Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L 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.
An estimated annual 1.3 billion dry tons of lignocellulosic biomass could support ethanol production and allow the U.S. to reduce its petroleum consumption by 30%.1 Although plant biomass hydrolysis yields sugar mixtures rich in glucose and xylose, fermentation inhibitors are generated by the chemical pretreatment necessary to break down hemicellulose and expose cellulose for enzymatic attack. Acetic acid, furfural, and hydroxymethylfurfural (HMF) are thought to be key components among many inhibitors that form during pretreatment. In order to move the lignocellulosic ethanol industry forward, research and procedures to allow the evolution of yeast strains capable of surviving and efficiently functioning to use both hexose and pentose sugars in the presence of such inhibitory compounds are needed. A significant additional weakness of traditional industrial yeast strains, such as Saccharomyces cerevisiae, is the inability to efficiently ferment the xylose available in hydrolyzates of plant biomass.
Pichia stipitis type strain NRRL Y-7124 (CBS 5773), recently renamed Scheffersomyces stipitis, is a native pentose fermenting yeast that is well known to ferment xylose to ethanol.2,3 The evolution of strain NRRL Y-7124 was pursued here because it has been documented to have the greatest potential of native yeast strains to accumulate economically recoverable ethanol exceeding 40 g/L with little xylitol byproduct.4,5,6 In optimal media, S. stipitis strain NRRL Y-7124 produces 70 g/L ethanol in 40 hr (1.75 g/L/hr) at a yield of 0.41 ± 0.06 g/g in high cell density cultures (6 g/L cells).7,8 Resistance to fermentation inhibitors ethanol, furfural, and HMF has also been reported,9 and S. stipitis has been ranked among most promising native pentose-fermenting yeasts available for commercial scale ethanol production from lignocellulose.10 Our objective was to apply diverse undetoxified lignocellulosic hydrolyzates and ethanol selection pressures to force evolution toward a more robust derivative of strain NRRL Y-7124 suitable for industrial applications. Key among improved features sought were faster sugar uptake rates in concentrated hydrolyzates, reduced diauxy for more efficient mixed sugar utilization, and higher tolerances of ethanol and inhibitors. The application of S. stipitis to undetoxified hydrolyzates was a key focus of the research to eliminate the added operating expense associated with hydrolyzate detoxification processes, such as overliming.
Two industrially promising hydrolyzates were applied to force evolution: enzyme saccharified ammonia fiber expansion-pretreated corn stover hydrolyzate (AFEX CSH) and dilute acid-pretreated switchgrass hydrolyzate liquor (PSGHL).11,12 AFEX pretreatment technology is being developed to minimize the production of fermentation inhibitors, while dilute acid pretreatment represents the current lowest cost technology most commonly practiced to expose cellulosic biomass for enzymatic saccharification. PSGHL is separable from the cellulose remaining after pretreatment and is characteristically rich in xylose from the hydrolyzed hemicellulose, but low in glucose. AFEX CSH and PSGHL compositions differ from one another in key aspects which were exploited to manage the evolution process. AFEX CSH is lower in furan aldehydes and acetic acid inhibitors but higher in amino acids and ammonia nitrogen sources compared with PSGHL (Table 1). PSGHL presents the additional challenge of xylose being the predominant sugar available. Thus PSGHL is appropriate to specifically enrich for improved xylose utilization in hydrolyzates, a weakness preventing commercial use of available yeast. Even among native pentose fermenting yeasts, the reliance on the suboptimal sugar xylose to support cell growth and repair becomes even more challenging in hydrolyzates because of a variety of reasons: nutrient deficiencies, inhibitors causing widespread damage to cell structural integrity, and disruption to metabolism due to redox imbalances.9 Nitrogen supplementation, especially in the form of amino acids, can represent a significant operating cost for fermentations. The impact of nitrogen supplementation on isolate screening and ranking was explored with switchgrass hydrolyzates.
Improved individuals were 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. Selection pressures were applied in parallel and in series to explore the evolution progress of S. stipitis toward desired derivatives able to grow and ferment efficiently in hydrolyzates (Figure 1). The repetitive culturing of functional populations in increasingly challenging hydrolyzates was accomplished in microplates employing a dilution series of either 12% glucan AFEX CSH or else PGSHL prepared at 20% solids loading. The application of ethanol-challenged growth on xylose in continuous culture further improved AFEX CSH adapted populations by enriching for phenotypes demonstrating less susceptibility to ethanol repression of xylose utilization. The latter feature was recently shown problematic to pentose utilization by strain NRRL Y-7124 following glucose fermentation.8 Enrichment on PSGHL was next explored to broaden hydrolyzate functionality.
