Automated UV-C Mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and Selection for Microaerophilic Growth and Ethanol Production at Elevated Temperature on Biomass Sugars

Abstract

The yeast Kluyveromyces marxianus is a potential microbial catalyst for fuel ethanol production from a wide range of biomass substrates. To improve its growth and ethanol yield at elevated temperature under microaerophilic conditions, K. marxianus NRRL Y-1109 was irradiated with UV-C using automated protocols on a robotic platform for picking and spreading irradiated cultures and for processing the resulting plates. The plates were incubated under anaerobic conditions on xylose or glucose for 5 mo at 46 °C. Two K. marxianus mutant strains (designated 7-1 and 8-1) survived and were isolated from the glucose plates. Both mutant strains, but not wild type, grew aerobically on glucose at 47 °C. All strains grew anaerobically at 46 °C on glucose, galactose, galacturonic acid, and pectin; however, only 7-1 grew anaerobically on xylose at 46 °C. Saccharomyces cerevisiae NRRL Y-2403 did not grow at 46 °C on any of these substrates. With glucose as a carbon source, ethanol yield after 3 d at 46 °C was higher for 8-1 than for wild type (0.51 and 0.43 g ethanol/g glucose, respectively). With galacturonic acid as a carbon source, the ethanol yield after 7 d at 46 °C was higher for 7-1 than for wild type (0.48 and 0.34 g ethanol/g galacturonic acid, respectively). These mutant strains have potential application in fuel ethanol production at elevated temperature from sugar constituents of starch, sucrose, pectin, and cellulosic biomass.

Keywords

thermotolerant ethanologenic yeast yeast UV-C mutagenesis Kluyveromyces marxianus yeast biomass sugars

A wide variety of biomass feedstocks will be needed to meet the increased requirements for total renewable fuel by 2015 mandated by the United States Environmental Protection Agency in the Renewable Fuel Standard (RFS2) program.¹ In the near future, these feedstocks will include lignocellulosic materials, such as agricultural and forestry residues, that are composed of cellulose, hemicellulose, and lignin.² Hydrolysis of these materials yields the hexose sugars (glucose, mannose, and galactose) and the pentose sugars (xylose and arabinose). Other possible biomass feedstocks are fruit- and coffee-processing wastes that contain pectin, a polymer of galacturonic acid.³ Saccharomyces cerevisiae is currently the most employed microbial catalyst in the biotechnology industry, but it is limited in its range of substrates for producing fuel ethanol, although genetic engineering has improved its utilization of some of the constituent sugars of lignocellulosic materials.^4–13

Recently, increasing attention has been directed toward developing microbial catalysts for ethanol production at elevated temperatures.^14–18 Fermentation processes conducted at elevated temperatures will significantly reduce cooling costs, improve efficiency of simultaneous saccharification and fermentation, allow continuous ethanol removal by evaporation under reduced pressure, and reduce risk of contamination, all of which would improve profitability of fuel ethanol production from biomass.^15,16,18,19 The temperatures suitable for conventional strains of S. cerevisiae are relatively low (25 to 30 °C), although some S. cerevisiae strains have been isolated that are able to produce ethanol efficiently at 40 °C or 42 °C.^20–22 The yeast Kluyveromyces marxianus has advantages that make it a promising candidate for development as a versatile, thermotolerant, industrial ethanologen. This species has been reported to grow at 47 °C and above²³ and to produce ethanol at temperatures greater than 40 °C.^15,16,24

Furthermore, K. marxianus offers other benefits compared with S. cerevisiae, including the ability to grow on a wide variety of substrates not used by S. cerevisiae such as xylose, xylitol, cellobiose, lactose, arabinose, and glycerol.^23,25 K. marxianus also grows on sucrose, raffinose, and inulin at 45 °C under a static condition even when glucose is present, unlike S. cerevisiae. This suggests that K. marxianus is useful for high-temperature fermentation using biomass composed of these sugars.²⁶ Because of these advantages, K. marxianus is currently being developed as a viable alternative to S. cerevisiae for ethanol production.²⁵ Some K. marxianus strains have generally-recognized-as-safe status,¹⁶ similar to S. cerevisiae and K. lactis; therefore, these strains are also acceptable for expression of high-value co-products for human or animal use in addition to producing fuel ethanol.

We describe two novel K. marxianus strains that were obtained by UV-C irradiation of wild-type K. marxianus NRRL Y-1109 cultures, followed by 5-mo anaerobic growth on glucose at 46 °C. UV-C irradiation is a standard technique^27–29 for inducing mutations in yeast. It produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Automated protocols were developed and implemented on a robotic platform for picking and spreading the irradiated cultures and for rapid sterile processing of the plates. The mutant strains were assessed for growth and ethanol production at elevated temperature and for carbon source utilization under aerobic and anaerobic conditions. The irradiation method is similar to that previously used by Hughes and coworkers²⁷ for producing mutagenized Scheffersomyces stipitis strains. However, the work described in this article extends the technique by increasing the radiation exposure time with the intention of maximizing the occurrence of mutations and by incubating the irradiated cells at an elevated temperature to produce strains with improved thermotolerance and enhanced ability to produce ethanol under microaerophilic conditions from a wide variety of substrates. These strains have potential application in industrial fuel ethanol production at elevated temperatures from sugar constituents of starch, sucrose, pectin, and cellulosic biomass.

