Brewing Beer in Microgravity: The Effect on Rate, Yeast, and Volatile Compounds

: The exploration of space is becoming more feasible, and with this comes the possibility of performing fermentations in microgravity. Our study explores the potential effects of microgravity on a standard brewing model fermentation. As the fermentation of barley wort has been studied for centuries, there is an established foundation of knowledge with which to compare any changes that occur under microgravity. A modified ASBC miniature fermentation protocol (Yeast-14) was conducted within a Yuri 2.0 microgravity chamber to examine the response of Saccharomyces pasto-rianus to simulated microgravity conditions. Our findings reveal that yeast exhibited accelerated growth rates under microgravity compared to standard conditions. Additionally, the microgravity environment led to significantly lower levels of volatile compounds such as isoamyl acetate and 2-phenethyl acetate in the final product. Further genetic analysis showed significant downregulation of the ATF2 gene in the control group, potentially the mechanism behind the observed decrease in volatile compounds. These results show that while fermentation under microgravity is likely to eventually be commonplace, there may be changes in rate and gene expression that are beneficial or detrimental to the final product depending upon the desired characteristics.


Introduction
The pursuit of space exploration has captivated the imagination of humankind for decades.One aspect of this endeavor will be eventually conducting fermentations in microgravity [1,2].While the concept may seem trivial, there are several mechanisms that have been shown to affect the ability of microorganisms to perform specific functions under microgravity.This paper aims to compare a control brewing fermentation to one conducted within a clinostat that simulated microgravity.The goal was to better understand the differences that occur during fermentation under microgravity and in the final product.Fermentation with Saccharomyces is used to produce various products in many industries, including the food, bio-fuel [3], pharmaceutical [4], chemical [5], and cosmetic [6] sectors.This is a continually evolving field where any manipulation of microorganism behavior or increase in efficiency can be exploited for numerous applications.The metabolism of yeast and bacteria is highly dependent upon physical and chemical environmental parameters, many of which are influenced by gravity [1].
Lager beer is the most consumed alcoholic beverage in the world; it makes up 94% of the world beer market [7,8] and is made using Saccharomyces pastorianus-an interspecific hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus [9].While this style is typically produced at low temperatures (~10-16 • C) to reduce the concentration of secondary metabolites, the optimum temperature for Saccharomyces pastorianus growth is 28-30 • C. Additionally, researchers are examining the potential for using lager strains at higher temperatures [10] for industrial purposes.The temperature and selection of yeast Beverages 2024, 10, 47 2 of 13 strains for brewing influence the rate of fermentation, the extent of fermentation, the flavor characteristics, and the overall quality of the finished beer [11].
While studies examining lager brewing under microgravity are relatively uncommon, the physical characteristics of Saccharomyces cerevisiae have been assessed over short (5-10 generations) and long-term microgravity experiments.During short-term microgravity studies, Saccharomyces cerevisiae was shown to exhibit a shortened lag phase and random budding patterns compared to the control group [12].The expression levels of genes associated with polarity, bipolar budding, and cell separation were also shown to be significantly altered.These findings suggest that low-shear environments, such as microgravity, can impact yeast gene expression and phenotype.Another study found that cytoskeleton alterations and activation of ion channels were possibly the mechanisms by which microgravity affected yeast [13].This was observed to result in changes to cell volume and metabolism, as well as the activation of specific cellular pathways.Another study used high-throughput image-processing techniques to analyze the morphological traits of yeast cells cultured in simulated microgravity [14].The study confirmed significant changes in bud direction, cell size ratio, daughter cell shape, mother cell size, and nucleus characteristics.The study also identified downregulated genes that contributed to these morphological changes.With respect to long-term (10+ generations) changes, a study on Saccharomyces cerevisiae in low-shear modeled microgravity conditions also found that yeast cells exhibited random budding compared to the usual bipolar pattern observed under normal gravity conditions [12].The study also identified changes in the expression of genes associated with cell polarity, bipolar budding, and cell separation.For behavior, a study on yeast colonies of Saccharomyces pastorianus found that physical factors such as colony rotation and microgravity can modulate ammonia convection and shear stress, leading to differences in cellular redox responses and potentially apoptosis (cell death) [15].
Most of the aforementioned studies that looked at fermentation under microgravity have focused on the physical characteristics of yeast.However, with respect to flavor, a large influence yeast can have on the final product is the production of volatile secondary metabolites such as organic acids, higher alcohols, aldehydes, esters, and ketones [16].In addition to yeast behaviors, this study also assessed the changes to the final product through the examination of volatile compounds.The formation of these compounds is in part governed by the alcohol acetyltransferase (ATF) family of genes, which includes ATF1, its counterpart Lg_ATF1, and ATF2 [17].These genes have been extensively studied for their involvement in the production of volatile esters during beer and wine fermentation.The regulation of these genes appears to also be intricately linked to growth rate [18].
Clinostats are devices used to emulate microgravity conditions in laboratory settings.They operate on the principle of continuous rotation, which constantly changes the orientation of cells or samples with respect to gravity [19].This rotation creates a vector-average reduction in the apparent gravity acting on the cells, simulating the effects of microgravity [19].One of the most unique attributes of bioprocessing in space may lie in the ability to keep cells suspended in the fluid medium without imparting the significant shear forces that often accompany agitation (stirred) terrestrial systems.As space-flight opportunities are infrequent (and expensive), ground-based methods that partially simulate the low-shear environment encountered in actual microgravity are useful in further exploring how cells respond to altered inertial conditions.However, there are also some limitations associated with clinostats as they do not fully replicate the lack of fluid convection experienced in microgravity.In space, the absence of gravity-driven fluid flow can impact cellular processes and interactions.Clinostats cannot fully mimic this aspect of microgravity, which may limit their ability to predict some space-related phenomena [20].
Agitated brewing is not normally performed in industrial operations [21] as CO 2 currents are generally sufficient to maintain sufficient cells in suspension [22].Boswell et al. [23] and others observed that agitation after a threshold of ~0.03 kW/m 3 increased brewing fermentation rates up to a point.Rollero et al. [21] found that nitrogen consumption increased with agitation, also after an agitation threshold (reported as 40 rpm).It was noted that the possible mechanisms of this observation were the increased number of yeast cells in suspension as well as the increased availability of nutrients.Microgravity is hypothesized to mimic the effects of low agitation as suspended cells would not settle during fermentation (similar to low levels of agitation), but without the benefit of reduced nutrient gradients found at higher levels of agitation.
The specific aim of this study was to assess how microgravity affected the fermentation characteristics, volatile production, and genetic expression of yeast during lager fermentation.In contrast to similar studies that focus on the physical characteristics of yeast, this study also examined fermentation kinetics and the properties of the final beverage.To achieve these aims, wort was fermented utilizing a modified version of the ASBC miniature fermentation method (Yeast 14) adjusted to be performed in a microgravity clinostat.Density, number of yeast cells in suspension, and yeast viability were assessed, and volatile analyses and genotyping were performed over the microgravity fermentation and compared to a control group fermented from the same inoculated wort under normal Earth gravity and otherwise identical environmental conditions.

