The Price of Survival: Yaks’ Adaptation to High Altitudes

Effect of individual and mixed live yeast culture feeding on growth performance, nutrient utilization and microbial crude protein synthesis in lambs

Peanut crop residues such as peanut vine are widely used to feed small ruminants as roughage in Asia, where the population of sheep and goat takes a large proportion in the world. Compared to peanut vine, straw is considered as a less nutritious but promising roughage source due to high availability and low price. Extrusion is a process to improve the palatability and digestibility of the feed. Here, we investigated the effects of replacing peanut vine with extruded rape straw on growth performance, apparent nutrient digestibility, energy metabolism, microbial crude protein (MCP) synthesis, and amino acid and fatty acid profiles in the longissimus thoracic (LT) of finishing lambs. Twenty-four 2-mo-old male Hu lambs of similar body weight (19.5 ± 1.0 kg) were fed two diets containing either peanut vine (CON, n = 12) or extruded rape straw (TRT, n = 12) as the only roughage source. The dietary concentrate to forage ratio was 70:30. Lambs were fed twice daily and had ad libitum access to feed and clean water. The experiment lasted 120 d, including a 30-d adaptation period. Lambs were weighed every 30 d. Digestibility trial was conducted during the 81 to 90 d of the experiment. At the end of the experiments, lambs were slaughtered for ruminal fluid and meat sample collection. Data were analyzed using mixed model with repeated measures, Student's t-test, or Wilcoxon signed-rank test. No difference in growth performance, apparent digestibility of nutrients, or energy utilization efficiency was observed, except for the apparent digestibility of ether extract, which was greater (P = 0.008) for lambs in the TRT group. In addition, the MCP estimated using urinary purine derivatives (P = 0.072) tended to be greater in the lambs of the TRT group. The concentration of most amino acids and fatty acids was not different in the LT sample of two groups of lambs, while the concentration of Met (P = 0.044) was greater, and that of myristoleic acid (C14:1) (P = 0.010), heptadecenoic acid (C17:1) (P < 0.001), trans-linoleic acid (C18:2n6t) (P = 0.003) and gamma-linolenic acid (C18:3n6) (P < 0.001) were lower in the LT sample of lambs from TRT group. In conclusion, extruded rape straw can effectively replace peanut vine in the diet of lambs without compromising health or nutrient utilization.

The objectives of this study were to evaluate changes in profiles of odd- and branched-chain fatty acids (OBCFA), including pentadecanoic acid (C15:0), 13-methyltetradecanoic acid (iso-C15:0), 12-methyltetradecanoic acid (anteiso-C15:0), 14-methylpentadecanoic acid (iso-C16:0), heptadecanoic acid (C17:0), 15-methylhexadecanoic acid (iso-C17:0), and 14-methylhexadecanoic acid (anteiso-C17:0) during in vitro fermentation of pure carbohydrates mixtures in the buffer-rumen fluid. The second objective was to correlate the changes in the OBCFA profile to the corresponding changes in ruminal fermentation parameters, microbial crude protein (MCP) synthesis, and bacterial populations. Five pure carbohydrates mixtures containing different cellulose: starch (C:S) ratios, i.e., 0:100, 25:75, 50:50, 75:25, and 100:0, were incubated for 6, 12, 18, and 24 h in vitro. The results showed that there was significant interaction (P < 0.05) between C:S and incubation time for changes in all OBCFA profiles, except iso-C17:0. The highest concentration of total OBCFA (3.94 mg/g dry matter; DM) was observed in the residues after 24 h of fermentation when the C:S was 0:100, while the lowest concentration of OBCFA (1.65 mg/g DM) was produced after 6 h of incubation when the C:S was 50:50. The correlation analysis revealed that the concentration of iso-C16:0 might be a potential marker for the estimation of total volatile fatty acids (ρ = 0.78) and MCP synthesis (ρ = 0.82) in the rumen. Compared to starch degrading bacteria, cellulolytic bacteria had stronger correlations with OBCFA concentrations, and the strongest correlation was found between the population of Ruminococcus flavefaciens with C15:0 concentration (ρ = 0.70). Notably, this is the first paper reporting relationship between OBCFA with rumen fermentation products and microbial protein synthesis based on fermentation of pure carbohydrates mixtures in vitro, and thus avoid confounding interference from dietary protein and fat presence in the in vivo studies. However, more in-depth experiments are needed to substantiate the current findings.

