We used six previously characterized kveik strains (Preiss et al., 2018) as well as four commonly used commercial ale yeast strains from the Beer 1 and Beer 2 beer yeast families in this study (Table 1). Cali Ale, Vermont Ale and Kölsch are popular, commonly used Saccharomyces cerevisiae ale strains of the Beer 1 family (Figure 1; Gallone et al., 2016), which are hypothesized to share a common ancestor with kveik strains (Preiss et al., 2018). St. Lucifer, a Belgian ale yeast of the Beer 2 family (Figure 1), possesses characteristics that are commonly associated with kveik strains, including a high degree of thermotolerance and greater production of fruity flavor compounds but is notably POF + (Gallone et al., 2016). The kveik strains were previously isolated from batch cultures (Preiss et al., 2018). Phylogenetically Hornindal1 and Laerdal2 are closer to each other and Hornindal2 to Ebbegarden1 (Figure 1). All beer yeast strains were supplied by Escarpment Laboratories (Guelph, Ontario). The commercially available Finnish baking and standard sahti brewing yeast Suomen Hiiva (Katina et al., 2007; Catallo et al., 2020; Magalhães et al., 2021) was used as a control in the trehalose and trehalase experiments.

Wort used for beer fermentations and yeast propagation was obtained from a commercial brewery, Wellington Brewery (Guelph, ON). The hopped wort was prepared using Canadian 2-row malt to an original gravity of 12.5°Plato (1.045 specific gravity). The wort was sterilized prior to use at 121°C for 20 min and incubated overnight to the desired fermentation or propagation temperature.

Colonies from YPD plates were inoculated into 100 mL of YPD in biological triplicate and grown at 30°C, 170 rpm until cultures reached the pre-diauxic phase of growth. Cell counts were performed using a haemocytometer and cells were transferred into 100 mL of sterilized wort to a targeted cell density of 5 × 10⁶ cells/mL and grown at 30°C, 170 rpm until pre-diauxic phase. These cultures were counted using a haemocytometer and inoculated at a rate of 1.2 × 10⁷ cells/mL into 200 mL of sterilized wort in 250 mL glass bottles fitted with airlocks. These small-scale fermentations were performed in triplicate at two colder temperatures (12 and 15°C), two standard ale fermentation temperatures (22 and 30°C), and four higher temperatures (35 37, 40, and 42°C). Incubations were for 5 days, except for the 12°C fermentations (10 days). The bottles were incubated without shaking to best approximate typical beer fermentation conditions. Fermentation profiles were acquired by collecting samples throughout the fermentation process and analyzing changes in specific gravity (SG) using a DMA35v4 portable densitometer (Anton-Paar).

Throughout the fermentation, samples were collected and filtered with 0.45 μm syringe filters prior to metabolite analysis. The wort sugar, ethanol and glycerol content were measured using HPLC and a refractive index (RI) detector. The samples collected at the indicated times throughout the fermentation were analyzed using an Aminex HPX-87H column, with 5 mM sulfuric acid as the mobile phase, under the following conditions: flow rate of 0.6 mL/min, 620 psi, and 60°C. Each sample contained 400 μL of filtered sample and 50 μL of 6% (w/v) arabinose as the internal standard.

Colonies were inoculated into 100 mL cultures of YPD and incubated at 30°C with shaking at 180 rpm. Cell samples were collected at the mid-exponential phase of growth and then every 2 h until after the diauxic shift to analyze the timing and quantity of trehalose production. Trehalose was extracted from the cells using 0.5 M cold TCA and analyzed via HPLC with an Aminex HPX-87H column at 30°C, 700 psi, and a flow rate of 0.4 mL/min. Concentration of trehalose was determined relative to a standard curve of known concentrations and expressed as milligrams of trehalose per gram of dry cell weight (DCW). Each sample contained 400 μL of unfiltered sample and 50 μL of 6% (w/v) arabinose as the internal standard.

