energy content of the stover. In stalk and leaf midribs, the increased histochemical staining and autofluorescence in the cell walls of the SbCCoAOMT overexpression lines also indicate increased phenolic deposition within cell walls, which is consistent with the chemical analyses of soluble and wall-bound hydroxycinnamic acids. The growth and development of overexpression lines were similar to wild-type plants. Likewise, RNA-seq and metabolite profiling showed that global gene expression and metabolite levels in overexpression lines were also relatively similar to wild-type plants. Our results demonstrate that SbCCoAOMT overexpression significantly altered cell wall composition through increases in cell wall associated hydroxycinnamic acids without altering lignin concentration or affecting plant growth and development.

Engineering Candida phangngensis-an oleaginous yeast from the Yarrowia clade-for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction.

Candida phangngensis is an ascomycetous yeast and a phylogenetic relative of the industrial workhorse Yarrowia lipolytica. Here, we report that genetic tools already established for use in the latter organism-including promoters, expression vectors, antibiotic resistance genes, a transformation protocol, and the Cre/lox system for marker recycle-can be transferred to the newer member of the Yarrowia clade with little or no need for modifications. Using these tools, we engineered C. phangngensis for improved cellulosic lipid production by introducing two heterologous yeast genes. First, overexpression of Saccharomyces cerevisiae ADH6 enhanced in situ detoxification of aldehyde fermentation inhibitors that are generated during biomass pretreatment (e.g. furfural). Subsequently, Y. lipolytica DGA1 expression boosted lipid accumulation in C. phangngensis by pulling additional carbon flux into the triacylglycerol synthesis pathway. In acid-pretreated switchgrass hydrolysate cultures, the final engineered strain JQCP04 showed a 58% decrease in lag time and a 32% increase in lipid titer as compared to wild-type PT1-17. Furthermore, we expect that this study will generate new interest in the highly oleaginous yeast C. phangngensis, which is closely related to a safe, industrial species, and is shown here to be quite amenable for genetic manipulation.

Fermentation of undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and mechanical refining pretreatment.

In this study, liquid hot water pretreatment was combined with disk milling for pretreatment of sugarcane bagasse. Sugarcane bagasse was pretreated using liquid hot water (LHW) at 140-180 °C for 10 min (20% w/w solids content) and then disk milled. Disk milling improved glucose release 41-177% and ethanol production from glucose/xylose cofermentation by 80% compared to only using LHW pretreatment. The highest ethanol conversion efficiency achieved was 94%, which was observed when bagasse was treated at 180 °C with LHW and disk milled. However, a small amount of residual xylose (3 g/L) was indicative that further improvement could be achieved to increase ethanol production.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates.

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes of fungal oxido-reductive pathway needed for xylose fermentation integrated into its genome (YRH1415) was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than YRH1415 strain and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting from the hydrolysis of lignocellulosic biomass (switchgrass). The rate of xylose consumption did not appear to be affected by the ploidy of strains or the presence of two copies of the xylose fermentation genes but by heterozygosity of alleles for xylose metabolism in YRH1415. Furthermore, inhibitor tolerance was influenced by the heterozygous genome of the industrial strain, which also showed a marked influenced on tolerance to increasing concentrations of toxic compounds, such as furfural. In this work, selection of haploid derivatives was found to be a useful strategy to develop efficient xylose-fermenting industrial yeast strains.

A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate.

Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates of Y. lipolytica identified, only a few of the strains have been studied extensively. Furthermore, 12 other yeast species were recently assigned to the Yarrowia clade, and most are not well characterized in terms of cell growth and lipid accumulation, especially in industrially relevant conditions. In the present study, we investigated biomass and lipid production by 57 yeast isolates, representing all 13 species in the Yarrowia clade, on a non-detoxified dilute acid-pretreated switchgrass hydrolysate under highly aerobic conditions. The objective was to compare yeast physiology during growth in an abundant, low-cost biomass feedstock and to expand diversity of genetically tractable, oleaginous yeasts available for lipid research. Screening of 45 Y. lipolytica isolates demonstrated considerable variation within the species in terms of lipid accumulation (min = 0.1 g/L; max = 5.1 g/L; mean = 2.3 g/L); three strains (NRRL YB-420, YB-419, and YB-392) were especially promising for cellulosic biomass conversion with average improvements of 43, 57, and 64%, respectively, in final lipid titer as compared to control strain W29. Subsequently, evaluation of strains from 13 distinct species in the Yarrowia clade identified Candida phangngensis PT1-17 as the top lipid producer with a maximum titer of 9.8 g/L lipid, which was over twofold higher than the second-best species in the clade (Candida hollandica NRRL Y-48254). A small set of the most promising strains from the screenings was further characterized to evaluate inhibitor tolerance, lipid production kinetics, and fatty acid distribution. We expect that the results of this study will pave the way for new biotechnological applications involving previously overlooked and under-characterized strains within the Yarrowia clade.

Erratum to: Promise of combined hydrothermal/chemical and mechanical refining for pretreatment of woody and herbaceous biomass.

[This corrects the article DOI: 10.1186/s13068-016-0505-2.].

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.