Castillo-Lopez, E., Klopfenstein, T. J., Fernando, S. C. and Kononoff, P. J. 2014. Effect of dried distillers’ grains and solubles when replacing corn or soybean meal on rumen microbial growth in vitro as measured using DNA as a microbial marker. Can. J. Anim. Sci. 94: 349–356. The objectives were to evaluate the use of rDNA markers to measure the effects of dried distillers’ grains with solubles (DDGS) and the potential treatment×time interaction on microbial crude protein (MCP) synthesis in vitro and secondly to measure the contribution of yeast based protein originating from DDGS. Treatments were: (1) CONT, control with no DDGS, but with alfalfa hay, corn silage, ground corn (GC) and soybean meal (SBM) included at 25% (DM basis); (2) LOWCORN, 20% DDGS (DM basis) replacing GC; (3) LOWSBM, 20% DDGS (DM basis) replacing SBM; and (4) LOWCORNSBM, 20% DDGS (DM basis) replacing 10% GC and 10% SBM. Treatments (0.5 g) were incubated in 50 mL of inoculum in duplicate. At 0, 4, 16, 32, 48 and 96 h of fermentation total DNA was extracted from each treatment and MCP was measured using rDNA markers. The sum of bacterial crude protein (BCP) and protozoal crude protein (PCP) was considered as MCP. Data were analyzed as a completely randomized design. The treatment×time interaction was tested and the SLICE option was included to evaluate the effect of treatment at each fermentation time point. There was a tendency to a treatment×time interaction (P=0.07) for MCP. Specifically, at 16 h, LOWCORNSBM yielded greater (P&lt;0.05) MCP compared to either CONT or LOWCORN with estimates of 68.5, 33.8 and 23.3±8.9 mg g–1DM, for LOWCORNSBM, CONT and LOWCORN, respectively. At 48 h, however, LOWCORN yielded greater MCP (P&lt;0.05) compared with LOWSBM with estimates of 72.2 and 32.5±8.9 mg g–1DM, for LOWCORN and LOWSBM, respectively. Yeast crude protein (YCP) was not affected (P=0.21) and averaged 0.04±0.02 mg g–1of substrate (DM basis). Overall, rDNA markers were effective for quantifying MCP, but further research on the methodology is needed. With DDGS inclusion, MCP was maintained; however, yeast cells were extensively degraded during fermentation.