Putative improved derivatives of S. stipitis NRRL Y-7124 were isolated from each phase of the evolution process using targeted enrichment under stress conditions and dilution plating to pick colonies from the most prevalent populations. Dimensionless relative performance indices (RPIs) were used to rank strains based on overall performance, where kinetic behavior was evaluated on the different hydrolyzate types and nutrient supplements applied. Although the successes of various adaptation procedures to improve the functionality of S. stipitis in lignocellulosic hydrolyzates have been previously documented, strains demonstrating economical ethanol production on undetoxified hydrolyzates have not been previously reported.13-17 Using the evolution procedures to be visualized in more detail here, Slininger et al.18 developed strains that are significantly improved over the parent strain NRRL Y-7124 and are able to produce >40 g/L ethanol in AFEX CSH and enzyme saccharified switchgrass hydrolyzate (SGH) appropriately supplemented with nitrogen sources. These novel strains are of future interest to the developing lignocellulose to ethanol industry and as subjects of additional genomics studies building on those of previously sequenced strain NRRL Y-11545.19 A genomics study of top strains produced during various phases of evolution diagramed in Figure 1 would elucidate the history of genetic changes that occurred during development as a prelude to further strain improvement research.
1. Prepare Starting Materials and Equipment for Assays
2. Enrich Robust Derivatives during Serial Transfer on AFEX CSH
3. Isolate Single Cell Tolerant Derivatives after Enrichment on AFEX CSH
4. Evaluate Performance of AFEX CSH Tolerant Derivatives Compared to Parent
5. Apply Continuous Culture to Select for Ethanol-challenged Xylose Utilization
6. Evaluate Glycerol Stock Populations and Identify Those with Improved Xylose Fermentation in the Presence of Ethanol
7. Isolate Single-cell Colonies That Utilize Xylose in PSGHL When Ethanol Is Present
8. Further Enrich Robust Evolved Strains during Serial Transfer on PSGHL, as for AFEX CSH
9. Isolate Single-cell Colonies Using PSGHL Gradients with or without Ethanol Challenge
10. In a Primary Screen, Eliminate Inferior Isolates by Comparing and Ranking Performances on PSGHL at Two Nutrient Conditions
11. Rank Isolates in the Primary PSGHL Screen Using Relative Performance Index (RPI)
12. In a Secondary Screen, Compare Top Primary Screen Performers on Multiple Complete Hydrolyzates (>100 g/L Mixed Sugars) to Reveal Highest Functioning Robust Strains
13. Rank the Performances of Isolates in the Secondary Screen Using RPI overall to Rate Use of Multiple Complete Hydrolyzates
S. stipitis was evolved using combinations of three selection cultures, which included AFEX CSH, PSGHL, and ethanol-challenged xylose-fed continuous culture. Figure 1 shows the schematic diagram of the evolution experiments performed along with the isolates found either to perform most effectively overall, or most effectively on one of the hydrolyzates tested. Table 3 shows the NRRL accession numbers of these superior isolates and summarizes the adaptation stresses applied in the process of achieving the enriched population from which each strain was isolated. Some isolates were seen to have superior relative performance on one or two hydrolyzate types, but 7 of 11 isolates performed well on all of the hydrolyzate types, even though most of these were exposed and challenged by only a single type during evolution. The successes of the various evolution approaches taken here are demonstrated in Figures 2–7 and Table 4.
Figure 2 depicts the distinct improvements seen after the first phase of adaptation in which robust derivatives of strain NRRL Y-7124 were enriched during serial transfer to increasing concentrations of AFEX CSH (Step 2). In cultures growing on 6% glucan AFEX CSH, the evolved population demonstrates more rapid accumulation and higher final titers of ethanol than the parent strain. More rapid glucose utilization also is seen along with more rapid xylose uptake immediately following the depletion of glucose. Additionally in Figure 3, the stability of the changed population features is demonstrated in the synthetic medium ODM with a mixture of 87 g/L glucose and 66 g/L xylose. In this case, both the enriched population (Figure 3B) and isolated colonies (Colony 1 and Colony 5, Figure 3C and 3D, respectively) are able to outperform the parent strain by using the xylose more efficiently to make ethanol more rapidly, reducing time to peak ethanol by at least 4 days. Significantly higher ethanol concentrations were accumulated by evolved strains on ODM (55-60 g/L) compared to the parent (40-45 g/L). The mutations, which led to a stable phenotype of reduced diauxy during the glucose-xylose transition and more efficient xylose fermentation, arose as the yeast population evolved in AFEX CSH.