Materials and Methods

Production of K. marxianus Mutant Strains 7-1 and 8-1 by Irradiation of K. marxianus NRRL Y-1109 and Evaluation of Growth

Duplicate 2 L Fernbach flasks were prepared by adding 1 L of YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone [Becton, Dickinson and Company, Franklin Lakes, NJ], and 1.0% dextrose [Sigma Aldrich, St. Louis, MO]) to each flask and inoculating with 20 mL of a culture of wild-type K. marxianus NRRL Y-1109 (USDA, ARS Culture Collection) grown on YM medium in a 100 mL flask at 28 °C for 2 d. The Fernbach flasks were incubated at 28 °C for 2 d at 100 rpm. The culture from each flask was divided into two Beckman 500 mL spin bottles and pelleted in a Beckman Avanti J20 centrifuge (Beckman Coulter, Inc., Indianapolis, IN) at 20 °C for 20 min at 2056 × g. Cell pellets were washed and resuspended in 50 mL of sterile water. A 25 mL aliquot was taken from each resuspension and placed into a Marsh RR-0014 deep-trough plate with baffled bottom (Marsh Biomedical Products, Inc., Rochester, NY). Before irradiation, a 10 µL sample was taken from the resuspension in the trough plates, diluted to 10⁻⁵, and evaluated using a Reichert Neubauer/Bright-Line hemacytometer (American Optical Corp., Buffalo, NY) to obtain an estimate of cell density. The cells were irradiated for 9 h with 234 nm UV-C radiation (UVP, LLC Light Table [inverted], Upland, CA) at a distance of 14 cm above the plates. Samples (100 µL) were taken every hour during irradiation, diluted to 10⁻⁴, and plated, and the surviving colonies were counted to determine the kill curve.

An automated protocol ( Fig. 1 ) on a robotic work cell³⁰ was used after irradiation to spread 600 µL aliquots from each trough plate onto 128 × 96 mm Omni Tray plates (Thermo Fisher Scientific, Waltham, MA) containing YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% Bacto Agar [Becton, Dickinson and Company], and 1.0% dextrose) or 2% xylose complete minimal medium plus all amino acids (1.4 g yeast synthetic dropout medium supplement, 0.06 g L-leucine, 0.04 g L-tryptophan, 0.02 g L-histidine, 0.02 g L-uracil; 20 g D-xylose [Sigma Aldrich], 20 g Bacto Agar; and 6.7 g yeast nitrogen base without amino acids [Sigma Aldrich] per liter). The plates from several passive and active stackers were moved to the liquid handler in a scheduled fashion, where they were spotted with medium and then with irradiated culture from the Marsh deep-trough plates on the deck. The spread plates (a total of 192 plates, 96 from each trough plate) were wrapped and placed into Mitsubishi anaerobic chambers (Mitsubishi Gas Chemical America, Inc., New York, NY) containing an AnaeroPack dry chemical system (Sigma Fluka, Buchs, Switzerland) at 46 °C for 5 mo. This lengthy incubation period at elevated temperature under stringently anaerobic conditions was chosen not only to ensure that surviving strains would grow well in these conditions but also to minimize background growth of strains that were less robust.

Figure 1.

Procedure for mutagenesis of Kluyveromyces marxianus NRRL Y-1109 to produce mutant strains 7-1 and 8-1 with a diagrammatic and photographic representation of the routine on the robotic platform.

Two YM plates, 7 and 8, each contained one growing colony when the spread plates were unwrapped. Duplicate samples were picked manually from these colonies, designated strains 7-1 and 8-1, respectively, and spread onto plates containing YM or YPD (1.0% yeast extract, 2.0% Bacto Peptone, 2.0% Bacto Agar, and 2.0% D-glucose [Sigma Aldrich]) medium or 2% xylose complete minimal medium plus all amino acids, and the plates were incubated at 46 °C for 2 wk anaerobically to isolate individual colonies. Four colonies were picked manually from each of the respread anaerobic plates onto plates containing YM, YPD, or xylose complete minimal medium plus all amino acids (one sample per plate, four samples per medium) and incubated aerobically at 28 °C for 3 d to confirm that strains 7-1 and 8-1 were still capable of aerobic growth.

Samples were also taken and plated on YPD medium for evaluation of growth at 30 °C and 47 °C to compare thermotolerance of mutant strains to that of wild-type K. marxianus NRRL Y-1109. The mutant strains were further evaluated for growth on various substrates aerobically and anaerobically at 46 °C in comparison to wild-type K. marxianus NRRL Y-1109 and to S. cerevisiae NRRL Y-2043. The cultures of wild-type K. marxianus used to inoculate the media containing the various substrates were not the cells from the primary screen but were taken from the ARS NRRL collection, as were the cultures of S. cerevisiae. Mutant strains 7-1 and 8-1 were passaged 10 times in YM medium before inoculation. All four strains were grown at 30 °C to an OD 600 of 0.1 before inoculation. Polymeric substrates—cellulose, pectin, starch, and guar—were untreated and used as received from the supplier (Sigma Aldrich).

Fermentation in 2 L Fernbach Flask

Fermentation experiments using wild-type K. marxianus NRRL Y-1109, mutant strains 7-1 and 8-1, and S. cerevisiae NRRL Y-2043 were performed in 2 L Fernbach flasks containing YM liquid medium maintained at 46 °C. A liquid preculture was grown in a 100 mL flask on YM medium for 2 d at 28 °C. The density of the preculture was adjusted to an absorbance equivalent to 1.0 at 660 nm (Beckman DU 800; Beckman Coulter, Inc.), and 20 mL was added to 1 L of YM medium in the Fernbach flask. The fermentation was carried out at 46 °C at 100 rpm for 30 h. The absorbance at 660 nm and ethanol production were measured at approximately 10 h intervals.