Preparation of Wort
Florida-grown "Copeland" 2-row was mashed following the ASBC standard regime [24] (Malt-4) and used to make 1000 mL of wort.A densitometer (Anton Paar, Graz, Austria) was used to assess the density of the extracted wort.The density was then diluted to 8 • Plato using sterile deionized water and supplemented with a 99.5% dextrose solution to a concentration of 12 Plato as per ASBC Yeast-4 [25].

Yeast Propagation
The yeast used for all the experiments was Diamond lager yeast (Saccharomyces pastorianus) from Lallemand (Montreal, QC, Canada).Yeast was inoculated into a 50 mL volume of YEPD medium and incubated at 30 • C in a water bath for 24 h to facilitate growth.Subsequently, the yeast culture was aliquoted into 50 mL tubes and centrifuged at 3000 RPM for 3 min to separate the yeast cells from the medium.The resulting yeast pellet underwent a washing step with deionized (DI) water to eliminate residual medium and contaminants.Following washing, the yeast cells were resuspended in 100 mL of fresh YEPD medium at a concentration of ~15 × 10 6 cells/mL and subjected to an additional 24 h incubation at 30 • C.After this incubation period, the yeast culture underwent another round of centrifugation and washing with deionized water to obtain a yeast slurry.A hemocytometer was then utilized to enumerate the yeast cells.The wort was then collectively inoculated at a rate of ~15× 10 6 cells/mL prior to separation for each treatment (control and microgravity).