Further improvements to Colony 5 were pursued by submitting it to natural selection in xylose-fed continuous culture in the presence of increasing levels of ethanol up to 50 g/L. Under this condition, the yeast population retained in the continuous culture must be able to induce enzymes for xylose utilization as sole carbon source in order for it to be able to grow at a rate high enough to populate the fermentor despite a steady dilution rate. In the parent strain, the induction of xylose enzymes begins to be inhibited at ethanol concentrations as low as 15-20 g/L.8 Derivatives of Colony 5 were captured in glycerol stocks at early and late time points of operation of the continuous culture, and Figure 4 shows the successful improvement in xylose utilization in the presence of 40 g/L ethanol observed in evolved derivatives of Colony 5 compared with the NRRL Y-7124 parent and the initial Colony 5 inoculated to the process.
Isolates arising from all phases of the adaptation scheme of Figure 1 were screened in PSGHL to identify those with superior ability to ferment xylose (Step 10.1). The isolates shown in Figure 5 are among the best performers on PSGHL out of ~150 ranked in this primary screen and in all secondary screens of performance as described below. To indicate improvement of evolved isolates relative to their parent, the performance of each isolate was expressed as the ratio of kinetic parameter values of isolate to parent strain. Ratio values of "one" occurred if the isolate performance was equivalent to the parent. Figures 5a and 5b summarize top isolate performances on 60% and 75% strengths of PSGHL with ODM or ODM+YM nutrient supplements. As the hydrolyzate concentration and harshness was increased, the performance ratios decreased. In the 60% strength PSGHL, five of seven top isolates exposed to PSGHL selection pressure performed many times better than NRRL Y-7124 (isolate 1). Four isolates performed better than Colony 5 (isolate 33), which had evolved during exposure to AFEX CSH but had no previous selective exposure to PSGHL. However, in the 75% strength of PSGHL, only 3 isolates significantly surpassed both the parent and Colony 5 (isolate 33) despite the added nutrients. Of these, superior isolates 15 and 16 were previously challenged with increasing concentrations of PSGHL as a selection pressure guiding evolution. Isolates 15, 14 and 3 were only exposed to PSGHL, while 11, 13 and 16 had multiple exposures. Isolates 3, 11, and 13 were from earlier time points during the evolution on PSGHL and so had somewhat less opportunity to develop tolerance compared to 14, 15 and 16. While it is evident in Figure 5 that serial transfer to increasing strengths of PSGHL served to develop strains robust to its inhibitory environment, it is also evident that exposure of the parent strain NRRL Y-7124 to AFEX CSH alone could potentially generate isolates such as Colony 5 (33) with cross tolerance to PSGHL. Thus, it was indicated that robust strains able to perform in multiple hydrolyzates could be found by enrichment of tolerance in one hydrolyzate followed by performance screening in another. Isolates obtained from the xylose-fed continuous culture were also screened on PSGHL 60% and 75% strengths with nutrients and also added glucose at 75 g/L in order to sustain the ethanol challenge and test diauxic lag on xylose following glucose utilization. While isolates 27, 28 and 30 were superior to others from this phase of adaptation, both yield and xylose uptake rate performance ratios were similar to the parent on 75% PSGHL with added ODM and YM nutrients, which is not necessarily surprising in that none had previous exposure to this hydrolyzate (see also Slininger et al.18 for additional data not shown here for the cultures provided 75 g/L glucose).
The best strains selected from the primary screen on xylose-rich PSGHL were subsequently screened on three complete hydrolyzates. These hydrolyzates (Table 1) included AFEX CSH and dilute-acid pretreated SG treated with commercial cellulases and supplemented at two different nitrogen levels (SGH-N1 and SGH-N2) to identify the most versatile isolates with respect to variations in inhibitor and nutritional environment. The relative performance indices (RPIs) were calculated for the fermentation of each isolate within each hydrolyzate (Figure 6A). Figure 6B shows the combined overall performance indices calculated for each isolate. Five isolates (3, 14, 27, 28, 33) had overall RPI above 60, which ranked them as the most robust to all of the variations of hydrolyzate and nutrient conditions combined. Reviewing Table 3, both isolates 3 and 14 were evolved in PSGHL while 27, 28, and 33 were evolved in AFEX CSH or AFEX CSH and ethanol-challenged continuous culture on xylose. None of the strains exhibiting superior multiple hydrolyzate use were evolved on more than one hydrolyzate.