Scanning Electron Microscopy (SEM) Analysis

Yeast cells from YPD (1.0% yeast extract, 2.0% Bacto Peptone, and 2.0% D-glucose [Sigma Aldrich]), 2% xylose (1.4 g yeast synthetic dropout medium supplement, 0.06 g L-leucine, 0.04 g L-tryptophan, 0.02 g L-histidine, 0.02 g uracil, 20 g D-xylose, and 6.7 g yeast nitrogen base without amino acids [Sigma Aldrich] per liter), or YPGA (1.0% yeast extract, 2.0% Bacto Peptone, and 2.0% galacturonic acid [Sigma Aldrich]) liquid medium incubated aerobically at 46 °C for 12 h were suspended in saline (0.85% NaCl) and centrifuged to remove residual medium. Following a modified procedure of Bang and Pazirandeh,³¹ the cell pellet was suspended and fixed in 2.5% glutaraldehyde prepared in 100 mM cacodylate buffer, pH 7.2, for 1 h on ice. To remove remaining glutaraldehyde, the cells were rinsed with the buffer twice and then with distilled water once, allowing several minutes for each step. The cells were dehydrated, respectively, in solutions containing 50%, 70%, 80%, and 100% ethanol successively for 15 min for each treatment. Cells were mounted on an aluminum stub and placed in a desiccator to dry overnight or until needed. The samples were subjected to SEM and analysis (Zeiss Supra 40 VP).

DNA Fingerprinting

Variable nucleotide tandem repeat (VNTR) PCR analysis was performed to detect differences in genomic DNA sequences using as PCR primer the 15-base pair (bp) 5 × CAG repeat sequence.³² Genomic DNA from wild-type K. marxianus NRRL Y-1109 and mutant strains 7-1 or 8-1 was isolated from a 1 mL sample of a 2-d 37 °C culture in YPD liquid medium in a 1.5-mL polypropylene tube. The samples were vortexed for 30 s and then centrifuged at 15 800 × g for 2 min (Thermo Micromax Microcentrifuge; Thermo Fisher Scientific). The supernatant was decanted, and an additional 1 mL of the culture was added to the tubes. The tubes were vortexed for 30 s and then centrifuged at 15 800 × g for 2 min. The supernatant was decanted, and 400 µL of water was added. The mixture was boiled for 10 min followed by addition of 400 µL of phenol solution (saturated, pH 6.6; AMRESCO LLC, Solon, OH). The tubes were vortexed for 30 s and then centrifuged at 15 800 × g for 2 min. The aqueous phase was transferred by pipette to new 1.5 mL tubes, and 400 µL of phenol:chloroform:isoamyl alcohol (25:24:1; tris buffer to pH 8.05; AMRESCO, LLC) was added. The tubes were vortexed for 30 s and then centrifuged at 15 800 × g for 2 min. The aqueous phase was transferred by pipette to new 1.5 mL tubes, and 400 µL of ethyl ether (water saturated; Avantor Performance Materials, formerly J.T. Baker, Phillipsburg, NJ) was added. The tubes were vortexed for 30 s and then centrifuged at 15 800 × g for 2 min. The organic phase was removed by pipette and discarded, and 40 µL of 3 M sodium acetate, pH 5.2 (Sigma Aldrich) was added to the remaining solution. The tubes were vortexed for 90 s, and 1.5 mL of cold 100% ethanol was added. The tubes were placed into a −80 °C freezer overnight. After removal from the freezer, the tubes were centrifuged for 10 min at 15 800 × g and the liquid decanted. One milliliter of cold 70% aqueous ethanol solution was added, and the tubes were centrifuged for 10 min at 15 800 × g. The liquid was removed with a pipette, leaving the clear pellet on the bottom. The material was dried in a Savant SPD 2010 SpeedVac System (Thermo Fisher Scientific) for 5 min at 45 °C and 8 psi. The tubes were removed from the dryer, 70 µL of water was added, and the tubes were allowed to stand for 10 min. The concentration of genomic DNA obtained was determined by densitometry using an AlphaImager 3400 (Alpha Innotech Corporation, San Diego, CA).

The PCR mixture contained 2 µL genomic DNA (0.5 mg/mL), 32.5 µL water, 10 µL 5X Phusion HF Buffer with MgCl₂, 1 µL 10 mM dNTPs, 4 µL (0.1 mg/mL) VNTR oligonucleotide primer (5′CAGCAGCAGCAGCAG3′), and 0.5 µL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). The PCR reaction was prepared in a Phenix MPC-3420 96-well PCR plate (Phenix Research Products, Candler, NC) on ice and was carried out in a PTC-225 Tetrad Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) using the following conditions: hold at 95 °C for 5 min, cycle at 95 °C for 1 min, 42 °C for 1 min, 72 °C for 1 min, repeated for 30 times, followed by 72 °C for 5 min and a 4 °C hold. The procedure amplified the genomic sequence between two VNTR sequences to determine alterations in the microsatellite or minisatellite regions in the genome. The amplified DNA was analyzed by gel electrophoresis on 1% (w/v) agarose gels stained with ethidium bromide.