Miniature Fermentation Assay
The inoculated wort was divided into 6 identical samples, for both the control and microgravity fermentation to be run in triplicate.The fermentation assays followed the ASBC Yeast-14 miniature fermentation method [25] with some adjustments to prevent spills or contamination during agitation in the microgravity chamber (YURI RPM 2.0).These adjustments include using sterile hydrophobic mesh caps (AMTAST) with silicone gaskets to seal the fermentors as opposed to foam bungs.This facilitated gas release while retaining the liquid from each fermentor.This change was also applied to the control.For each fermentation (3 control, 3 microgravity), inoculated wort was subdivided into 10 fermentors which were fermented at standard ambient temperature and pressure and destructively sampled at each time point (as per Yeast-14 [25]).Samples were taken from the top portion of sampled fermentors at 0, 3, 15, 18, 21 24, 39, 42, 45, 48, and 72 h, and were monitored for density, yeast counts, and yeast viability.
Considering the limited space within the microgravity testing environment (15 cm × 15 cm × 15 cm cube), a 3D-modeled fermentor rack, Figure 1, was designed and printed.This rack ensured each fermentation tube was secured.The lid of the rack was made to have holes on the top to allow the samples to release carbon dioxide during fermentation.
monitored for density, yeast counts, and yeast viability.
Considering the limited space within the microgravity testing environment (15 cm × 15 cm × 15 cm cube), a 3D-modeled fermentor rack, Figure 1, was designed and printed.This rack ensured each fermentation tube was secured.The lid of the rack was made to have holes on the top to allow the samples to release carbon dioxide during fermentation.
The control fermentation was conducted next to the microgravity machine to negate differences in temperature (ambient 22-25 °C) and other environmental conditions between the two groups.

Clinostats
The 3D-printed fermentation tube receptacle was placed within the YURI 2.0 clinostat.The clinostat ran in random modes of speed and direction to achieve a microgravity environment of 0-0.01 g

Density
At each time point, the density from each fermentation (3 microgravity, 3 control) was measured using ASBC Beer 2 methods for density measurement of beer [26].The samples were run through a Whatman 4 filter to degas prior to measurement with a densitometer (Anton Paar, Graz, Austria).

Yeast in Suspension
At each sampling time, the clinostat was briefly stopped, and a 1 mL sample was taken from the top of the test tube immediately to prevent settling or agitation.Yeast in suspension was enumerated using the ASBC Yeast-4 [27] method for cell counting.

Total Yeast and Viability
The entire contents of each 15 mL fermentor tube was subsequently vortexed to resuspend any flocculated yeast.The sample underwent centrifugation at 3000 RPM for 3 min with the supernatant being preserved for subsequent analysis.The pellet obtained from centrifugation was resuspended using 1.5 mL of a 20% glycerol solution to maintain the viability of the yeast cells.
To ensure accurate measurements, a 10 × dilution was prepared by using 0.1 mL of the resuspended pellet, while the remaining solution was stored for further analysis.To assess the viability and total count of yeast cells, the diluted solution was further mixed with 0.9 mL of methylene blue.Enumeration of the yeast cells and viability was conducted using a hemocytometer.The total cell count was determined using Equation (1), while the viability was calculated using Equation (2).The control fermentation was conducted next to the microgravity machine to negate differences in temperature (ambient 22-25 • C) and other environmental conditions between the two groups.

Clinostats
The 3D-printed fermentation tube receptacle was placed within the YURI 2.0 clinostat.The clinostat ran in random modes of speed and direction to achieve a microgravity environment of 0-0.01 g

Density
At each time point, the density from each fermentation (3 microgravity, 3 control) was measured using ASBC Beer 2 methods for density measurement of beer [26].The samples were run through a Whatman 4 filter to degas prior to measurement with a densitometer (Anton Paar, Graz, Austria).

Yeast in Suspension
At each sampling time, the clinostat was briefly stopped, and a 1 mL sample was taken from the top of the test tube immediately to prevent settling or agitation.Yeast in suspension was enumerated using the ASBC Yeast-4 [27] method for cell counting.