Patricia Louvandini1,2*, Brenda L. A. Silva.1, João P. Duarte, Livia M. Presuto1, Henrique Guedes1, Adibe L. Abdalla 1,2, Helder Louvandini1,2 1 University of São Paulo, Center for Nuclear Energy in Agriculture, Laboratory of Animal Nutrition, Piracicaba, SP, Brazil.2 University of São Paulo, Piracicaba, São Paulo, Brazil - Center for Carbon Research in Tropical Agriculture (CCARBON) This study was conducted to evaluate the influence of Zinc Oxid Nanoparticle (Zn-NP) dietary supplementation during pregnancy and lactation on rumen microbiota of ewe’s offspring. The rumen microbiota plays an important role in host nutrition and enhancing it in the early stages of life can improve health and performance. Offspring’s microbiota is strongly shaped by the maternal microbiota. Zinc supplementation, as an essential mineral, has shown a positive effect on ruminal fermentation in ruminants. Nanoparticle (NP) minerals, due to the smaller size and easier transport through the gastrointestinal tract, provide a more efficient residence time, ensuring a greater uniformity of distribution and improving absorption in mucosal tissues and cells. Here, during the gestation and lactation, thirty primiparous ewes were randomly divided into three groups: Placebo (no supplementation), micro (zinc oxid microparticle) and nano (zinc oxid nanoparticle). After sixty days of delivery (weaning period), rumen fluid samples were taken by esophageal tube from ewes and their offspring lambs and immediately frozen for DNA extraction. Copy number of total Bacteria, Archaea, Fungi, Protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminoccocus albus, Clostridium sp, Prevotella ruminicola, Ruminobacter amilophylum, Selenomonas ruminantium and mcrA (methyl-coenzyme M reductase) were quantified by real-time quantitative PCR analysis (qPCR) (QuantStudio5, Applied Biosystems, USA) and Pearson’s analysis was performed to calculate the correlation between qPCR data from maternal microbiota and their offspring lambs. The results showed no significant difference for the abundance of microbial taxa and functional mcrA gene, however, we observed a strong correlation between the nano ewes’ group and their offspring lambs, for Archaea (Pearson Correlation coefficient of 0.616, p = 0.043), Protozoa (Correlation coefficient of 0.619, p = 0.041) and Prevotella ruminicolla (Correlation coefficient of 0.738, p = 0.009). Our study indicates that maternal Zn-NP supplementation may influence the early postnatal rumen microbiota in a positive way, which in turn may serve as a mechanism to intervene in the development of the offspring rumen microbiota.

Simple SummaryThe Tianzhu white yaks is a unique species living on the Qinghai–Tibet Plateau and has strong adaptability to high altitude and low oxygen. These yaks mainly rely on natural pasture as the source of nutrients. However, because of low temperature and insufficient pasture in winter, the Tianzhu white yak lose weight. The ruminal microbiota of yaks can efficiently acidify and hydrolyze fiber to produce volatile fatty acids (VFAs). In this study, we investigated how the ruminal microbiota affects the growth performance of yaks after supplementing straw feed and assessed the changes in ruminal microbiota and VFAs. Correlation analysis revealed a correlation among the ruminal microbiota, VFAs, and daily weight gain (ADG) of yaks. Mainly, the abundance of cellulose bacteria that can hydrolyze straw feed increased to produce more levels of VFAs, which were absorbed by the yaks, increasing their ADG.The Tianzhu white yaks (Bos grunniens) live on the Qinghai–Tibet Plateau. During winter, a lack of resources and low nutritional levels seriously affect their growth performance. In this study, we aimed to explore the effect of supplementation straw pellets on the growth performance and ruminal microbiota of yaks. Overall, at 6 (6M, n = 24), 18 (18M, n = 26), 30 (30M, n = 20), 42 (42M, n = 24), and 54 (54M, n = 22) month old Tianzhu white yaks were selected (total n = 116) and divided into the mixed straw + grazing (MSG), corn straw + grazing (CSG), and the grazing control (G) groups according to age and gender. Their growth performance was measured as per different dietary treatments. The rumen microbial community structure and levels of VFAs were analyzed from the 6M, 30M, and 54M male yaks from each group. The supplementary diets led to an increase in the ADG, which was the highest in the MSG group. The MSG group exhibited the highest level of acetate and total VFAs (TVFAs) among the three groups (p < 0.05). In addition, the 16S rRNA sequencing results proved that the microbial composition was dominated by the members of Firmicutes and Bacteroidetes. Christensenellaceae R-7 group was significantly abundant in the CSG and MSG groups compared to the G group (p < 0.05). Principal coordinate analysis (PCoA) revealed that the bacterial community structure of rumen in the MSG and CSG groups was considerably different from that in the G group; 6M samples exhibited different rumen microbial diversity compared with the other samples. Correlation analysis revealed that Christensenellaceae_R-7_group was positively correlated with the levels of acetate, TVFAs, and ADG. These results demonstrated that mixed straw pellets improved the growth performance of yaks, increased the abundance of Christensenellaceae R-7_group involved in cellulose degradation in the rumen, and produced large amounts of VFAs, which were absorbed by yaks, thus increasing their ADG. This study provides new insights into the effects of straw pellet supplementation on the changes in the rumen microbiota and growth performance of yaks.