Growth on Pectin and on Mono-, Di-, and Tri-Galacturonic Acids

To each of four test tubes containing a mini magnetic stirrer were added 1 mL water, 0.005 g peptone, 0.0025 mg yeast extract, and 0.005 g agar. To tubes 1, 2, 3, and 4 were added, respectively, 0.005 g pectin, 0.005 g D-galacturonic acid, 0.005 g di-galacturonic acid, and 0.005 g tri-galacturonic acid (Sigma Aldrich). The mixtures were stirred until the solids were dissolved, and the solutions were autoclaved for 10 min. Tubes were slanted while cooling. A sample composed of nine loops of a culture of K. marxianus mutant strain 7-1 (only one strain was tested because of reagent expense; strain 7-1 gave the highest ethanol yield on galacturonic acid in the Braun reactor) was added to 4 mL of sterile-filtered water and mixed. After mixing, 0.5 mL of the solution was added to each of the four slant media tubes, and the tubes were placed inside an Innova 4230 Incubator Shaker (New Brunswick Scientific, Enfield, CT) at 47 °C for 15 d at 100 rpm. Images of the samples were captured using the AlphaImager 3400 system and analyzed using AlphaEase FC software (Alpha Innotech Corporation).

Fermentation in Braun Reactor

Fermentations with wild-type K. marxianus NRRL Y-1109 and mutant strains 7-1 and 8-1 were performed in a B. Braun Sartorius Biostat B reactor (B. Braun Biotech International GmbH [now Sartorius BBI Systems GmbH], Melsungen, Germany). Liquid precultures in YPD medium were incubated at 30 °C for 2 d at 100 rpm. The density of the preculture was adjusted to an absorbance equivalent to 1.0 at 660 nm (Beckman DU 800; Beckman), and 20 mL was added to 1 L of medium in a 1.5 L culture vessel.

In the first stage of the fermentation, ethanol production was measured from YPD medium. The reactor was maintained at 46 °C with stirring (100 rpm) for 3 d with 1 mL/min sparge of filtered nitrogen (dissolved oxygen reading was 0) at a constant pH of 5.5. Samples were collected for ethanol, glucose, melanin, and 2,3-butanediol analysis, and the cells were allowed to settle. Spent YPD medium was removed, and YPGA medium was added. In the second stage of the fermentation, ethanol production was measured from YPGA medium. The reactor was maintained at 46 °C with stirring (100 rpm) for 7 d with 1 mL/min sparge of filtered nitrogen (dissolved oxygen reading was 0) at a constant pH of 5.5. After the second stage, samples were collected for ethanol, galacturonic acid, melanin, and 2,3-butanediol analysis.

Ethanol, Glucose, Galacturonic Acid, and 2,3-Butanediol Analysis

Ethanol, glucose, galacturonic acid, and 2,3-butanediol concentrations were determined by high-performance liquid chromatography (HPLC) using a 300 mm Aminex HPX-87H column (Bio-Rad Laboratories, Inc.) on an HP 1100 Series HPLC system equipped with a refractive index detector (Agilent Technologies, Santa Clara, CA). Samples (10 µL) were injected onto a heated column (65 °C) and eluted at 0.6 mL/min using an isocratic 5 mM H₂SO₄ mobile phase.

Melanin Analysis

Melanin concentrations were determined via an enzyme-linked immunosorbent assay (ELISA) assay (CUSABIO 96-well Human Melanin ELISA Kit; CUSABIO Biotech Co. Ltd, Wuhan, Hubei Province, P.R. China) using a polyclonal human melanin antibody conjugated to clear-bottom white-walled microplates to which 100 µL of a melanin standard or 100 µL of the fermentation sample from the Braun reactor were added, followed by biotin anti-melanin antibody (1×) and then horseradish peroxidase–avidin antibody (1×) according to the manufacturer’s instructions. Tetramethyl benzidine substrate was added, and the plate was incubated at 37 °C for 15 min protected from the light. Stop solution was added to each well, and the plates were evaluated within 5 min in a Bio-Rad Ultramark Microplate Absorbance Reader (Bio-Rad Laboratories Inc.) at 450 nm. A reading at 570 nm was subtracted from the reading at 450 nm. This subtraction will correct for optical imperfections in the microplates. If the microplate reader has wavelength correction available, it should be set at 540 or 570 nm. Readings made directly at 450 nm without correction may be higher and less accurate.

Results

Number of Cells Irradiated and Robotic Selection

Approximately 20% of the genome of K. marxianus has been sequenced, and about 1300 novel genes encoding proteins were identified.³³ Based on this information and on the results from sequencing of the K. lactis genome regarding the number of genes (about 5331) and the average length of a gene transcript (approximately 1800),³⁴ it can be roughly estimated that tens of millions of cells would need to be irradiated to maximize mutation of the K. marxianus genome. The culture samples of wild-type K. marxianus NRRL Y-1109 taken prior to irradiation contained more than 100 million cells.

An automated process on a robotic platform was developed and implemented to analyze the number of samples needed.³⁰ A diagrammatic and photographic representation of this routine on the robotic platform is shown in Figure 1 . The automated protocols for picking and spreading the irradiated cultures enabled rapid sterile processing of six 128 mm × 96 mm Omni Tray plates per run ( Fig. 1 , step 1). Up to 270 plates can be stacked on the work cell for continuous processing, and the stacks can be readily refilled as needed. The SoftLink software moved the plates from several passive and active stackers to the liquid handler in a scheduled fashion, where they were spotted with medium and then with irradiated culture from the Marsh deep-trough plates on the deck. The plates with the irradiated samples were then selected for anaerobic growth at elevated temperature ( Fig. 1 , step 2).