Total Yeast and Viability
The entire contents of each 15 mL fermentor tube was subsequently vortexed to resuspend any flocculated yeast.The sample underwent centrifugation at 3000 RPM for 3 min with the supernatant being preserved for subsequent analysis.The pellet obtained from centrifugation was resuspended using 1.5 mL of a 20% glycerol solution to maintain the viability of the yeast cells.
To ensure accurate measurements, a 10 × dilution was prepared by using 0.1 mL of the resuspended pellet, while the remaining solution was stored for further analysis.To assess the viability and total count of yeast cells, the diluted solution was further mixed with 0.9 mL of methylene blue.Enumeration of the yeast cells and viability was conducted using a hemocytometer.The total cell count was determined using Equation (1), while the viability was calculated using Equation (2).
where the number of cells per square is averaged (A) and (B) is the dilution factor.

Viability = ((
where the nonviable cell count (B) was subtracted from the total count (A) and subsequently divided by it to obtain a percentage of live cells.

Volatile Extraction
Extraction and concentration of the volatile and semi-volatile compounds for the experimental and control wort and beer samples were performed using solid-phase microextraction (SPME).A 50/30 µm thick Divinylbenzene/Carboxen/Polydimethylsiolxane (DVB/Carboxen/PDMS) fiber (Supelco, Inc., Bellefonte, PA, USA) was exposed to the headspace above 10 mL of sample beer spiked with 25 µL 2-octanol (20 mg/L) used as the internal standard and 30% w/v of salt in 20 mL headspace vials with Teflon-lined silicone septa (Chromacol, Fisher Scientific, Waltham, MA, USA) for 30 min at 40 • C with an agitation speed of 250 RPM.Samples were equilibrated at 40 • C for two minutes prior to fiber exposure.

Gas Chromatography (GC-MS)
Volatile compounds were desorbed for five minutes in the injection port of a Shimadzu gas chromatograph (GC) 2010 Plus Series coupled with a QP2010 SE mass spectrometer detector (MSD) (Shimadzu, Tampa, FL, USA).The injection port was set to 250 • C, and all injections were performed in spitless mode using a narrow bore, deactivated glass insert.Volatile compounds were separated using a nonpolar ZB-5MS (ZB; 30 m × 0.25 mm id × 0.25 µm film thickness) with helium (He 2 ) used as the carrier gas at a flow rate of 2.0 mL/min (linear velocity 53.8 cm/sec).The GC oven temperature program had an initial temperature of 35 • C which was held for 5 min and then increased to 225 • C at a rate of 6 • C/min.Once the final temperature of 225 • C was reached, it was maintained for 10 min.The ion source was maintained at 200 • C, while the transfer line was set to 250 • C. Sample masses were scanned in the range of 40-800 m/z.Peaks were identified using standardized retention time (retention index (RI) values), pure compounds, and fragmentation spectra of standards and the Wiley 2014 mass spectral library [28].

Volatile Identification
Volatile compounds were identified based on their retention index (RI) values using nonpolar (ZB-5MS) columns (30 m × 0.25 mm i.d., 0.25 µm film; J&W, Folsom, CA, USA).The RI values were compared to literature values.Aliphatic hydrocarbon standards were analyzed in the same manner using a ZB-5MS column to calculate RI as per Equation (3): where N is the carbon number of the lowest alkane and n is the difference between the carbon numbers of the two n-alkanes that are bracketed between the compound; t Rn , t RN , and t R(N+n) are the retention times of the unknown compound, the lower alkane, and the upper alkane [28] 2.11.Free Amino Nitrogen (FAN) The free amino nitrogen (FAN) was measured using the NOPA method from the UC Davis cooperative extension [29].

RNA Extraction and Gene Expression Analysis
Total RNA was extracted from yeast after fermentation using the Monarch Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions.The concentrations of the isolated total RNA samples were quantified using a DS-11 Series RNA Spectrophotometer (DeNovix Inc., Wilmington, DE, USA).RNA samples were stored at −80 • C until they were used for reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis.
Three target genes (ATF1, ATF2, and Lg-ATF1) of Saccharomyces pastorianus were selected for gene expression analysis related to volatile ester and alcohol synthesis [30].
Sequences of genes ATF1 and ATF2 were obtained from the Saccharomyces Genome Database (https://www.yeastgenome.org),while the sequence for Lg-ATF1 was retrieved from the nucleotide database at the National Center for Biotechnology Information (https:// www.ncbi.nlm.nih.gov).Primers for the target genes were designed using the online PrimerQuest™ Tool (Integrated DNA Technologies, Coralville, IA, USA) and purchased from Integrated DNA Technologies.The primers used in this study are listed in Table S1.
RT-qPCR analysis was performed with the Luna ® Universal One-Step RT-qPCR Kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer's protocol.Reaction mixtures were prepared as detailed in Table S2.Gene expression levels were quantified based on SYBR Green fluorescence using the PikoReal 24 real-time system (ThermoFisher Scientific, Waltham, MA, USA) with the cycling parameters provided in Table S3.The gene expression levels were reported as quantitative cycle (Cq) values.The Cq values were analyzed using an ANOVA test to assess whether there are statistically significant differences in the expression levels of the target genes under microgravity conditions compared to the control group.