Culture conditions for trehalase activity assays were identical to those previously described for the trehalose assays. Cell samples were collected 2 h post maximum trehalose levels, when trehalase activity is predicted to be highest in all strains. The enzyme kinetics of the hydrolysis of trehalose by neutral trehalase was measured using a stopped assay. Trehalase activity assay was adapted from a previously described assay (Pernambuco et al., 1996). Briefly, cell pellets were collected and resuspended in ice-cold 50 mM MES/KOH (pH 7) + 50 μM CaCl₂ and crude cell extract was prepared using the glass bead lysis method. Overnight dialysis was performed using a 22 μm Cellu Sep H1 Dialysis Membrane and the dialyzed extract was utilized in stopped trehalase activity assays. The quantity of glucose liberated was determined using a Glucose GOD-PAP Kit (Roche/Hitachi). Trehalase activity was standardized to total protein content using the DC protein quantification technique according to the manufacturer’s recommendations (Bio-Rad).

The genomes of the six kveik strains, Cali and Vermont were previously sequenced (Preiss et al., 2018), while those of the Kölsch and St. Lucifer control strains were sequenced here. DNA was extracted from Kölsch and St. Lucifer as previously described (Preiss et al., 2018). Whole-genome sequencing was performed by the TCAG sequencing facility at Sick Kids (Toronto, ONT, Canada). Briefly, an Illumina TruSeq TL paired-end 150 bp library was prepared for each strain and sequenced using an Illumina HiSeq X instrument. This data and WGS data previously generated for the kveik, Cali and Vermont strains (Preiss et al., 2018) were further analyzed. FastQC version 0.11.8 (Andrews, 2010) and fastp version 0.20.1 (Chen et al., 2018) were used to quality-analyze the sequencing reads. Low quality reads and nucleotides were trimmed and filtered using fastp. Filtered reads were aligned against the latest S. cerevisiae version R64-3-1 genomic reference sequence, which is also known as S288C, using BWA version 0.7.17 (Li and Durbin, 2009). PCR duplicates were marked up and the alignments were sorted and indexed via sambamba version 0.7.1 (Tarasov et al., 2015). Per base depth were calculated via mosdepth version 0.3.0 (Pedersen and Quinlan, 2018) and FreeBayes version 1.2.0 (Garrison and Marth, 2012) was used to perform variant analysis on aligned reads. Variant effect prediction and the gene names were assigned with SnpEff version 5.0e (Cingolani et al., 2012). Copy number variations of genes were estimated based on coverage with Control-FREEC version 11.6 (Boeva et al., 2011). Wilcoxon Rank Sum test (p < 0.05) was performed using the significance script provided by Control-FREEC to evaluate significant copy number variations. Allele frequencies and read depth were plotted to predict the ploidy and aneuploidies for each chromosome.

Phylogenetic analysis was performed using consensus genome sequences generated from variant calls with vcftools (Danecek et al., 2011), which were then aligned to the R64-3-1 genome with minimap2 (Li, 2018). Single nucleotide polymorphisms in the whole-genome alignments were called with paftools.js (included within minimap2), and these were filtered and concatenated using bcftools and bedtools (Quinlan and Hall, 2010). The concatenated SNP matrix was converted into a PHYLIP alignment using vcf2phylip for tree inference with FastTree (Price et al., 2010; Ortiz, 2019). Trees were visualized and annotated in R using the ggtree package (Yu et al., 2017).

All experiments were performed in biological triplicate and the data was processed and visualized using the ggplot2 and pheatmap packages in R v4.1.2¹. One-way ANOVA with Tukey’s Honest Significant Difference (HSD) post hoc test was performed on all data to ascertain significant differences between treatment means using the agricolae package in R v4.1.2. Threshold of statistical significance was set to p < 0.05 and compact letter display was used to report the results of statistically significant pairwise comparisons. Means assigned with a common letter are not significantly different by the HSD-test at the 5% level of significance.