Yak (Bos grunniens) is a unique large ruminant species in the Qinghai-Tibetan Plateau (QTP). Changing the energy levels of their rations can significantly improve their growth performance. Therefore, studying the effects of dietary energy levels on the rumen microflora and metabolites of yak is crucial for enhancing the development of the yak industry. Currently, there is a lack of understanding regarding the impact of feeding energy diets on rumen fermentation parameters, microbial functions, and metabolites. This study was designed to determine the appropriate energy level for feeding yak. Three test diets with metabolizable energy levels of 7.57 MJ/kg, 9.44 MJ/kg, and 11.9 MJ/kg were used and the concentration of volatile fatty acids (VFA) in rumen fluid was measured. The microbial communities, functions, and metabolites in yaks were studied by 16S rRNA sequencing, metagenome, and LC-MS non-targeted metabolomics to investigate the relationships among rumen fermentation parameters, microbial diversity, and metabolites. Ration energy levels significantly affect total VFA, acetate, propionate, butyrate, iso-valerate, valerate, and acetate/propionate (p < 0.05). At the phylum level, the dominant phyla in all three treatment groups were Bacteroidota, Firmicutes, and Actinobacteriota. At the genus level, the abundance of the unclassified_o__Bacteroidales, norank_f_Muribaculaceae, Lachnospiraceae_NK4A136_group, and Family _XIII_AD3011_group showed significant differences (p < 0.05) and were significantly correlated with differential metabolites screened for phosphatidylcholine [PC(16:0/0:0), PC(18:3/0:0)], uridine 3'-monophosphate, and adenosine monophosphate, etc. CAZymes family analysis showed that GHs and CEs differed significantly among the three groups. In addition, differential metabolites were mainly enriched in the pathways of lipid metabolism, nucleotide metabolism, and biosynthesis of other secondary metabolites, and the concentrations of differential metabolites were correlated with microbial abundance. In summary, this study analyzed the effects of ration energy levels on rumen microorganisms and metabolites of yaks and their relationships. The results provided a scientific basis for the selection of dietary energy for yaks in the house feeding period in the future.

Background: In high-altitude animals, brain cell resilience against hypoxia stress is one critical evolutionary step that has promoted individual survival and species adaptation to the environment. Aquaporin-4 (AQP4) is implicated in a number of physiopathological processes, particularly in the development of brain edema, and other functions such as the regulation of extracellular space volume, potassium buffering, waste clearance, and calcium signaling. Still, the role of AQP4 in the adaptation to high-altitude hypoxia remains unknown. The yak (Bos grunniens) is the only large mammal that is currently known to have adapted to the high-altitude hypoxic environment of the Qinghai-Tibet Plateau (>4000 m above sea level). Methods: In this study, we cloned the complementary DNA (cDNA) for yak AQP4 and analyzed structural differences of AQP4 between yak and cattle. We used reverse transcription quantitative polymerase chain reaction and western blot to investigate whether the expression of AQP4 mRNA and protein was different in brain of yak and cattle. In addition, immunohistochemistry was use to analyze the localization and expression of AQP4 in brain of yak and cattle. Results: Immunohistochemical results have shown that AQP4 is expressed in many regions of the yak brain, and both protein and messenger RNA (mRNA) levels are significantly lower than those of low-altitude cattle (Bos taurus). Phylogenetic analysis revealed that yak AQP4 is evolutionarily conserved. Interestingly, a substitution of Ala (cattle) to Ser in position 82, and eight additional amino acid residues composing an α-helix region are present in yak AQP4 protein. These sequence modifications potentially modulate the function of AQP4 in distinct environments. Conclusions: Our findings suggest that AQP4 may have an important role in the resistance to cerebral edema through low expression and maintenance of normal physiological function in the yak brain.