Statistics
Mean separation was completed using ANOVA comparison with Tukey's HSD test (p ≤ 0.05) to assess significant differences between treatments.Fermentability curves were compared using Prism Version 9.4.1 (Graphpad, San Diego, CA, USA).Individual statistical comparisons were completed using a one-way analysis of variance (ANOVA) with α = 0.05.Samples were analyzed in triplicate (n = 3).

Yeast Growth Kinematics
The triplicate fermentations were sampled periodically over the 3-day run for density, number of suspended yeast cells, and cell viability.These data are presented in Figure 2. The fermentation midpoint, defined as the time when 50% of the initial sugars have been consumed [31], indicated a higher fermentation rate under microgravity.The microgravity fermentations reached their midpoint at 21.2 h compared to 28.2 h for the control, representing a 33% increase in fermentation rate (Figure 2a).
As shown in Figure 2b, the number of yeast cells in suspension immediately began to diverge.This was expected as the initial number of yeast cells in suspension in most fermentations typically falls both at the beginning and end of fermentation as yeast settles due to gravity [32].During the most active portion of standard fermentations, evolving CO 2 maintains circulating currents which keep all but the most flocculent yeast suspended.It was hypothesized that under microgravity, the lack of gravitational pull would prevent cells from settling, thus maintaining a higher number of yeast cells in suspension than in the control (as was observed).
During the fermentations, neither the viability (Figure 2c) nor the total number of yeast cells (Figure 2d) was significantly different between treatments.These findings confirm that Saccharomyces pastorianus was not adversely affected by microgravity.Instead, the fermentation rate increased, likely due at least in part to the constant suspension of yeast cells, which maximizes nutrient availability by preventing settling.In contrast with agitated fermentations where the constant agitation tends to have negative effects on cell viability [23], a loss of viability (and possible activation of stress responses) was not observed.yeast cells, which maximizes nutrient availability by preventing settling.In contrast with agitated fermentations where the constant agitation tends to have negative effects on cell viability [23], a loss of viability (and possible activation of stress responses) was not observed.

Free Amino Nitrogen and Volatile Compound Analysis
Free amino nitrogen utilization has often been linked to higher fermentation rates during beer and winemaking fermentations.Differences have been observed in agitated fermentations at high rates [21], but not at low agitation rates.The initial FAN concentration was over 100 mg/L, which has been shown to be above the minimum concentration that is expected to result in a satisfactory fermentation [33,34].As shown in Table 1, there was no statistical (p < 0.05) difference observed in the FAN levels between the microgravity and the standard fermentation.Our results are comparable to those found by Rollero et al. [21] in that no differences were observed between low-agitation fermentations and controls.There were no statistical (p < 0.05) differences observed between the different volatile compound groups except for esters (Table 2), which typically have a very low threshold in beer [35].The ratio of the higher alcohol group to esters varies in lager beers from 3-4:1 [36]; according to Cui et al. [36], "A higher ratio results in a dry taste, and a less aromatic characteristic of the beer.".The control beer had a ratio of 1.4:1 while the microgravity beer had a ratio of 4.6:1, indicating that the microgravity beer was less aromatic by this measure.Two esters that showed significant differences between treatments were isoamyl acetate and 2-phenethyl acetate.Both of these compounds were above the minimum threshold for detection, with isoamyl acetate having a threshold level of 1.2-2 mg/L and 2-phenethyl acetate having a threshold of 0.2-3.8mg/L in lager beer [37].Our results showed that these two compounds had a multiple-fold decrease in concentration for the microgravity fermentation compared to the standard fermentation.An increase in these compounds would likely result in a "banana" or "fruity" flavor in the final product.High levels of isoamyl acetate have been identified as having an impact on the final aromatic profile of beer [35,37,38].Similar to ethyl acetate, isoamyl acetate has been described as being 'solvent-like', 'fruity', or 'sweet' [39].If esters are found in excess, they can give beer a fruity characteristic that is undesirable to most consumers [40].These compounds are often observed in higher-temperature fermentations (as was conducted in this study).Depending upon the brewery, these compounds may be desirable; however, the presence of these compounds above a detection threshold would usually be considered a defect.In this study, the microgravity fermentation resulted in a final product that would be considered higher quality due to the reduced esters.Once again, these results are comparable to fermentations conducted under mild agitation [21].This implies that the differences in volatile compounds observed may be linked to the physical phenomenon of maintaining yeast in suspension.As ester production in yeast is controlled through a small number of genes, the gene expression of yeast from each treatment was examined to determine if this could be the mechanism behind the observed differences in ester production.