While kveik strains are known to be strong fermenters with increased thermotolerance (Preiss et al., 2018), their fermentation efficiencies at a range of temperatures are poorly characterized. We monitored the specific gravities of small-scale wort fermentations at eight different temperatures (Supplementary Figure 1) to gain insight into the temperature-dependent fermentation characteristics of six kveik strains. Variance analyses of the specific gravity (SG) measurements at specific timepoints during these fermentations clearly distinguished the Beer 1 controls from the kveik strains especially at the higher temperatures (Figure 2). With a final gravity (FG) ≤ 1.01 as the target for ales (Parker, 2008), all the control strains displayed their most efficient attenuation at 22 and 30°C (Figure 2D). The Beer 1 control strains reached SG < 1.01 after only 3 days supporting moderate temperatures as optimal for fermentation (Figure 2C), St. Lucifer (Beer 2) never reached an FG < 1.01, regardless of temperature.

As most traditional kveik fermentations occur at ∼30°C or higher (Garshol, 2014), we anticipated their optimal fermentation efficiencies would be at higher temperatures. 5/6 kveik strains reached a SG < 1.01 within 5 days at fermentation temperatures between 22–40°C (Figure 2D). In most instances, a SG < 1.01 was already achieved within 3 days at temperatures between 30–40°C (Figure 2C), with Hornindal1 (35°C; 37°C), Hornindal2 (35°C; 37°C) and Laerdal2 (30°C) doing so in 2 days (Figure 2B). The kveiks initiated fermentation faster and notably so at higher temperatures; 5/6 kveik strains achieved ∼30% or more attenuation within 12 h at 30–40°C, while the control fermentations were still lagging (Figure 2A). These accelerated attenuation rates were sustained throughout the fermentation (Figures 2A–C) resulting in FG < 1.01 at 120 h (Figure 2D). Increasing the fermentation temperature to 42°C showed only Hornindal1, Granvin1 and Voss1 approaching FG ∼ 1.01 or lower (Figures 2D, 3). By contrast, Beer 1 control strains displayed increasingly weaker attenuation efficiencies as the temperature increased and essentially stopped fermenting after 48 h at 40 and 42°C, while St. Lucifer’s attenuation rate was only impacted at 42°C (Figures 2C,D, 3).

In addition to strong fermentation capabilities at higher temperatures, our data also demonstrates that some kveik strains possess fermentation efficiencies similar to Beer 1 control strains at lower temperatures. While most kveik strains attenuated similar to the Vermont or St. Lucifer controls at 15°C, Hornindal1 and Laerdal2 completed fermentations similar to the Kölsch and Cali controls, respectively (Figure 2D). Again, some kveik strains and in particular Hornindal1 and Hornindal2, rapidly initiated fermentation at 15°C (Figures 2A–C). In addition, Hornindal1 was the only kveik strain that completed a 12°C fermentation within 10 days along with the Beer 1 controls (Supplementary Figure 3).

Statistical analyses identified variation in the fermentation profiles between kveik strains across all timepoints and temperatures tested. For example, Hornindal1 generally fermented faster and produced beers with the lowest SG, while Laerdal2 and Ebbegarden1 fermented slower to generate beers with higher FG. Furthermore, our analyses of attenuations by individual strains identified distinct temperature-dependent fermentation efficiencies for kveik strains (Figure 3). For example, Hornindal1 shows efficient attenuation at 15–42°C, while Laerdal2 does so at 22–37°C.

We next focused on wort sugar metabolism to gain further insight into the accelerated attenuation of the kveik strains. We observed differential sugar consumption, and ethanol and glycerol production rates between strains and temperatures (Figure 4 and Supplementary Figure 2). Sugar consumption (e.g., glucose and maltose) and ethanol production was most efficient at the preferred temperatures and slower metabolism were observed at the less preferred temperatures. Kveik generally performed well in the 30–40°C range, while the Beer 1 controls did so in the 22–30°C range (Figure 4).