Some strains were "specialists" performing better on either SGH-N1/2 or AFEX CSH. Those isolates performing best as specialists on SGH hydrolyzates (11, 16 and 9) were obtained by evolution on PSGHL as the final or only challenge. For isolates 11 and 16, which were initially enriched via increasing AFEX CSH challenge, the ability to efficiently utilize AFEX CSH was not actively selected during the lengthy final enrichment in PSGHL, and key genetic factors supporting its use were evidently lost. Conversely, isolates 14, 25, 27, 30 and 33 were superior performers on AFEX CSH, and all except isolate 14 were evolved on AFEX CSH with or without ethanol challenge. So as expected, directed evolution on AFEX CSH or PSGHL tended to select for yeast well adapted to the selection hydrolyzate. The one exception in this regard was isolate 14. Isolate 14 originated from the PSGHL only challenge and was isolated from YM agar dilution plating of the final PSGHL enrichment culture. Slininger et al.18 showed this isolate to have superior capability of xylose enzyme induction in glucose-grown cells in the presence of 5-15 g/L acetic acid and reduced diauxic lag on ODM with 75 g/L each of glucose and xylose, despite >30 g/L ethanol occurring prior to the glucose-xylose transition point.
The superior kinetics of evolved strains relative to the parent strain S. stipitis NRRL Y-7124 were demonstrated as shown in Table 4, representing the results of low level aeration flask cultures inoculated to initial A620 8.4 ± 2.5 75 ml SGH-N2 (pH 6.2) incubated in 125 ml flasks with silicone sponge caps at 25 °C, 150 rpm (1"orbit), and in Figure 7, representing moderate aeration flask cultures inoculated to initial A620 0.5 in 23 ml SGH-N2 per 50 ml flask.18 These conditions represent two different types of operation suggested by the literature as being potentially commercially promising for ethanol production by S. stipitis.8,22 In the first instance of low level aeration, the high cell density provides for rapid fermentation to begin immediately. Whereas in the second instance, the low cell density and higher level aeration lead to logarithmic growth to build the population and accelerate growth-associated sugar conversion to ethanol. These data demonstrate that the evolved strains have significant kinetic advantages over the parent strain: more rapid glucose uptake rate (Table 4), more rapid specific xylose uptake rate (Table 4), more rapid ethanol productivity on both glucose and xylose and overall (Table 4), shorter lag preceding growth (Figure 7), and shorter diauxic glucose to xylose transitional lag. These improvements allowed ethanol to accumulate to over 40 g/L and to peak days earlier (Figure 7) than was seen for the parent NRRL Y-7124. Higher overall ethanol productivity (Table 4) was achieved at 1.5 to 5 times that of the parent strain (Table 4), depending on evolved strain.
Figure 1: Scheffersomyces stipitis adaptation flow chart. The diagram shown indicates the order of the stresses applied during the adaptation process and the points of recovery of superior isolates (numbers in parenthesis). See also Table 3 isolate key as reference for strain identities. To provide time orientation, the numbers in red indicate the number of days in each phase of adaptation. For the serial transfer phases in AFEX CSH and xylose-rich PSGHL, each day of adaptation represents approximately 2-4 generations. For the continuous culture phase (205 days total), the dilution rate D was variable at ~ 0-0.1 hr-1 during 125 d of operation with pH-actuated feeding. In the next 80 days, operation was at a continuous flow with D at 0.012 hr-1, providing a generation time (ln 2)/(D) of 58 hr, or 1 generation per 2.4 d at steady state. Next a sample of the adapted population from the 205-day continuous culture was mutagenized with UV light and inoculated to a continuous culture operated with D at 0.012 hr-1. (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Figure 2: Improved batch fermentation of 6% glucan AFEX CSH. Scheffersomyces stipitis NRRL Y-7124 parent strain fermentation of 6% glucan AFEX CSH (A) is compared with adapted Colony 5 fermentation of 6% glucan AFEX-pretreated corn stover hydrolyzate (B). Symbols designate biomass (red square), glucose (black circle with dashed line), xylose (blue circle with solid line), ethanol (green triangle), and xylitol (purple diamond). (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Figure 3: Reduced diauxic lag in defined medium with mixed sugars. Fermentation performances are compared in ODM with 66 g/L glucose and 87 g/L xylose for parent strain S. stipitis NRRL Y-7124 (A), the AFEX CSH adapted population derived from Y-7124 (B), single cell Colony 1 isolated from the adapted S. stipitis population (C), single cell Colony 5 isolated from the adapted population (D). Symbols designate biomass (red square), glucose (black circle with dashed line), xylose (blue circle with solid line), ethanol (green triangle), xylitol (purple diamond), and adonitol (gold diamond with black edge). (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Figure 4: Ethanol resistant derivatives of Colony 5. Hydrolyzate tolerant Colony 5 was further developed by continuous culture selection on ODM containing xylose as sole carbon source and high levels of ethanol. Two derivative glycerol stock populations obtained early in the selection process (2A.1.53R, orange triangle and dashed line) and after UV irradiation of continuous culture inocula (2A.1.30R.2, purple circle and dashed line) are shown in comparison with the NRRL Y-7124 parent strain (green circle with solid line) and AFEX CSH tolerant Colony 5 (black triangle with solid line). Xylose uptake by dense populations of glucose-grown yeast (A620 = 50) in ODM with 40 g/L ethanol indicated that all adapted strains surpassed the unadapted parent in the ability to induce xylose metabolism. (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Figure 5: Ratio of performance improvement of tolerant isolate compared to parent. The performances of superior tolerant isolates are summarized relative to the control parent strain NRRL Y-7124 for each formulation of PSGHL (A, B). Performances were assessed in terms of xylose uptake rate (blue bars representing ratios of isolate to parent) and ethanol yield per sugar supplied (green bars representing ratios of isolate to parent). (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Figure 6: Isolate ranking based on RPI. The relative performance index (RPI) concept was applied to the performance results of the secondary screen in order to rank 33 isolates within each hydrolyzate type based on xylose uptake rate and ethanol yield per sugar supplied. (A) The relative ranking of any given isolate depended on the hydrolyzate type (P <0.001): SGH-N1(blue bars), SGH-N2 (red bars) and AFEX CSH (green bars). (B) The overall RPI calculated across all hydrolyzate types (light blue bars) indicated superior strains with most robust performance across different hydrolyzate conditions. (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Figure 7: Comparative SGH fermentations of superior adapted isolates of S. stipitis. Superior adapted isolates and their parent strain NRRL Y-7124 are compared fermenting enzymatic hydrolyzates of dilute acid-pretreated switchgrass (20% solids loading) at 25 °C and initial pH 6.2 at low initial cell density. Time courses of biomass (red squares), glucose (black circles and dashed line), xylose (blue circles and solid line), and ethanol (green triangles) are shown. Error bars represent the range about the mean value marked by symbols. (Reproduced from Slininger et al.18) Please click here to view a larger version of this figure.
Table 1: Compositions of hydrolyzates used in cultivations. (Reproduced from Slininger et al.18) Please click here to download this table.
Table 2: Optimal defined medium for Scheffersomyces stipitis NRRL Y-71247 Please click here to download this table.
Table 3: Summary of superior tolerant Scheffersomyces stipitis strains for fermentation of hydrolyzates of plant biomass. (Reproduced from Slininger et al.18) Please click here to download this table.
Table 4: Comparative kinetics of isolates on switchgrass hydrolyzate SGH-N2 inoculated to initial A620 = 8.4 ± 2.5. Rates are normalized rates per unit absorbance during glucose or xylose consumption. (Reproduced from Slininger et al.18) Please click here to download this table.
Several steps were critical to the success of the evolution process. First, it is key to choose appropriate selection pressures to drive the population evolution toward the desired phenotypes that are needed for successful application. The following selective stresses were chosen for S. stipitis development and applied at appropriate times to guide enrichment for the desired phenotypes: increasing strengths of 12% glucan AFEX CSH (which forces growth and fermentation of diverse sugars in the presence of acetic acid and low levels of furan aldehydes and other inhibitors); xylose fed continuous cultures with increasing ethanol concentration (which forces xylose enzyme induction to reduce diauxic lag); and increasing strengths of 20% solids loading PSGHL (which forces growth and fermentation of xylose in the presence of high acetic acid, furans, and other inhibitors). Second, it is important to preserve the evolving yeast populations by freezing glycerol stocks of population samples as the enrichment process progresses. Such snapshots of the population can be stored for periodic functional testing to document evolution progress and to allow subsequent isolations as desired, or to restart evolution processes after a hiatus. A third key step in the evolution procedure was to recover exceptional