The objective of this study was to investigate the effects of dietary neutral detergent fiber (NDF) to non-fibrous carbohydrate (NFC) ratios on in vitro rumen fermentation, methane production, and microbiota in dairy cows. In vitro rumen fermentation was conducted with five dietary groups: R0.48 (NDF/NFC = 0.48), R0.57 (NDF/NFC = 0.57), R0.70 (NDF/NFC = 0.70), R0.90 (NDF/NFC = 0.90), and R1.12 (NDF/NFC = 1.12). As the NDF/NFC ratios increased, total gas production decreased linearly. The degradation rates of dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) decreased, showing a quadratic response (p = 0.018). Methane production per unit of DM was not significantly affected (p > 0.05), whereas methane production per unit of degraded DM increased (p < 0.05). The concentrations of acetate, lactate, and the acetate-to-propionate ratio, as well as pH, increased linearly (p < 0.05), whereas the concentrations of propionate, isobutyrate, isovalerate, and total volatile fatty acids (TVFA) decreased linearly (p < 0.05). Microbial crude protein (MCP) production was greater in the low NDF/NFC groups (p = 0.003). Quantitative real-time PCR analysis revealed that anaerobic fungi were more abundant in the high NDF/NFC groups (p = 0.001), whereas bacterial and archaeal abundances did not differ significantly among groups (p > 0.05). Illumina MiSeq PE250 sequencing revealed that the alpha diversity of both bacterial and archaeal communities was influenced by NDF/NFC (p < 0.05). Principal coordinate analysis further indicated that composition of bacterial (R2 = 0.298, p = 0.001) and archaeal (R2 = 0.470, p = 0.001) communities differed significantly among groups. Differences in bacterial communities were primarily driven by Firmicutes (e.g., Ruminococcus, Pseudobutyrivibrio) and Proteobacteria (e.g., Succinivibrio, Ruminobacter), whereas variations in archaeal communities were associated with Methanobacteriales and Methanomicrobiales. In conclusion, increasing dietary NDF/NFC ratios led to higher methane production per unit of degraded DM, reduced nutrient degradation, and lower TVFA concentrations during in vitro rumen fermentation, accompanied by distinct shifts in bacterial and archaeal community composition.

Rumen microbiota is pivotal for nutrient metabolism and physiological adaptation in ruminants. This study investigated the rumen microbial community, fermentation parameters, and serum biochemistry of three Cervid species-Sika deer (Cervus nippon), Reindeer (Rangifer tarandus), and Milu deer (Elaphurus davidianus) (n = 5/group)-fed an identical diet. Using 16S rRNA sequencing and biochemical analyses, we found that while Bacteroidota, Firmicutes, and Proteobacteria were dominant phyla across species. Sika deer and Milu deer exhibited significantly higher microbial diversity and abundance of carbohydrate-digesting genera (e.g., Butyrivibrio, Saccharofermentans), and pathways of carbohydrate digestion and absorption, starch and sucrose metabolism compared to Reindeer. Conversely, Reindeer showed increased abundances of Lachnospiraceae ND3007 and butyrate metabolism pathway, and significantly elevated rumen volatile fatty acid concentrations, particularly acetate and butyrate. Serum profiling revealed that Milu deer had significantly higher lipid levels (CHO, TG, LDL-C) but lower total protein and AST levels compared to other species. Notably, WGCNA linked these blood lipid traits to host genes enriched in PI3K-Akt, MAPK, and bile secretion pathways. These findings demonstrate distinct species-specific rumen fermentation patterns and host metabolic adaptations, suggesting a coordinated regulation between the rumen microbiome and host genetics in Cervid.