Glucose was rapidly consumed by all strains at their preferred temperature optima with most kveiks noticeably faster (50% after 6 h; >90% after 12 h) than most of the controls (Figures 5A,B and Supplementary Figure 2). Also, most of the kveik strains rapidly depleted most of the glucose at higher temperatures (Figure 5B), while glucose consumption was slower by all strains at cooler temperatures (Figures 5A,B).

As maltose is the main sugar in wort, we expected the faster fermentation rates of kveik would be explained by faster maltose consumption. Generally, the control and kveik strains consumed maltose faster at their respective fermentation temperature optima (Figures 5C,D). The kveik strains depleted maltose noticeably faster than the controls with most kveiks consuming at least 30% of this sugar after 12 h and >70% after 24 h of fermentation in the 30–37°C range (Figures 5C,D). However, variability exists in maltose consumption among the kveik yeasts earlier in the fermentation with Laerdal2 and Ebbegarden1 showing slower consumption rates (Supplementary Figure 2 and Figures 5C,D). Finally, all kveik yeasts depleted maltose at 40°C, while Hornindal1, Granvin1 and Voss1 also did so at 42°C (Figure 6A). While St. Lucifer could deplete maltose at 40°C, the Beer 1 controls were deficient in maltose consumption at these higher temperatures.

While kveiks are traditionally used for higher temperature fermentations, we also found most of the kveik yeasts consumed maltose similar to the controls at cooler temperatures after 120 h at 15°C (Figure 6A) and with prolonged incubation (10 days) at 12°C (Supplementary Figure 3). Hornindal1 was notably the most efficient maltose consumer at these colder temperatures (Figure 6A and Supplementary Figure 3).

Maltotriose consumption efficiency at a range of temperatures in kveik strains is not known. Here, maltotriose was consumed slower and less completely than the other wort sugars by all strains and fermentation temperature had a pronounced impact. While none of the kveik strains depleted maltotriose completely after 120 h of fermentation (Figures 6B, 7B), Hornindal1 and Hornindal2 initiated maltotriose usage the fastest and at the widest temperature range (30–42°C) (Supplementary Figure 4), and, along with Ebbegarden1 (30–40°C), consumed the most maltotriose (Figures 6B, 7B and Supplementary Figure 4). Statistical analyses confirmed kveik strains have differential abilities to consume maltotriose at their fermentation optima (30–40°C) with Laerdal2, Granvin1 and Voss1 displaying weaker maltotriose consumption (Figure 7B). At 42°C, 4/6 kveik strains consumed >50% of the maltotriose with Hornindal1 being most efficient (Figure 6B). At the colder temperatures, maltotriose consumption was slower. Again, the kveik strains showed some variation in maltotriose consumption at 12°C after a 10-day fermentation with Hornindal1 being the most efficient (Supplementary Figure 3). Thus, while the kveik strains can achieve a FG ∼1.01 after 10 days in a 12°C fermentation, most, with the exception of Hornindal1, will leave a significant amount of maltotriose at the end of the fermentation. Beer 1 control strains consumed maltotriose effectively at 22–30°C, but showed inefficient consumption outside their fermentation temperature optima (Figures 6B, 7B). By contrast, St. Lucifer used ∼25% of the available maltotriose at 22–30°C within the first 12 h and then stalled consumption at all temperatures (Figures 6B, 7B).

The kveik strains produced ethanol and glycerol faster than the controls through the first 24 h of the fermentation, with clear differences present in the 35–40°C temperature range (Supplementary Figure 5). After 120 h all the strains tested displayed similar lower levels of glycerol at the 12–30°C fermentation range, while the control strains, with the exception of St. Lucifer, produced lower levels of glycerol at temperatures beyond 37°C. By contrast, 5/6 kveik strains produced higher levels of glycerol at 35–42°C after 120 h (Figure 6D). The kveik strains produced their highest amounts of ethanol at 35–40°C (Figure 6C). Again, statistical analyses showed temperature-dependent variation in glycerol production by kveik strains. For example, Ebbegarden1 produced increasing amounts of ethanol and glycerol at 35–40°C, but less at 42°C. By contrast, Hornindal1 and Voss1 produced almost double these amounts at 42°C (Figures 6C,D, 7C,D). In combination this data support differential capabilities of the kveik strains at a wide temperature range.