Growth Characteristics of Mutant Strains Selected for Anaerobic Growth at Elevated Temperature (Isolates 7-1 and 8-1)

K. marxianus NRRL Y-1109 wild-type strain was subjected to 234 nm UV-C irradiation to achieve a target mortality of about 80%, which was expected to eliminate any background growth. The progress of the irradiation was monitored by taking samples every hour during irradiation, diluting and plating them, and counting the surviving colonies to determine the kill curve. The results show that 80% mortality is obtained after 9 h of irradiation ( Fig. 2 ). After the wild-type strain was irradiated for 9 h, followed by incubation on YM or xylose medium for 5 mo at 46 °C in an anaerobic chamber, only two YM medium plates contained growing colonies when the spread plates were unwrapped. Samples from these surviving colonies were spread onto plates containing YM medium and incubated at 46 °C for 2 wk anaerobically to isolate individual colonies ( Fig. 1 , step 2). Samples of these colonies were then spread on YM, YPD, and xylose plates and incubated aerobically at 28 °C for 3 d. The colonies grew, demonstrating that mutant strains 7-1 and 8-1 retained capacity for aerobic growth on these substrates.

Figure 2.

Cell kill curves for Kluyveromyces marxianus NRRL Y-1109 wild-type (WT) strain irradiated at 14 cm for 9 h with UV-C 234 nm. Cell counts were determined on the cell pellet resuspension in one trough plate (represents half the total cells from the Fernbach flask). Each point is the average of two determinations.

Comparison of Growth of K. marxianus NRRL Y-1109 Wild-Type Strain and Mutant Strains 7-1 and 8-1 at 30 °C and 47 °C

Growth levels of K. marxianus NRRL Y-1109 wild-type strain and mutant strains 7-1 and 8-1 were compared at 30 °C and 47 °C to examine thermotolerance of these strains. The results after the strains were spread on YPD plates and incubated for 5 d at 30 °C and 47 °C are shown in Figure 3 . All strains grew well at 30 °C, but only the mutant strains 7-1 and 8-1 grew at 47 °C. Irradiation of the wild-type strain and selection at elevated temperature produced mutant K. marxianus strains with increased thermotolerance.

Figure 3.

Kluyveromyces marxianus mutant strains 7-1 and 8-1 grown on glucose medium at 30 °C and 47 °C for 5 d under aerobic conditions compared with K. marxianus NRRL Y-1109 wild type (WT).

Cell Growth and Ethanol Production in Fernbach Flask at 46 °C with Wild-Type K. marxianus NRRL Y-1109, Mutant Strains 7-1 and 8-1, and S. cerevisiae NRRL Y-2043

Ethanol production (g/L) and cell growth (absorbance at 660 nm) were monitored during a 30 h fermentation experiment in a 2 L Fernbach flask in YM medium (10 g/L glucose) at 46 °C for wild-type K. marxianus NRRL Y-1109, mutant strains 7-1 and 8-1 and S. cerevisiae NRRL Y-2043 ( Fig. 4 ). S. cerevisiae NRRL Y-2043 did not grow at 46 °C, and therefore no ethanol was produced. Cell growth for wild-type K. marxianus NRRL Y-1109 and mutant strain 8-1 rose from initial OD 660 values of about 0.16 up to 0.55 at 5 h then to 1.57 at 15 h and remained essentially at that level for the remainder of the 30 h experiment. Cell growth for mutant strain 7-1 showed a delayed entry into log phase with the OD 660 value remaining at 0.18 until 5 h, then rising to 1.22 at 15 h, showing a growth rate (based on slope) similar to that of wild type and 8-1. Growth was still increasing at 15 h, and the OD 660 value reached 1.51 at 26 h, similar to wild type and 8-1, where it remained to the end of the experiment at 30 h. For all 3 strains, ethanol production started at 5 h. The ethanol level for wild-type K. marxianus NRRL Y-1109 reached a maximum of 4.7 g/L at 15 h, after which it decreased steadily to 3.1 g/L at 30 h. The ethanol level for mutant strain 8-1 rose more slowly than for the wild-type strain, reaching a maximum of 3.9 g/L at 25 h, after which it decreased steadily to 2.9 g/L at 30 h. Ethanol production for mutant strain 7-1 increased more slowly than mutant strain 8-1 but was still rising at the end of the experiment (30 h), where it was 3.4 g/L.

Figure 4.

Ethanol production (g/L) and cell growth (absorbance at 660 nm) for wild-type (WT) Kluyveromyces marxianus NRRL Y-1109, K. marxianus mutant strains 7-1 and 8-1, and Saccharomyces cerevisiae NRRL Y-2043 for 30 h at 46 °C in 1 L YM medium (10 g/L glucose) under microaerophilic conditions in a 2 L Fernbach flask.

SEM Analysis of Wild-Type K. marxianus NRRL Y-1109 and Mutant Strains 7-1 and 8-1

Cells from cultures of K. marxianus wild-type and mutant strains grown using glucose, xylose, or galacturonic acid as substrates were examined using SEM ( Fig. 5 ). The SEMs of the wild-type K. marxianus NRRL Y-1109 and mutant strains grown on glucose show that the cells were generally similar in size, shape, and surface features. On the other hand, the micrographs of cells from these strains grown on xylose show that the cells of mutant strain 7-1 are larger than those of the wild-type strain and that more of the 7-1 cells have cratered surfaces compared with the wild-type cells. The cells of mutant strain 8-1 grown on xylose are smaller than those of mutant strain 7-1; however, the surfaces are not only cratered but also wrinkled, and the shapes of the cells are flattened and distorted compared with the shapes of wild type and 7-1 cells. The micrographs of cells from strains grown on galacturonic acid show that most of the cells of the wild-type strain have a relatively smooth, noncratered surface and are urn shaped with budlike projections at the tops. In contrast, the cells of mutant strains 7-1 and 8-1 are larger and their surfaces are more cratered than those of the wild-type strain. The shapes of most of the cells of strain 8-1 grown on galacturonic acid are round with dimples, which are notably different from the urn-shaped cells with budlike projections observed in the micrograph for the wild-type strain.