Gene Expression
To determine if the differences in volatile compounds were related to physical or genetic differences, RT-qPCR was employed to determine the Cq values for three target genes (ATF1, ATF2, and Lg-ATF1) responsible for regulating the alcohol and esters in question.The Cq value represents the cycle number at which the fluorescence generated within a reaction crosses the threshold, signifying the point at which the amount of amplified target reaches a detectable level [41].The Cq value serves as an indicator of gene expression levels.A high Cq value represents a low expression level [42,43].Figure 3 displays the boxplot of Cq values of ATF1, ATF2, and Lg-ATF1 between microgravity and control groups.In the control group, the mean Cq values of ATF1, ATF2, and Lg-ATF1 were 25.66, 21.90, and 21.99, with standard deviations of 0.22, 0.86, and 0.27, respectively.In the microgravity group, ATF1, ATF2, and Lg-ATF1 yielded mean Cq values of 25.51, 22.69, and 22.21 with standard deviations of 0.62, 0.68, and 0.17.These results indicate that all three genes (ATF1, ATF2, and Lg-ATF1) could be expressed in Saccharomyces pastorianus under both microgravity and control groups, which are linked to the results in Table 2, where alcohol and esters were detected in these two groups.Under both control and microgravity conditions, ATF1 exhibited the lowest expressed levels, with higher mean Cq values, compared to ATF2 and Lg-ATF1.This difference in transcriptional regulation between the ATF1 genes (ATF1 and Lg-ATF1) and the ATF2 gene is one of the most distinctive features of the ATF gene family [44].
within a reaction crosses the threshold, signifying the point at which the amount of amplified target reaches a detectable level [41].The Cq value serves as an indicator of gene expression levels.A high Cq value represents a low expression level [42,43].Figure 3 displays the boxplot of Cq values of ATF1, ATF2, and Lg-ATF1 between microgravity and control groups.In the control group, the mean Cq values of ATF1, ATF2, and Lg-ATF1 were 25.66, 21.90, and 21.99, with standard deviations of 0.22, 0.86, and 0.27, respectively.In the microgravity group, ATF1, ATF2, and Lg-ATF1 yielded mean Cq values of 25.51, 22.69, and 22.21 with standard deviations of 0.62, 0.68, and 0.17.These results indicate that all three genes (ATF1, ATF2, and Lg-ATF1) could be expressed in Saccharomyces pastorianus under both microgravity and control groups, which are linked to the results in Table 2, where alcohol and esters were detected in these two groups.Under both control and microgravity conditions, ATF1 exhibited the lowest expressed levels, with higher mean Cq values, compared to ATF2 and Lg-ATF1.This difference in transcriptional regulation between the ATF1 genes (ATF1 and Lg-ATF1) and the ATF2 gene is one of the most distinctive features of the ATF gene family [44].Under microgravity conditions, no significant difference in the expression levels of ATF1 and Lg-ATF1 was observed compared to the control group.However, the gene ATF2 was significantly downregulated under microgravity conditions compared to the control.These findings illustrate that microgravity conditions exerted varying impacts on gene expression, contingent upon the gene type.Genes ATF1 and ATF2, encoding alcohol acetyltransferase I and II enzymes, are responsible for diverse ester synthesis, including isoamyl acetate and 2-phenethyl acetate [37,45,46].According to the abovementioned concentrations of esters listed in Table 2, the total esters, isoamyl acetate, and 2-phenethyl acetate levels in microgravity-brewed beer were significantly reduced compared to the control, likely due to the downregulation of ATF2 expression.As indicated in Table 2, the concentrations of isoamyl alcohol and phenethyl alcohol were higher in microgravitybrewed beer than in the standard beer.This observation could also be attributed to ATF2 downregulation.Isoamyl acetate and phenethyl acetate are produced, respectively, through the reactions between isoamyl alcohol and activated acyl-coenzyme A, and between phenethyl alcohol and activated acyl-coenzyme A, in which ATF2 plays a role [30,37].The reduced expression levels of ATF2 expression level in microgravity-brewed beer might hinder these reactions, leading to the accumulation of isoamyl alcohol and Under microgravity conditions, no significant difference in the expression levels of ATF1 and Lg-ATF1 was observed compared to the control group.However, the gene ATF2 was significantly downregulated under microgravity conditions compared to the control.These findings illustrate that microgravity conditions exerted varying impacts on gene expression, contingent upon the gene type.Genes ATF1 and ATF2, encoding alcohol acetyltransferase I and II enzymes, are responsible for diverse ester synthesis, including isoamyl acetate and 2-phenethyl acetate [37,45,46].According to the abovementioned concentrations of esters listed in Table 2, the total esters, isoamyl acetate, and 2-phenethyl acetate levels in microgravity-brewed beer were significantly reduced compared to the control, likely due to the downregulation of ATF2 expression.As indicated in Table 2, the concentrations of isoamyl alcohol and phenethyl alcohol were higher in microgravity-brewed beer than in the standard beer.This observation could also be attributed to ATF2 downregulation.Isoamyl acetate and phenethyl acetate are produced, respectively, through the reactions between isoamyl alcohol and activated acyl-coenzyme A, and between phenethyl alcohol and activated acyl-coenzyme A, in which ATF2 plays a role [30,37].The reduced expression levels of ATF2 expression level in microgravity-brewed beer might hinder these reactions, leading to the accumulation of isoamyl alcohol and phenethyl alcohol.These results suggest that microgravity had a potential impact on secondary metabolite production.This difference could yield positive or negative outcomes depending upon the industry application for fermentation under microgravity.
When compared to standard fermentations, microgravity has been shown to affect yeast behavior and secondary metabolite production.This behavior is likely linked to physical phenomena such as continual suspension given how closely the results mimic lightly agitated fermentations.These results show promise for the future application of fermentation under variable gravity conditions and provide data for a better understanding of the complex behaviors of yeast in terrestrial industries.