The kveik strains were clearly metabolically more active while the Beer 1 controls showed significant deficiencies at higher temperatures (Figures 2, 6). As higher temperatures impact yeast cell viability (Lindquist, 1986; Davidson et al., 1996), we measured the survival of these yeasts at 35–42°C. The Beer 1 control strains were impacted at all the temperatures tested as they showed decreased cell viabilities of <75% at 37°C and above, but variable susceptibilities to higher temperatures; e.g., Kölsch showed 50% viability at 40°C, while Cali were <5% viable (Figure 8). St. Lucifer was much more temperature tolerant, only displaying significant cell death at 42°C. By contrast, all the kveik strains, except for Laerdal2, showed no noticeable cell death at 35–37°C. Interestingly, as the temperature increased further, we observed variation in the thermotolerance of kveik strains. The Hornindal1, Granvin1 and Voss1 strains showed more cell survival (>90% at 40°C and >75% at 42°C) than the remaining kveik strains. Hornindal2 still had ∼75% viability at 40°C, but this decreased substantially to only 25% at 42°C (Figure 8). Interestingly, chromosomal copy number analyses revealed the three most tolerant kveik strains had 4n + 1 aneuploidies of Chr IX (Supplementary Table 2). In combination, our data revealed differential tolerances among kveik strains to high temperatures.

Trehalose starts to accumulate as a storage carbohydrate in the diauxic phase when glucose becomes depleted as cells transition into stationary phase or is produced as a stress protectant, even in abundant glucose, to combat cold, heat and ethanol stress (Eleutherio et al., 2015). To gain further insight into the tolerance of the kveik strains to the higher fermentation temperatures, we measured trehalose accumulation as a function of time in selected kveik and control yeasts grown at optimal (30°C) and increased (37°C) temperatures. Trehalose was undetectable during the early and mid-exponential phases of growth at 30°C. When cells approached the diauxic shift and most available glucose in the media was depleted, intracellular trehalose rapidly increased (Figure 9A). Not only did the kveik strains produce >1.5-2-fold more trehalose, they also did so much faster than the control beer strains (Figures 9A,C). The kveik strains all produced >90 mg trehalose/g DCW after 22 h of growth, before the control strains produced any detectable trehalose (Figure 9A).

Yeast cells are known to hydrolyse trehalose in stationary phase (Lillie and Pringle, 1980; Parrou et al., 1999). We observed different patterns of trehalose hydrolysis and re-accumulation among the strains analyzed. The control strains displayed a significant hydrolysis of its accumulated trehalose followed by varying efficiencies to re-accumulate trehalose as the cells progressed further into stationary phase (Figures 9A,C). By contrast, the kveik strains consistently maintained high levels of trehalose throughout prolonged growth, but with some variation. While Hornindal1, Hornindal2 and Voss1 showed a reduction of its initial intracellular trehalose shortly after synthesis, trehalose re-accumulation in the Hornindal strains recovered to its original levels during prolonged growth (Figures 9A,C). Interestingly, Granvin1 showed no reduction in intracellular trehalose levels, but strikingly continued to accumulate trehalose throughout post-diauxic growth into stationary phase where it ultimately reached a maximum concentration of 148 mg trehalose/g DCW after 72 h, equivalent to nearly 15% of the dry cell weight. When yeast strains transferred from late exponential phase at 30 to 37°C for further growth, trehalose accumulation peaked within the first 6 h of heat treatment (Figures 9B,C). The Beer 1 control yeasts again accumulated trehalose to lower levels and degraded the accumulated trehalose to levels below detection (Figure 9C). However, St. Lucifer, the more heat tolerant Beer 2 control strain, showed ∼33% reduction in trehalose after 4–6 h of heat exposure, followed by a steady increase in trehalose re-accumulation (Figure 9B). By contrast, the kveik strains and Suomen Hiiva accumulated trehalose within 6 h of the heat treatment to substantially higher levels than the controls (Figures 9B,C). Furthermore, some kveik (Voss1, Granvin1 and Hornindal2) maintained high levels of trehalose, while Hornindal1 and Suomen Hiiva degraded between 40–50% of its accumulated trehalose by 48 h into the heat treatment (Figures 9B,C). In combination, these data show high levels of trehalose accumulation by kveik and the heat tolerant St. Lucifer strains after 48 h at 37°C.