Figure 5.

Scanning electron micrographs of Kluyveromyces marxianus mutant strains 7-1 and 8-1 compared with K. marxianus NRRL Y-1109 wild type (WT). The scale bar represents 2 µm, except in the bottom middle micrograph, where the bar represents 10 µm, but the scale is the same for all three micrographs of strains grown on glucose.

VNTR PCR from Genomic DNA for Wild-Type K. marxianus Strain NRRL Y-1109 and Mutant Strains 7-1 and 8-1

The PCR products amplified from the genomic DNA of K. marxianus NRRL Y-1109 wild-type strain and mutant strains 7-1 and 8-1 using a VNTR primer produced different banding patterns (fingerprints) when analyzed on an agarose gel ( Fig. 6 ). The arrow on the left at approximately 275 bp points out a band that is present in the fingerprint of the wild-type strain but not detectable in the fingerprints of the mutant strains 7-1 and 8-1. The arrow on the right at approximately 750 bp points to a band present in mutant strain 7-1 but not detectable in the wild-type strain or mutant strain 8-1. The PCR products from the genomes of the mutant strains are different from each other and from the products from the genome of the wild-type strain.

Figure 6.

Variable nucleotide tandem repeat fingerprint of genomic DNA of Kluyveromyces marxianus mutant strains 7-1 and 8-1 compared with K. marxianus NRRL Y-1109 wild type (WT). PCR products were generated from genomic DNA using the CAG repeat primer and analyzed on a 1% (w/v) agarose gel. Bionexus DNA bp markers are identified by length on the left side of the figure. Bands present or absent in mutagenized strains 7-1 and 8-1 compared with wild-type K. marxianus NRRL Y-1109 are indicated by arrows. Electrophoresis was performed at 80 V on a Bio-Rad Power Pac 3000, and a high-resolution digital image file was generated with an AlphaImager 3400 using a trans-UV light.

Anaerobic and Aerobic Growth on Various Substrates at 46 °C of Mutant Strains 7-1 and 8-1 Compared with Wild-Type K. marxianus NRRL Y-1109 and S. cerevisiae NRRL Y-2043

The aerobic and anaerobic growth of mutant strains 7-1 and 8-1 compared with wild-type K. marxianus NRRL Y-1109 and S. cerevisiae NRRL Y-2043 was examined on various substrates of industrial interest, including galacturonic acid, pectin, glucose, arabinose, xylose ( Fig. 7 ), cellulose, starch, guar, and galactose ( Fig. 8 ). S. cerevisiae NRRL Y-2043 did not grow at 46 °C on any of these substrates. None of the strains tested grew on untreated cellulose or starch. Wild-type K. marxianus NRRL Y-1109 and mutant strain 8-1 grew on all other substrates tested, except essentially no growth was detected anaerobically on arabinose, xylose, or guar. Mutant strain 7-1 showed essentially the same results, except that it also grew to a detectable extent anaerobically on xylose and guar. Growth on hexose substrates was better than on pentose substrates. The results of the tube assay ( Table 1 ) demonstrate that mutant strain 7-1 has the ability to grow on pectin and to use the resulting mono-, di-, and tri-galacturonic acids to the same extent as the individual acids tested separately.

Figure 7.

Anaerobic and aerobic growth on selected substrates at 46 °C for 7 d of mutant strains 8-1 (top position on plate) and 7-1 (right) compared with wild-type Kluyveromyces marxianus NRRL Y-1109 (bottom) and Saccharomyces cerevisiae NRRL Y-2043 (left).

Figure 8.

Anaerobic and aerobic growth on additional substrates at 46 °C for 7 d of Saccharomyces cerevisiae NRRL Y-2043 (top right position on plate), wild-type Kluyveromyces marxianus NRRL Y-1109 (bottom right), and mutant strains 7-1 (bottom left) and 8-1 (top left).

Table 1.

Growth of Mutant Strain 7-1 in a Slant-Tube Assay Using Pectin as Substrate Compared with Mono-, Di-, and Tri-D-Galacturonic Acids as Substrates at 47 °C for 15 d at 100 rpm.

Sample	IDV^a	% Density of Pectin Standard (Based on IDV)	% Adjusted Color Density (Relative to 20 g/mL Pectin Standard)
Blank (no strain added)	11 424	0.3
Pectin	1 109 448	100.0	29.2
Mono-galacturonic acid	853 944	76.9	22.4
Di-galacturonic acid	940 680	84.7	24.7
Tri-galacturonic acid	846 940	76.3	22.2

IDV = integrated density value.

Images were captured using the AlphaImager 3400 system and analyzed using AlphaEase FC software.