Metabolic adaptation in natural bacterial populations supplemented with selected amino acids

Ovarian cancer is the eighth most common malignancy among women worldwide. Despite advances in treatment, rising cases of chemoresistance and associated morbidity continue to pose significant challenges, highlighting the urgent need to identify novel therapeutic targets. The tumor microenvironment presents a hostile context for cancer cells, compelling them to undergo metabolic rewiring and adaptation to ensure survival. Acetate-dependent acetyl-CoA synthase 2 (ACSS2), a critical cytosolic enzyme, translocates to the nucleus in response to cellular stress. Given its essential metabolic and regulatory roles, ACSS2 has emerged as a key player in promoting cancer progression, particularly under metabolic stress conditions such as hypoxia. However, its role in ovarian cancer remains unexplored. This study aims to elucidate the role of ACSS2 in ovarian cancer progression under hypoxic conditions. Immunohistochemistry was performed on ovarian cancer tissue samples, including High Grade Serous Ovarian Carcinoma (HGSOC), and control tissues, to evaluate ACSS2 expression and localization. Ovarian cancer cell lines (Skov-3 and PA-1) were treated with an ACSS2 inhibitor under normoxic and hypoxic conditions and functional assays, including proliferation, colony formation, migration, invasion, and three-dimensional spheroid growth, were conducted. The impact of ACSS2 inhibition on key signaling pathways, including PI3K and HIF, was analyzed using RT-qPCR and Western blot. We observed that ACSS2 was overexpressed and predominantly localized to the nucleus in HGSOC tissues, with significantly higher H-scores compared to low-grade and normal tissues. ACSS2 inhibition impaired proliferation, colony formation, and spheroid growth in both cell lines, with greater sensitivity observed under hypoxic conditions. Migration and invasion assays revealed that ACSS2 inhibition significantly reduced cellular motility and invasiveness, particularly in hypoxia. Mechanistic studies demonstrated that ACSS2 inhibition modulates the PI3K pathway and downregulates HIF-responsive genes, suggesting its role in metabolic adaptation and tumor progression. ACSS2 is a critical regulator of ovarian cancer growth and invasiveness, particularly under hypoxic stress. Targeting ACSS2 may represent a promising therapeutic strategy for managing HGSOC. Further studies are warranted to evaluate its clinical potential.

In the seasonally dry tropics the effects of three times of weaning and three nutritional regimes on the changes in liveweight (LW) and body condition score (BCS) of grazing Bos indicus × Bos taurus breeder cows (n = 210) and their calves were examined through an annual cycle, commencing in the early dry season in April 1998. Most of the cows (n = 180) were lactating initially, and were weaned in April (W1), July (W2) or September (W3) to represent the expected early, mid and late dry season. In addition, cows that had not lactated for 11 months before the experiment commenced (NOCALF treatment; n = 30) were examined. The seasonal break occurred in late August, 3.5 months earlier than average for the site. The nutritional regimes consisted of a native pasture (LOW), another native pasture augmented with Stylosanthes spp. legumes (MEDIUM), or this latter pasture supplemented during the dry season with molasses-urea (HIGH). These nutritional regimes were imposed from the commencement of the experiment in April 1998 until February 1999, except that for the HIGH treatment the supplement was fed only during the dry season. Near-infrared reflectance spectroscopy of faeces (F.NIRS) was used to estimate the contents of non-grass, crude protein (CP) and DM digestibility of the diet selected, and also DM intake and metabolisable energy (ME) intake. Diet quality was in accord with the expected seasonal cycle, and was consistently lower (P &amp;lt; 0.05) for the LOW than for the MEDIUM treatment. Concentrations of CP and CP/MJ ME in the diet, and of N in faeces, indicated that the cows grazing the LOW treatment were deficient in rumen degradable protein during the dry season. There was no interaction (P &amp;gt; 0.05) between the nutritional regime and the time of weaning on changes in conceptus-free liveweight (CF.LW) or BCS during the dry season. Weaning increased breeder CF.LW, relative to lactating breeders, by 0.42 kg/day in the early dry season (April–July; the difference between the W1 and W2 treatments), and 0.18 kg/day in the usual mid dry season (July–September; the difference between the W2 and W3 treatments). The NOCALF treatment cows were initially 79 kg heavier than lactating cows, and lost more LW during the dry season. Microbial CP synthesis was 21 and 29% greater (P &amp;lt; 0.05) in lactating than in non-lactating cows in the late dry season and shortly after the seasonal break (August and September), respectively. Calf growth was not affected (P &amp;gt; 0.05) by nutritional regime during the early dry season (April–July), but was lower for the LOW nutritional regime during the usual mid dry season (July–September); this indicated that the LOW nutritional regime cows mobilised sufficient additional body reserves to maintain milk production during the former, but not the latter, interval. All cows that were lactating at the commencement of the experiment gained CF.LW rapidly from September 1998 following the seasonal break. In conclusion, although nutrition affected LW change of both cows and calves, there was a much larger effect of weaning than of the nutrition treatments examined on conservation of body reserves in breeder cows during the dry season. The observation that the effects of weaning on conservation of cow body reserves were similar across a wide range of nutrition is important for management to achieve appropriate targets for breeder cow body reserves.