isolates by enriching selection culture populations or glycerol stocked populations on a convenient selective media (such as agar or micro-plates containing a stress gradient presenting a series of hydrolyzate and/or ethanol concentrations). Then surviving colonies growing under the most stressful condition can be picked to preserve for characterization later. These three basic steps can be repeated to pursue each additional desired phenotype selection pressure, or, alternatively, multiple pressures in a single cycle if appropriate. When the native parent yeast population is exposed to the various stresses, genetic diversity is expected to arise through natural or induced mutations, and continued exposure will allow natural selection to enrich for the individuals with the most beneficial mutations supporting competitive survival. It is expected that the selected phenotype will occur as a result of multiple genetic mutations. In the protocol above, mutations may be induced during UV treatment or potentially during exposure of yeast to hydrolyzates. Hydrolyzates are known to contain three classes of compounds damaging to microorganisms: carboxylic acids, aldehydes and phenolics.22 Reactive aldehydes, such as furfural and 5-hydroxymethylfurfural, can damage cells and cause an elevation of reactive oxygen species (ROS), generated typically in mitochondria. ROS are well known to cause DNA mutations in eukaryotic cells.23,24 The phenolics present in hydrolyzates, though not previously known to be genotoxic, may promote and synergize the mutagenic effects of aldehydes and mutagenic ROS.25
The stepwise strategy of progressively challenging environments is expected to build evolved strains with a series of phenotype improvements, both targeted and also non-targeted. It is possible that certain non-targeted phenotypes may arise that are undesirable or unstable. In order to capture the most highly functioning and robust strains, an additional key step that must be taken is to evaluate and compare selected isolates for commercially relevant traits, reaching for both targeted and non-targeted phenotypes. To do this, isolate performances should be compared in a variety of application-oriented stress conditions, such as in hydrolyzates with different inhibitor challenges and nutrient formulations, allowing selection of best overall strains that are stable and robust to broad industrial lignocellulosic substrate variation. Additionally, a strain stability challenge can be incorporated into the screen as was done in the example by including preculturing steps involving initial yeast growth on two nonselective media, YM agar for several generations followed by the synthetic ODM with 50 g/L xylose for an additional 6-7 generations, giving opportunity for destabilization of desired culture traits prior to exposure to the challenge of performance in hydrolyzate. Based on significant performance features, such as ethanol yield and xylose uptake rate, isolates are then ranked in each different stress condition, such as each hydrolyzate environment tested, which may be different from the isolate's enrichment culture medium. The dimensionless RPI can be used to calculate the average overall relative performance and rank of isolates being compared. A dimensionless factor is appropriate to reflect relative performance in different types of hydrolyzates and based on different performance assessments, such as yields versus rates. This ranking procedure identifies strains that perform consistently well across broad variations in growth conditions and hydrolyzates and are apt to be both robust and genetically stable.
Various iterations and modifications of these basic steps may be necessary to accommodate the evolution of any microbial strain capable of specific or unique performance features on lignocellulosic hydrolyzates or other substrates of interest. In the S. stipitis example, it is important to note that supplementation of nutrients, including low-cost commercial sources, to hydrolyzates prepared at high solids loading was key to controlling the dynamic range of isolate performances for improved statistical separation during screening. The importance of nutrients to successful fermentation of concentrated inhibitory hydrolyzates has also been reported previously and is thought to be due to an elevated need for amino acids and other nutrients required for cell maintenance, redox balancing, and repair as a result of such stress conditions, especially during xylose utilization.9,26,27 Additionally, if nutrients are too sparse or too profuse, differences in isolate performances may be hard to detect statistically due to all isolates doing exceedingly poorly, or all doing exceedingly well, respectively. Isolates capable of performing well under diverse conditions are expected to be reliable, or robust to variations in industrial conditions.