Cell Growth, Substrate Use, and Ethanol Yield of Mutant Strains 7-1 and 8-1 Compared with Wild-Type K. marxianus NRRL Y-1109 with Glucose or Galacturonic Acid Medium in Braun Fermentor at 46 °C

The results presented in Table 2 show that with glucose as the carbon source, the ethanol yield after 3 d at 46 °C was 19% higher for mutant strain 8-1 than for wild-type K. marxianus NRRL Y-1109 (0.51 and 0.43 g ethanol/g glucose, respectively). Cell growth of mutant strain 8-1 on glucose as measured by OD 660 was 2.7 times greater than that of the wild-type strain. The cell growth and ethanol yield on glucose were the same for mutant strain 7-1 and wild type. With galacturonic acid as the carbon source, the ethanol yield after 7 d at 46 °C was 41% higher for mutant strain 7-1 than for wild-type K. marxianus NRRL Y-1109 (0.48 and 0.34 g ethanol/g galacturonic acid, respectively). Cell growth of mutant strain 7-1 on galacturonic acid as measured by OD 660 was 1.3 times greater than that of wild-type strain. The cell growth and ethanol yield on galacturonic acid were the similar for mutant strain 8-1 and wild type.

Table 2.

Cell Growth, Substrate Consumption, and Production of Ethanol, 2,3-Butanediol, and Melanin Using Mutant Strains 7-1 and 8-1 Compared with Wild-Type Kluyveromyces marxianus NRRL Y-1109 (WT) in a Braun-Sartorius Biostat B Fermentor at 46 °C under Microaerophilic Conditions.

Sample	OD 600	Sugar Remaining (g/L)	Sugar Consumed (g/L)^a	Ethanol Produced (g/L)	Yield (g Ethanol/g Sugar)	2,3-Butanediol (g/L)	Melanin (µg/L)
Sample	OD 600	n = 3	Sugar Consumed (g/L)^a	n = 3	Yield (g Ethanol/g Sugar)	n = 3	n = 2
Glu control	0.029	44.0 ± 0.1	0.0	0.0 ± 0.0		0.00 ± 0.00	0.54 ± 0.04
Glu WT	0.069	3.4 ± 0.1	40.6	17.5 ± 1.5^b	0.43	1.93 ± 0.02	0.67 ± 0.14
Glu 7-1	0.067	0.0 ± 0.0	44.0	20.0 ± 7.7	0.45	1.25 ± 0.04	1.04 ± 0.48
Glu 8-1	0.190	0.0 ± 0.0	44.0	22.5 ± 1.3^b	0.51	1.95 ± 0.06	0.84 ± 0.38
GA control	0.089	42.2 ± 0.02	0.0	0.0 ± 0.0		0.00 ± 0.00	0.54 ± 0.07
GA WT	0.407	1.5 ± 0.01	40.7	13.9 ± 0.2^c	0.34	1.56 ± 0.03	0.64 ± 0.07
GA 7-1	0.523	0.0 ± 0.0	42.2	20.5 ± 0.2^c	0.48	1.11 ± 0.06	1.67 ± 0.28
GA 8-1	0.351	0.0 ± 0.0	42.2	14.8 ± 0.2	0.35	1.49 ± 0.11	1.78 ± 0.67

Fermentation proceeded for 3 d using glucose (Glu) as a carbon source. Spent medium was removed, galacturonic acid (GA) medium was added, and fermentation was continued for 7 d using GA as carbon source.

For difference between WT and 8-1, p = 0.014 calculated using two-tailed type 2 t test.

For difference between WT and 7-1, p < 0.00001 calculated using two-tailed type 2 t test.

Discussion

Patterns of Cell Growth and Ethanol Production at 46 °C for Wild-Type K. marxianus NRRL Y-1109 and Mutant Strains 7-1 and 8-1

The pattern of cell growth for mutant strain 8-1 was similar to that of wild-type K. marxianus NRRL Y-1109 with cell density rising from initial OD 600 values about 0.16 rising to a maximum of 1.57 at 15 h. However, the growth of mutant strain 7-1 (initial OD 660 of 0.12) showed a delayed entry into log phase, with the OD 660 value remaining at 0.18 until 5 h, then rising to 1.22 at 15 h, showing a growth rate (based on slope) similar to that of wild type and 8-1. Mutant strain 7-1 initiated ethanol production at 5 h, the same time as mutant strain 8-1 and wild-type strain, and similar ethanol production levels were observed for all strains at the end of the experiment (30 h). At that point, ethanol production was decreasing for wild-type and 8-1 strains but appeared to be increasing for strain 7-1. This suggests that mutant strain 7-1 might be better adapted to ethanol production under extended microaerophilic conditions than mutant strain 8-1 or wild-type strain.

SEM and VNTR Analysis for Wild-Type K. marxianus NRRL Y-1109 and Mutant Strains 7-1 and 8-1 Strains

The SEM results indicated that the two K. marxianus strains that were selected for growth at elevated temperature under anaerobic conditions are unique strains because their morphologic features differ from each other and from those of the wild-type K. marxianus strain when grown on xylose or galacturonic acid. Most notably, the micrographs of cells from strains grown on galacturonic acid show that most cells of the wild-type strain have relatively smooth, noncratered surfaces and are urn shaped with budlike projections at the tops, whereas the cells of mutant strains 7-1 and 8-1 are larger and their surfaces have more indentations. The shapes of most of the cells of strain 8-1 grown on galacturonic acid are round with dimples in striking contrast to the urn-shaped cells with budlike projections at the tops observed in the micrograph for the wild-type strain.

The differences observed in the PCR products amplified from the genomic DNA of K. marxianus NRRL Y-1109 wild-type strain and mutant strains 7-1 and 8-1 using the VNTR sequence as PCR primer demonstrate that the mutant strains are different from each other and from the wild-type strain. These differences in the PCR products can be used to distinguish differences in the genome.^35,36 VNTR analysis of wild-type and mutant strains 7-1 and 8-1 indicated mutations had occurred in the wild-type strain to produce strains 7-1 and 8-1. The exact nature of these mutations will need to be investigated by comparative DNA sequencing.