Lastly, neutral trehalases are primarily responsible for intracellular trehalose degradation (Nwaka et al., 1995; Elbein et al., 2003; Magalhaes et al., 2018). The consistently higher trehalose accumulation in the kveik strains suggested impaired neutral trehalase activity. We collected samples for trehalase activity assays 2 h after the maximum trehalose spike in post-diauxic growth. The Beer 1 control strains possessed significantly higher neutral trehalase activities (Figure 10). This coincides with the rapid hydrolysis of trehalose after its initial maximum accumulation (Figure 9A). Strikingly, the kveik strains displayed much lower neutral trehalase activities, ranging from 10–18% of the activity supported by Cali (Figure 10).

Intracellular trehalose accumulation is controlled by the interplay between its biosynthesis, hydrolysis, and export (Singer and Lindquist, 1998; Eleutherio et al., 2015). All six kveik strains possess SNPs in genes known to be involved with trehalose biosynthesis (Supplementary Table 1). While trehalose biosynthetic genes TPS1 and TPS2 both carry SNPs, the regulatory genes TPS3 and TSL1 contained more variants. Specifically, TPS3 have four unique SNPs of which two are present in all strains; 2500A>G (Ser834Gly) is homozygous in all six strains, while 238G>A is homozygous in 3/6 strains (Supplementary Table 1).

Also, the high-affinity trehalose H⁺-symporter Agt1 is important in combatting environmental stress (Da Costa Morato Nery et al., 2008; Ratnakumar et al., 2011) as it is proposed to export intracellularly accumulated trehalose to protect the plasma membrane (Eleutherio et al., 1995; Magalhaes et al., 2018). Our SNP analyses of AGT1 identified two additional heterozygous frameshift mutations (1175_1176insT in Ebbegarden1 and Laerdal2, and 1772delA in Voss1 and Hornindal2), and gained stop codon mutations [491AAG>TTA (Leu164*) again in Laerdal2 and Ebbegarden1, and 1236C>G (Tyr412*) heterozygous in Hornindal2] (Supplementary Table 1), suggesting heterozygous loss-of-function of AGT1 in 4/6 kveik strains.

We screened the three yeast trehalases for potential mutations and identified several SNPs in all three genes (Supplementary Table 1). NTH1 possesses several missense mutations, some homozygous, near the catalytic site in Nth1. In particular, 1213C>T (Leu405Phe) is homozygous in 4/6 kveik strains. NTH2 carries three unique homozygous SNPs (Supplementary Table 1) in Granvin1, the strain that continuously accumulated trehalose in our assay (Figure 9). Lastly, ATH1 carries seven unique SNPs, of which three are present in 3/6 or more kveik strains (Supplementary Table 1). These latter three mutations (3394A>G, Asn1132Asp; 3434A>G, Ser1145Asn; 3596A>G; Asn1199Ser) are either homozygous or present in high copy number in Granvin1. In combination, these observations suggest increased trehalose biosynthesis, impaired trehalose export, along with deficient trehalase could lead to the rapid accumulation and maintenance of high levels of intracellular trehalose in kveik.