The main limitation of this protocol is that adaptive evolution and screening are very literal in outcome, in that "one will get what one enriches and screens for". Thus, the enrichment and screen conditions need to be designed to amplify and identify individuals, respectively, with the desirable changes in phenotypes while still retaining desirable pre-existing traits. Evolution may affect non-selected for traits that are critical for industrial performance (e.g., growth factor requirements). For this reason, population enrichment progress for one trait needs to be periodically monitored for other traits as a method of troubleshooting for undesirable changes. For example, one of the unique traits of S. stipitis is its native ability to grow on and ferment xylose. All enrichment and screening substrates included xylose as sole or key contributing carbon source in the presence of inhibitors. Consequently, a common feature of all of the enrichment cultures in this example was that they directly selected for more rapid xylose utilizing strains, whose populations became more and more enriched in serial or continuous culturing because they were better competitors. As an intended result, improved strains were all capable of more rapid xylose uptake, but improvements to ethanol yield were much less dramatic than improvements in xylose uptake rate. The latter trend likely came about since ethanol yield by the parent strain was relatively high at around 0.3 g/g, or 60% of theoretical (0.51 g/g), as cell growth became stationary in hydrolyzates, leaving less room for improvement. In cultures, higher ethanol yields could be possibly selected against because ethanol is a growth inhibitor, which necessitated performance monitoring of the evolved population for this trait. Ethanol concentrations kept below the level for growth inhibition would tend to neutralize this as a selection factor. For S. stipitis NRRL Y-7124, 40 g/L ethanol halves specific growth rate while 64 g/L prevents growth entirely.28 In ethanol challenged continuous cultures fed xylose, aeration was kept low and dilution rates were also kept low enough with ample xylose present to foster fermentation rather than respiration of ethanol and prevent enrichment for an unwanted ethanol use property. Additionally, nutrient supplements to the hydrolyzates used in performance screens were thoughtfully designed so as to avoid selecting for strains needing a costly rich nutrient supply, such as yeast extract. Supplements always included lowest cost commercial sources available, such as soy flour and urea, although richer components such as in YM were applied in tandem cultures for comparative testing, such as in the formulations of 60% and 75% PSGHL which are normally deplete of nitrogen. The ODM synthetic medium, used in preculture and performance culture screens, was originally designed for optimal performance of the parent strain S. stipitis NRRL Y-71247 in accordance with the culture medium optimization routine described by Traders Protein20 which recommends defined ingredients, including vitamins, minerals, purines and pyrimidines, and amino acid sources to supply nutrients compatible with cost-effective industrial scale-up using commercial sources. A final recommendation in designing relevant strains, is to keep the design of the enrichment and screen conditions as relevant as possible to the expected industrial process conditions.
Cost-competitive renewable ethanol has long been pursued to reduce dependence on fossil fuels. To improve the ability of S. stipitis NRRL Y-7124 to tolerate, grow and ferment inhibitory hydrolyzates of lignocellulose, several researchers have used repetitive culturing in the xylose-rich liquor resulting from dilute acid-pretreatment of lignocellulosic biomass.13,14,16,17 Over-liming to detoxify inhibitors was still needed to allow a reasonable level of performance of early adapted strains on hardwood and wheat straw pretreatment liquors.13,14 Multiple repeated rounds of UV mutagenesis and genome shuffling have been applied to develop derivatives of S. stipitis NRRL Y-7124 with improved inhibitor tolerance and growth in hardwood spent sulfite liquor (HWSSL).16,17 However, ethanol concentrations produced by these strains in HWSSL were under 10 g/L after 6 to 7 days. Using the techniques shown and described here, new evolved strains of S. stipitis NRRL Y-7124 were developed to meet phenotype goals needed for economical commercial use: fermentation of hydrolyzates at pH 5-6 without prior detoxification measures, such as over-liming, which leads to costly waste disposal; greatly reduced growth and diauxic lags; ethanol accumulations high enough for commercial distillation (>40 g/L ethanol); more rapid growth and ethanol productivity than previously reported for native yeast strains fermenting undetoxified hydrolyzates; and performance in diverse hydrolyzates at high solids loading, including appropriately soy nitrogen-supplemented SGH (which is otherwise nitrogen poor) and unsupplemented AFEX CSH. The improved strains developed using the novel aggressive approaches to evolution as embodied in the key steps summarized above, are expected to benefit the economics of producing ethanol from agricultural biomass by lowering operating costs, capital costs, and energy and water inputs. This is the first strain evolution plan reported to yield adapted strains of S. stipitis demonstrating economically recoverable ethanol production on undetoxified hydrolyzates.
The novel strains of S. stipitis arising from each phase of the evolution process (Figure 1) are candidates for future studies to determine the genetic changes associated with specific selection pressures applied and the mechanisms underlying key attributes of improved hydrolyzate fermentation, such as inhibitor tolerance and reduced diauxic lag. Such valuable new knowledge will aid the engineering of next generation yeast biocatalysts and processes for conversion of lignocellulose to biofuels and other products. As new strains are derived, it will likely be beneficial to again pass the derivative population through a strain evolution protocol similar to that described here in order to select the most useful transformants for commercial use in hydrolyzates. Similarly, as new microbial strains are discovered with the potential to make a myriad of useful new products from renewable biomass, the evolution process could be further applied to improve strain robustness and productivity in hydrolyzates, potentially allowing novel bio-catalytic processes and products to be made available.
The authors have nothing to disclose.
We would like to express our sincere appreciation to Drs. Kenneth Vogel, Robert Mitchell and Gautam Sarath, Grain, Forage, and Bioenergy Research Unit, Agricultural Research Service, Lincoln, NE for their kind supply of switchgrass for this project. We also thank U.S. Department of Energy for funding to VB through the DOE Great Lakes Bioenergy Research Center (GLBRC) Grant DE-FC02-07ER64494.