Two trials were conducted to investigate the effects of disodium fumarate (DSF; 0.00, 8.00, 10.00 and 12.00 mM) and thyme essential oil (TEO; 0.00, 100.00, 200.00, 300.00 and 400.00 µL L-1) solely and simultaneously (10.00 mM DSF along with 100.00, 200.00, 300.00 and 400 µL L-1 TEO) on in vitro ruminal fermentation of a 50:50 alfalfa hay to concentrate diet. The DSF and TEO did not affect crude protein disappearance, gas production, microbial crude protein synthesis and hydrogen recovery. The DSF addition linearly increased partitioning factor (PF) and molar proportion of propionate and decreased acetate: propionate ratio and methane production. Moreover, 100.00 µL L-1 of TEO decreased ammonia nitrogen, total volatile fatty acids concentration and methane production and increased PF compared to the control. Results of the present study demonstrated that simultaneous use of DSF and TEO can cause a further decrease in methane production and linearly increase in the molar proportion of propionate and efficiency of feed use compared to DSF and TEO solely.

Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable the efficient conversion of poorly water-soluble gaseous substrates, thereby offering significant potential to advance bioremediation and bioproduction. However, microorganisms in the gas phase are exposed to various environmental stresses, mainly due to the absence of bulk water. While survival strategies of microorganisms in gaseous environments have been studied in environmental microbiology, the metabolic adaptations that sustain bacterial cell activity remain poorly understood. In this study, we elucidated the comprehensive metabolic alterations of a highly adhesive bacterium Acinetobacter sp. Tol 5 degrading toluene under gas- and aqueous-phase conditions. An integrated approach combining metabolomics, lipidomics, and transcriptomics revealed significant differences in metabolic profiles between cells under these conditions. Under the gas-phase condition, the degradation of amino acids and nucleic acids was significantly promoted, and the intracellular glutamate pool was maintained at high levels. Notably, citrulline was found to accumulate specifically under the gas-phase condition, representing a stress response similar to that reported in Cucurbitaceae plants during drought. Furthermore, lipidomics revealed the lipid composition of Tol 5 and demonstrated a shift in response to environmental conditions. Specifically, the degradation of intracellular storage lipids was promoted under gas-phase conditions, suggesting a crucial link to bacterial survival in water-limited environments. These findings provide critical insights into the adaptation strategies of bacteria adapting to gaseous environments, offering fundamental information for the rational design of robust gas-phase bioprocesses and a deeper understanding of environmental microbiology.