Figure 1 .
Figure 1.Three-dimensional model of the fermentation tube receptacle placed within the YURI 2.0 clinostat.

Figure 1 .
Figure 1.Three-dimensional model of the fermentation tube receptacle placed within the YURI 2.0 clinostat.

Figure 2 .
Figure 2. Fermentation kinetics and yeast characteristics throughout the fermentation process: (a) density readings in °Plato, and modeled alcohol; (b) concentration of cells in suspension duringFigure 2. Fermentation kinetics and yeast characteristics throughout the fermentation process: (a) density readings in • Plato, and modeled alcohol; (b) concentration of cells in suspension during fermentation, (c) yeast cell viability during fermentation, (d) total yeast concentration in the fermentation tube (including settled cells).

Figure 2 .
Figure 2. Fermentation kinetics and yeast characteristics throughout the fermentation process: (a) density readings in °Plato, and modeled alcohol; (b) concentration of cells in suspension duringFigure 2. Fermentation kinetics and yeast characteristics throughout the fermentation process: (a) density readings in • Plato, and modeled alcohol; (b) concentration of cells in suspension during fermentation, (c) yeast cell viability during fermentation, (d) total yeast concentration in the fermentation tube (including settled cells).

Figure 3 .
Figure 3. Boxplot of Cq values of ATF1, ATF2, and Lg-ATF1 between microgravity and control groups.White circles represent the mean values of Cq.Symbol * represents the significant difference.

Figure 3 .
Figure 3. Boxplot of Cq values of ATF1, ATF2, and Lg-ATF1 between microgravity and control groups.White circles represent the mean values of Cq.Symbol * represents the significant difference.

Table 1 .
Free amino nitrogen (FAN) levels in the wort and finished beer over the control and microgravity fermentations.

Table 2 .
Volatile and semi-volatile concentrations of samples using GC-MS.Concentrations of each functional group are bolded.