Cellic Ctec, Contains Xylanase (endo-1,4-) | Novozymes | No product number | www.novozymes.com, 1-919-494-3000 |
Cellic Htec, Contains Cellulase and Xyalanase | Novozymes | No product number | www.novozymes.com, 1-919-494-3000 |
Toasted Nutrisoy Flour | Archer Daniels Midland Co. (ADM) | 63160 | ADM, 4666 Faries Parkway, Decatur, IL 1800-37-5843 |
Pluronic F-68 (Surfactant) | Sigma-Aldrich | P1300 | Sigma-Aldrich |
Difco Vitamin Assay Casamino Acids | Becton Dickinson and Company | 228830 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
D,L-tryptophan | Sigma-Aldrich | T3300 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
L-cysteine | Sigma-Aldrich | C7352 | multiple suppliers: e.g. Fisher Scientific, Sigma-Aldrich |
Bacto Agar | Becton Dickinson and Company | 214010 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
Bacto Malt Extract | Becton Dickinson and Company | 218630 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
Bacto Yeast Extract | Becton Dickinson and Company | 212750 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
Peptone Type IV from soybean | Fluka | P0521-500g | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
Adenine, > 99% powder | Sigma-Aldrich | A8626 | CAS 73-24-5, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
Cytosine, > 99% | Sigma-Aldrich | C3506 | CAS 71-30-7, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
Guanine, SigmaUltra | Sigma-Aldrich | G6779 | CAS 73-40-5, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
Thymine, 99% | Sigma-Aldrich | T0376 | CAS 65-71-4, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
Uracil, 99% | Sigma-Aldrich | U0750 | CAS 66-22-8, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
Dextrose (D-Glucose), Anhydrous, Certified ACS | Fisher Chemical | D16-500 | CAS 50-99-7, Could use other brands. Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich |
D-Xylose, assay > 99% | Sigma-Aldrich | X1500 | CAS 58-86-6, Could use other brands. Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich |
96-well, flat bottom plates | Becton Dickinson Falcon | 351172 | multiple suppliers: e.g. Thermo-Fisher, VWR, Daigger |
Wypall L40 Wiper | Kimberly-Clark | towel in microplate boxes to absorb water for humidification; multiple suppliers: e.g. Thermo-Fisher, uline, Daigger | |
Corning graduated pyrex flask, 125-mL, narrow opening (stopper #5) | Corning Life Science Glass | 4980-125 | multiple suppliers: e.g. Thermo-Fisher, VWR, Daigger |
Innova 42R shaker/incubator, 2.5 cm (1") rotation | New Brunswick Scientific (1-800-631-5417) | M1335-0016 | multiple suppliers: e.g. Eppendorf, Thermo-Fisher. Other shaker/incubators with a 2.5 cm (1") throw could be used. |
Duetz Cover clamp for 4 deepwell MTP plates | Applikon Biotechnology | Z365001700 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
Duetz System sandwich cover for 96 deepwell plates | Applikon Biotechnology | Z365001296 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
Duetz System silicone seal (0.8mm black low evap) for 96 deep well plate cover | Applikon Biotechnology | V0W1040027 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
Blue microfiber layer for Duetz system sandwich cover | Applikon Biotechnology | V0W1040001 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
96 well, 2 mL square well pyramid bottom plates, natural popypropylene | Applikon Biotechnology | ZC3DXP0240 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
Bellco 32mm silicon sponge plug closures, pk of 25 for 125-mL flasks | Bellco | 1924-00032 | Thomas Scientific, their Catalog number is 1203K27 |
Bellco Spinner Flask, 1968-Glass Dome, Sealable Flange Type, 100-mL working volume. This design no longer manufactured. | Bellco | 1968-00100 (original Cat. No.) | Jacketed vessels have lower inlet & upper outlet ports for temp. control with circulating water bath. Vessels are 75mm in outer diam and 200mm in height. There are four side ports at ~45o angles and one top port. Port openings appropriate size for size 0 neoprene stoppers (21-22mm inner diameters on ports). |
Mathis Labomat IR Dryer Oven | MathisAg | Typ-Nbr BFA12 215307 | Werner Mathis U.S.A. Inc. usa@mathisag.com, 704-786-6157 |
Dual Channel Biochemistry Analyzer | YSI Life Sciences | 2900D-UP | www.ysi.com, robotic system for rapid sugars assay in 96-well microplate format |
PowerWave XS Microplate Spectrophotometer | Bio-Tek Instruments, Inc | MQX200R | www.biotek.com |