Growth of Mutant Strains 7-1 and 8-1 on Substrates of Potential Use for Renewable Fuels

The mutant strains 7-1 and 8-1 grew on substrates that are constituents of the sugar, starch, or biomass materials commonly considered as feedstocks for renewable fuels.^1,37,38 The sources may be sugar cane, corn starch, sugar beet pulp, fruit waste, dedicated plants, plant waste, forestry by-products, or urban waste.^39,40 As expected, S. cerevisiae NRRL Y-2043 did not grow at 46 °C. None of the strains tested grew on untreated cellulose or starch. Interestingly, although no colonies were isolated from xylose medium after irradiation, one strain selected for elevated temperature and anaerobic growth that initially survived on glucose medium showed anaerobic growth on xylose substrate at 46 °C. The mutant strain that grew on xylose did not grow as well on the pentose substrate as on the hexose substrates.

Guar is a plant gum of considerable commercial interest. It consists of a main chain of β-1,4-linked D-mannopyranose units, with D-galactopyranose units linked α-1,6 to the mannose backbone. It is commonly used as a thickener in foods and as a thickener and suspending agent in oilfield applications.⁴¹ Therefore, enzymatic modification and biodegradation of guar are of potential interest. To use guar as a carbon source, an organism would most likely need to produce at least an α-galactosidase and quite likely also an endo-β-D-mannanase and a β-mannosidase, although these enzymes have not been reported for K. marxianus.¹⁶ It is expected that some of these enzymes would need to be secreted into the growth medium because of the macromolecular nature of the guar polymer. Growth of the K. marxianus strains on guar indicates that galactose and mannose are being used by these strains aerobically and by mutant strain 7-1 also anaerobically.

The results of the tube assay confirm that mutant strain 7-1 has the ability to depolymerize pectin and to use the resulting mono-, di-, and tri-galacturonic acids to the same extent as the individual acids tested separately, reflecting endopolygalacturonase activity typically produced by K. marxianus.³ Endopolygalacturonases are an industrially important subgroup of pectinases because they promote a rapid decrease in the molecular weight of pectin and hence in the viscosity of pectin-containing solutions. Currently, commercial preparations are a mixture of pectic enzymes that includes some enzymes with undesirable activities. K. marxianus is being evaluated as a source because it produces almost exclusively endopolygalacturonase. It has been shown that during alcoholic fermentation by K. marxianus CCEBI 2011, endopolygalacturonase accumulates together with ethanol,³ which contributes favorably to the economy of the bioprocess.³ The dark color of the colonies on the pectin plates and especially on the galacturonic acid plates indicates the production of an unknown pigment. The identity of the pigmented material is under investigation.

Cell Growth, Substrate Use, Ethanol Yield, and Melanin and 2,3-Butanediol Production by Mutant Strains 7-1 and 8-1 Compared with Wild-Type K. marxianus NRRL Y-1109 with Glucose or Galacturonic Acid Medium in Braun Fermentor at 46 °C

Using glucose as substrate, strain 8-1 gave a higher ethanol yield after 3 d at 46 °C than wild type or 7.1 (0.51, 0.43, and 0.45 g ethanol/g glucose, respectively). Using galacturonic acid as substrate, strain 7-1 gave a higher ethanol yield after 7 d at 46 °C than wild type or 8-1 (0.48, 0.34, and 0.35 g ethanol/g galacturonic acid, respectively).

Rodrussamee and coworkers²⁵ obtained an ethanol yield of 0.48 g ethanol/g glucose after 12 h for K. marxianus DMKU3-1042 on YPD medium at 45 °C under a shaking condition, compared with 0.43 g ethanol/g glucose for wild-type K. marxianus NRRL Y-1109 at 46 °C after 72 h in our work. Pang and coworkers²⁹ used UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine to mutate K. marxianus GX-15. They obtained a yield of 0.46 g ethanol/g glucose for the best mutant strain, K. marxianus GX-UN120, and this strain completely consumed all glucose at 60 h. In our work, the mutant strains 7-1 and 8-1 completely consumed all the glucose after 72 h and all the galacturonic acid after 168 h in comparison with the wild-type strain, which consumed 92% of the glucose and 96% of the galacturonic acid.

On galacturonic acid, the production of 2,3-butanediol was inversely correlated with ethanol production. Mutant strain 7-1 on galacturonic acid had the highest ethanol level and the lowest 2,3-butanediol level, whereas wild type and 8-1 had lower ethanol levels but higher 2,3-butanediol levels. No correlation was observed for the strains on glucose substrate. Although melanin was not found to be a major product, melanin levels for strains 7-1 and 8-1 were higher on galacturonic acid than on glucose and also higher than wild-type K. marxianus NRRL Y-1109 on both substrates.

The robust thermotolerant K. marxianus mutant strains resulting from the intense multigene mutagenesis strategy used in this work appear to have traits that have potential application in industrial fuel ethanol production at elevated temperature from biomass hydrolysates and fruit and coffee-processing wastes. Growth and high levels of ethanol production at elevated temperature provide an advantage over S. cerevisiae in industrial fermentations. These mutant strains have an additional advantage in their ability to produce ethanol from a wide variety of substrates, including constituents of starch and sucrose as well as biomass feedstocks such as wet mill corn pericarp waste or fruit- and coffee-processing wastes.

Footnotes

Acknowledgements

We thank Karen Hughes for critical reading and formatting of the manuscript. We acknowledge the assistance of Nathane Orwig in obtaining the PCR primer.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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